**Meet the editor**

Dr. David Wagner, PhD is an Associate Professor in the Department of Medicine, Division of Pulmonary Sciences at the University of Colorado Denver. He is also the Immunology Section Head of the Webb-Waring Center at UCD. Training includes a PhD in Biomedical Sciences from The Quillen College of Medicine at East Tennessee State University and post doctoral fellowships at The

National Jewish Medical Research Center in Immunology and Diabetes. Professional memberships include the American Diabetes Association, Immunology of Diabetes Society of the Federation of Clinical Immunological Societies (FOCIS) and American Association of Immunologists as well as a member of the Society for Luekocyte Biology. Invited Lectures include regular attendance at the Aegean Conferences: Mechanisms and Treatments for Autoimmunity. He has numerous publications in diabetes research and immunologic functions focusing on T cell development, TCR revision and pathogenic T cells.

Contents

**Preface IX** 

**Part 1 Diabetes Onset 1** 

Chapter 3 **Islet Endothelium:** 

Chapter 5 **Hypoglycemia as a** 

Chapter 1 **Genetic Determinants of** 

Chapter 2 **Early and Late Onset Type 1 Diabetes:** 

Laura Espino-Paisan, Elena Urcelay,

Chapter 4 **Type 1 Diabetes Mellitus and Co-Morbidities 85** 

**Pathological Result in Medical Praxis 109** 

**Focus on the Vascular Endothelium 157** 

**Pediatric Patients with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 143**  Miguel Ángel García Cabezas and Bárbara Fernández Valle

Adriana Franzese, Enza Mozzillo,

Chapter 6 **Autoimmune Associated Diseases in** 

**Part 2 Cardiovascular Complications 155** 

Petru Liuba and Emma Englund

Chapter 7 **Etiopathology of Type 1 Diabetes:** 

**Microvascular Complications in Type 1 Diabetes 3**  Constantina Heltianu, Cristian Guja and Simona-Adriana Manea

Emilio Gómez de la Concha and Jose Luis Santiago

**One and the Same or Two Distinct Genetic Entities? 29** 

**Role in Type 1 Diabetes and in Coxsackievirus Infections 55**  Enrica Favaro, Ilaria Miceli, Elisa Camussi and Maria M. Zanone

Rosa Nugnes, Mariateresa Falco and Valentina Fattorusso

G. Bjelakovic, I. Stojanovic, T. Jevtovic-Stoimenov, Lj.Saranac, B. Bjelakovic, D. Pavlovic, G. Kocic and B.G. Bjelakovic

### Contents

#### **Preface XIII**

#### **Part 1 Diabetes Onset 1**


Chapter 3 **Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 55**  Enrica Favaro, Ilaria Miceli, Elisa Camussi and Maria M. Zanone

	- **Part 2 Cardiovascular Complications 155**

Contents VII

Chapter 18 **Cell Replacement Therapy: The Rationale for** 

Stephen J. M. Skinner, Paul L. J. Tan,

**Part 5 Diabetes and Oral Health 409**

Chapter 19 **Dental Conditions and Periodontal** 

Chapter 20 **Impact of Hyperglycemia on** 

Chapter 21 **The Effect of Type 1 Diabetes**

M.G.K. Albrecht

S. Mikó and M. G. Albrecht

Luciana Reis Azevedo-Alanis

**Encapsulated Porcine Islet Transplantation 391**

**Xerostomia and Salivary Composition and Flow** 

Carlos Cesar Deantoni, Rosângela Réa and

**Mellitus on the Craniofacial Complex 437**  Mona Abbassy, Ippei Watari and Takashi Ono

Chapter 22 **The Role of Genetic Predisposition in Diagnosis and Therapy** 

**of Periodontal Diseases in Type 1 Diabetes Mellitus 463** 

Olga Garkavenko, Marija Muzina, Livia Escobar and Robert B. Elliott

**Disease in Adolescents with Type 1 Diabetes Mellitus 411** 

**Rate of Adolescents with Type 1 Diabetes Mellitus 427**  Ivana Maria Saes Busato, Maria Ângela Naval Machado, João Armando Brancher, Antônio Adilson Soares de Lima,


#### **Part 3 Retinopathy 279**


#### **Part 4 Treatment 321**


Chapter 18 **Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 391**  Stephen J. M. Skinner, Paul L. J. Tan, Olga Garkavenko, Marija Muzina, Livia Escobar and Robert B. Elliott

#### **Part 5 Diabetes and Oral Health 409**

VI Contents

Chapter 8 **Cardiovascular Autonomic Dysfunction in Diabetes** 

Chapter 9 **Microvascular and Macrovascular Complications in**

Chapter 10 **Type 1 Diabetes Mellitus: Redefining the Future of** 

Aleksandar N. Jovanovic and Radojica V. Stolic

Alessandra Saldanha De Mattos Matheus and

Chapter 13 **Review of the Relationship Between Renal and Retinal** 

Pedro Romero-Aroca , Juan Fernández-Ballart, Nuria Soler, Marc Baget-Bernaldiz and

Chapter 15 **Perspectives of Cell Therapy in Type 1 Diabetes 323** Maria M. Zanone, Vincenzo Cantaluppi,

Enrica Favaro, Elisa Camussi, Maria Chiara Deregibus and

**Adult Pancreas: Self-Renewal Versus Neogenesis 367** 

Chapter 14 **Ocular Complications of Type 1 Diabetes 293** Daniel Rappoport, Yoel Greenwald, Ayala Pollack and Guy Kleinmann

Chapter 16 **Prevention of Diabetes Complications 353** 

Chapter 17 **The Enigma of -Cell Regeneration in the** 

M. Trucco and R. Bottino

A. Criscimanna, S. Bertera, F. Esni,

**Microangiopathy in Type 1 Diabetes Mellitus Patients 281** 

Chapter 12 **Understanding Pancreatic Secretion in Type 1 Diabetes 261**

Anwar B. Bikhazi, Nadine S. Zwainy, Sawsan M. Al Lafi, Shushan B. Artinian and

Chapter 11 **Diabetic Nephrophaty in Children 245**  Snezana Markovic-Jovanovic,

Mirella Hansen De Almeida,

Giovanna A. Balarini Lima

Isabel Mendez-Marin

**Part 3 Retinopathy 279** 

**Part 4 Treatment 321**

Giovanni Camussi

Nepton Soltani

Yu-Long Li

Suzan S. Boutary

**as a Complication: Cellular and Molecular Mechanisms 167**

**Cardiovascular Complications with Novel Treatments 219**

**Children and Adolescents with Type 1 Diabetes 195** Francesco Chiarelli and M. Loredana Marcovecchio


Preface

treatment strategies.

focus on diabetes complications.

This book is a compilation that includes reviews on type 1 diabetes onset, complications of cardio, vascular, retinal, oral health and potential treatment options. Authors have reviewed current literature on each of these topics to provide an excellent compendium on current understanding of how type 1 diabetes evolves and progresses with more emphasis on diabetic complications and on the current status of

The etiology of diabetes remains a mystery. There is discussion about the genetic predisposition and more detailed complications including neural, nephropathy and co-morbidity in youth. The autoimmune nature of the disease including CD4+ and CD8+ T cells have been extensively explored; yet why these cells become pathogenic and the underlying causes of pathogenesis are not fully understood. This book is an excellent review of the most current understanding on development of disease with

Webb-Waring Center and Department of Medicine, University of Colorado,

**David Wagner,**

USA

### Preface

This book is a compilation that includes reviews on type 1 diabetes onset, complications of cardio, vascular, retinal, oral health and potential treatment options. Authors have reviewed current literature on each of these topics to provide an excellent compendium on current understanding of how type 1 diabetes evolves and progresses with more emphasis on diabetic complications and on the current status of treatment strategies.

The etiology of diabetes remains a mystery. There is discussion about the genetic predisposition and more detailed complications including neural, nephropathy and co-morbidity in youth. The autoimmune nature of the disease including CD4+ and CD8+ T cells have been extensively explored; yet why these cells become pathogenic and the underlying causes of pathogenesis are not fully understood. This book is an excellent review of the most current understanding on development of disease with focus on diabetes complications.

> **David Wagner,**  Webb-Waring Center and Department of Medicine, University of Colorado, USA

**Part 1** 

**Diabetes Onset** 

**Part 1** 

**Diabetes Onset** 

**1** 

*Romania* 

**Genetic Determinants of Microvascular** 

*1Institute of Cellular Biology and Pathology "N. Simionescu", Bucharest,*

Constantina Heltianu1, Cristian Guja2 and Simona-Adriana Manea1

*2Institute of Diabetes, Nutrition and Metabolic Diseases "Prof. NC Paulescu", Bucharest,*

Diabetes mellitus is one of the most prevalent chronic diseases of modern societies and a major health problem in nearly all countries. Its prevalence has risen sharply worldwide during the past few decades (Amos et al., 1997; Shaw et al., 2010). Moreover, predictions show that diabetes prevalence will continue to rise, reaching epidemic proportions by 2030: 7.7% of world population, representing 439 million adults worldwide (Shaw et al., 2010). This increase is largely due to the epidemic of obesity and consequent type 2 diabetes (T2DM). However, the incidence of type 1 diabetes (T1DM) is also rising all over the world (DiaMond Project Group, 2006; Maahs et al., 2010). Recent data for Europe (Patterson et al., 2009) predict the doubling of new cases of T1DM between 2005 and 2020 in children

younger than 5 years and an increase of 70% in children younger than 15 years, old.

Despite major progresses in T1DM treatment during the past decades, mortality in T1DM patients continues to be much higher than in general population, with wide variations in mortality rates between countries. In Europe, these variations are not explained by the country T1DM incidence rate or its gross domestic product, but are greatly influenced by the presence of its chronic complications, especially diabetic renal disease (Groop et al., 2009; Patterson et al., 2007). In fact, much of the health burden related to T1DM is created by its chronic vascular complications, involving both large (macrovascular) and small

Many genetic, metabolic and hemodynamic factors are involved in the genesis of diabetic vascular complications. However, major epidemiological and interventional studies showed that chronic hyperglycemia is the main contributor to diabetic tissue damage (DCCT Research Group, 1993). If the degree of metabolic control remains the main risk factor for the development of diabetic chronic complications, an important contribution can be attributed to genetic risk factors, some of them common for all microvascular complications (diabetic retinopathy, neuropathy, and renal disease) and some specific for each of them (Cimponeriu et al., 2010). Additional factors are represented by some accelerators such as hypertension

In the following pages, we present briefly the pathogenesis type 1 diabetes and its chronic microvascular complications. The main information of the genetic background in T1DM with particular focus on gene variants having strong impact on endothelial dysfunction as the key

factor in the development of microvascular disorders are also summarized.

**1. Introduction** 

(microvascular) blood vessels.

and dyslipidemia.

**Complications in Type 1 Diabetes** 

### **Genetic Determinants of Microvascular Complications in Type 1 Diabetes**

Constantina Heltianu1, Cristian Guja2 and Simona-Adriana Manea1 *1Institute of Cellular Biology and Pathology "N. Simionescu", Bucharest, 2Institute of Diabetes, Nutrition and Metabolic Diseases "Prof. NC Paulescu", Bucharest, Romania* 

#### **1. Introduction**

Diabetes mellitus is one of the most prevalent chronic diseases of modern societies and a major health problem in nearly all countries. Its prevalence has risen sharply worldwide during the past few decades (Amos et al., 1997; Shaw et al., 2010). Moreover, predictions show that diabetes prevalence will continue to rise, reaching epidemic proportions by 2030: 7.7% of world population, representing 439 million adults worldwide (Shaw et al., 2010). This increase is largely due to the epidemic of obesity and consequent type 2 diabetes (T2DM). However, the incidence of type 1 diabetes (T1DM) is also rising all over the world (DiaMond Project Group, 2006; Maahs et al., 2010). Recent data for Europe (Patterson et al., 2009) predict the doubling of new cases of T1DM between 2005 and 2020 in children younger than 5 years and an increase of 70% in children younger than 15 years, old.

Despite major progresses in T1DM treatment during the past decades, mortality in T1DM patients continues to be much higher than in general population, with wide variations in mortality rates between countries. In Europe, these variations are not explained by the country T1DM incidence rate or its gross domestic product, but are greatly influenced by the presence of its chronic complications, especially diabetic renal disease (Groop et al., 2009; Patterson et al., 2007). In fact, much of the health burden related to T1DM is created by its chronic vascular complications, involving both large (macrovascular) and small (microvascular) blood vessels.

Many genetic, metabolic and hemodynamic factors are involved in the genesis of diabetic vascular complications. However, major epidemiological and interventional studies showed that chronic hyperglycemia is the main contributor to diabetic tissue damage (DCCT Research Group, 1993). If the degree of metabolic control remains the main risk factor for the development of diabetic chronic complications, an important contribution can be attributed to genetic risk factors, some of them common for all microvascular complications (diabetic retinopathy, neuropathy, and renal disease) and some specific for each of them (Cimponeriu et al., 2010). Additional factors are represented by some accelerators such as hypertension and dyslipidemia.

In the following pages, we present briefly the pathogenesis type 1 diabetes and its chronic microvascular complications. The main information of the genetic background in T1DM with particular focus on gene variants having strong impact on endothelial dysfunction as the key factor in the development of microvascular disorders are also summarized.

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 5

Several mechanisms explain the contribution of chronic hyperglycemia to the development of endothelial dysfunction and chronic diabetes complications. An unifying mechanism was proposed by Michael Brownlee, suggesting that overproduction of superoxide anion (O2

the mitochondrial electron transport chain might be the key element (Brownlee, 2005). According to this theory, hyperglycemia determines increased mitochondrial production of reactive oxygen species (ROS). Increased oxidative stress induces nuclear DNA strand breaks that, in turn, activate the enzyme poly ADN-ribose polymerase (PARP) leading to a cascade process that finally activates the four major pathways of diabetic complications: (1) *Increased aldose reductase* activity and activation of the *polyol pathway* lead to increased sorbitol accumulation with osmotic effects, NADPH depletion and decreased bioavailability of nitric oxide (NO). (2) *Activation of protein kinase C* with subsequent activation of NF-kB pathway and superoxide-producing enzymes. (3) *Advanced glycation end-products* (AGEs) generation with alteration in the structure and function of both intracellular and plasma proteins. (4) *Activation of the hexosamine pathway* leads to a decrease in endothelial NO synthase (NOS3) activity as well as an increase in the transcription of the transforming growth factor (TGF-β) and the

In Europe, the prevalence of DN was estimated at 31%, DR was diagnosed in 35.9% of patients while proliferative DR in 10.3%. Apart hyperglycemia, the most important risk factor was the duration of the disease. Thus, the prevalence of proliferative DR is null before 10 years diabetes duration but 40% after 30 years duration while the prevalence of DN is null before 5 years diabetes duration but reaches 40% after 15 years of diabetes (EURODIAB IDDM Complications Study Group, 1994). Similar data were provided by the diabetes control and complications trial (DCCT) study in USA. Thus, after 30 years of diabetes, the cumulative incidence of proliferative DR and DN was 50% and 25%, respectively, in the

DN in T1DM can be defined by the presence of increased urinary albumin excretion rate (UAER) on at least two distinct occasions separated by 3–6 months (Mogensen, 2000). DN is usually accompanied by hypertension, progressive rise in proteinuria, and decline in renal function. According to several guidelines, normal UAER is defined as an excretion rate below 30 mg/24 h; microalbuminuria represents an UAER between 30-300 mg/24 h while more than 300 mg/24 h defines overt proteinuria. In T1DM, five DN stages have been proposed (Mogensen, 2000). Stage 1 is characterized by renal hypertrophy and hyperfiltration, being frequently reversible with good metabolic control. Stage 2 is typically asymptomatic and lasts for an average of 10 years. Typical histological abnormalities include diffuse thickening of the glomerular and tubular basement membranes as well as glomerular hypertrophy. About 30% of subjects will progress towards microalbuminuria. Stage 3 (incipient DN) develops 10 years after the onset of diabetes. Microalbuminuria, the earliest clinically detectable sign, is well correlated with histological findings of nodular glomerulosclerosis. About 80% of subjects will progress to overt proteinuria. This proportion may decrease with tight glycemic control, hypoproteic diet and early treatment with angiotensin I-converting enzyme (ACE) inhibitors or angiotensin II receptor (Ang II R) blockers. Stage 4 (clinical or late DN) occurs on average 15–20 years after diabetes onset and is characterized by macroalbuminuria. The glomerular filtration rate (GFR) declines progressively, UAER increases usually to more than 500 mg/day and blood pressure starts

plasminogen activator inhibitor-1 (PAI-1) as reported (Brownlee, 2005).

**3.1 Diabetic nephropathy** 

DCCT conventional treatment group (DCCT/EDIC Research Group et al., 2009).


### **2. Type 1 diabetes mellitus**

T1DM is a common, chronic, autoimmune disease characterized by the selective destruction of the insulin secreting pancreatic beta cells, destruction mediated mainly by the T lymphocytes (Eisenbarth, 1986). The destruction of the insulin secreting pancreatic beta cells is progressive, leading to an absolute insulin deficiency and the need for exogenous insulin treatment for survival. The pathogenic factors that trigger anti beta cell autoimmunity in genetically predisposed subjects are not yet fully elucidated, but there is clear evidence that it appears consequently to an alteration of the immune regulation. The destruction of the beta cells in T1DM is massive and specific and it is associated with some local (Gepts, 1965) or systemic (Bottazzo et al., 1978) evidence of anti-islet autoimmunity.

It is currently considered that, on the "background" of genetic predisposition, some putative environmental trigger factors will initiate the autoimmune process that will finally lead to T1DM. Identifying these triggers proved to be difficult, mainly due to a long period of time elapses between the intervention of the putative environmental trigger and the clinical onset of overt diabetes. The most important factors seem to be non-genetic external (environmental) ones. However, from environmental factors repeatedly associated with T1DM, the most important were viral infections, dietary and nutritional factors, nitrates and nitrosamines, etc. (Akerblom et al., 2002).

*Genetic factors in the pathogenesis of T1DM in humans*. T1DM is a common, complex, polygenic disease, with many predisposing or protective gene variants, interacting with each other in generating the global genetic disease risk (Todd, 1991). The study of candidate genes identified several susceptible genes for T1DM (Concannon et al., 2009): IDDM1 encoded in the HLA region of the major histocompatibility complex (MHC) genes on chromosome 6p21 and genes mapped to the *DRB1, DQB1* and *DQA1* loci , IDDM2 encoded by the insulin gene on chromosome 11p15.5 and mapped to the *VNTR* 5' region, IDDM12 encoded by the cytotoxic T lymphocite associated antigen 4 (*CTLA4*) gene on chromosome 2q33, the lymphoid tyrosine phosphatase 22 (*PTPN22*) gene on chromosome 1p13 and the *IL2RA/CD25* gene on chromosome 10p15. The genome wide linkage (GWL) analysis strategies (Morahan et al., 2011) or genome wide association (GWA) techniques (Todd et al., 2007) led to the identification of other T1DM associated loci, for most of which the causal genes are still not elucidated.

#### **3. Chronic complications in T1DM**

T1DM is characterized by the slow progression towards the generation of some specific lesions of the blood vessels walls, affecting both small arterioles and capillaries (microangiopathy) and large arteries (macroangiopathy). The "classical" diabetes microvascular complications are represented by *diabetic retinopathy* (DR) the main cause of blindness, *diabetic nephropathy* (DN) also known as renal disease, the main cause of renal substitution therapy (dialysis or renal transplantation) in developed countries, and *diabetic neuropathy* (DPN) as reported (IDF, 2009). As we already mentioned, chronic hyperglycemia represents the key determinant in the development of T1DM chronic microvascular complications. Meanwhile, considerable biochemical and clinical evidence (Hadi & Suwaidi, 2007) indicated that endothelial dysfunction is a critical part of the pathogenesis of vascular complications both in T1DM and T2DM.

Several mechanisms explain the contribution of chronic hyperglycemia to the development of endothelial dysfunction and chronic diabetes complications. An unifying mechanism was proposed by Michael Brownlee, suggesting that overproduction of superoxide anion (O2 - ) by the mitochondrial electron transport chain might be the key element (Brownlee, 2005). According to this theory, hyperglycemia determines increased mitochondrial production of reactive oxygen species (ROS). Increased oxidative stress induces nuclear DNA strand breaks that, in turn, activate the enzyme poly ADN-ribose polymerase (PARP) leading to a cascade process that finally activates the four major pathways of diabetic complications: (1) *Increased aldose reductase* activity and activation of the *polyol pathway* lead to increased sorbitol accumulation with osmotic effects, NADPH depletion and decreased bioavailability of nitric oxide (NO). (2) *Activation of protein kinase C* with subsequent activation of NF-kB pathway and superoxide-producing enzymes. (3) *Advanced glycation end-products* (AGEs) generation with alteration in the structure and function of both intracellular and plasma proteins. (4) *Activation of the hexosamine pathway* leads to a decrease in endothelial NO synthase (NOS3) activity as well as an increase in the transcription of the transforming growth factor (TGF-β) and the plasminogen activator inhibitor-1 (PAI-1) as reported (Brownlee, 2005).

In Europe, the prevalence of DN was estimated at 31%, DR was diagnosed in 35.9% of patients while proliferative DR in 10.3%. Apart hyperglycemia, the most important risk factor was the duration of the disease. Thus, the prevalence of proliferative DR is null before 10 years diabetes duration but 40% after 30 years duration while the prevalence of DN is null before 5 years diabetes duration but reaches 40% after 15 years of diabetes (EURODIAB IDDM Complications Study Group, 1994). Similar data were provided by the diabetes control and complications trial (DCCT) study in USA. Thus, after 30 years of diabetes, the cumulative incidence of proliferative DR and DN was 50% and 25%, respectively, in the DCCT conventional treatment group (DCCT/EDIC Research Group et al., 2009).

#### **3.1 Diabetic nephropathy**

4 Type 1 Diabetes Complications

T1DM is a common, chronic, autoimmune disease characterized by the selective destruction of the insulin secreting pancreatic beta cells, destruction mediated mainly by the T lymphocytes (Eisenbarth, 1986). The destruction of the insulin secreting pancreatic beta cells is progressive, leading to an absolute insulin deficiency and the need for exogenous insulin treatment for survival. The pathogenic factors that trigger anti beta cell autoimmunity in genetically predisposed subjects are not yet fully elucidated, but there is clear evidence that it appears consequently to an alteration of the immune regulation. The destruction of the beta cells in T1DM is massive and specific and it is associated with some local (Gepts, 1965) or systemic (Bottazzo et al., 1978) evidence of anti-islet

It is currently considered that, on the "background" of genetic predisposition, some putative environmental trigger factors will initiate the autoimmune process that will finally lead to T1DM. Identifying these triggers proved to be difficult, mainly due to a long period of time elapses between the intervention of the putative environmental trigger and the clinical onset of overt diabetes. The most important factors seem to be non-genetic external (environmental) ones. However, from environmental factors repeatedly associated with T1DM, the most important were viral infections, dietary and nutritional factors, nitrates and

*Genetic factors in the pathogenesis of T1DM in humans*. T1DM is a common, complex, polygenic disease, with many predisposing or protective gene variants, interacting with each other in generating the global genetic disease risk (Todd, 1991). The study of candidate genes identified several susceptible genes for T1DM (Concannon et al., 2009): IDDM1 encoded in the HLA region of the major histocompatibility complex (MHC) genes on chromosome 6p21 and genes mapped to the *DRB1, DQB1* and *DQA1* loci , IDDM2 encoded by the insulin gene on chromosome 11p15.5 and mapped to the *VNTR* 5' region, IDDM12 encoded by the cytotoxic T lymphocite associated antigen 4 (*CTLA4*) gene on chromosome 2q33, the lymphoid tyrosine phosphatase 22 (*PTPN22*) gene on chromosome 1p13 and the *IL2RA/CD25* gene on chromosome 10p15. The genome wide linkage (GWL) analysis strategies (Morahan et al., 2011) or genome wide association (GWA) techniques (Todd et al., 2007) led to the identification of other T1DM associated loci, for most of which the causal

T1DM is characterized by the slow progression towards the generation of some specific lesions of the blood vessels walls, affecting both small arterioles and capillaries (microangiopathy) and large arteries (macroangiopathy). The "classical" diabetes microvascular complications are represented by *diabetic retinopathy* (DR) the main cause of blindness, *diabetic nephropathy* (DN) also known as renal disease, the main cause of renal substitution therapy (dialysis or renal transplantation) in developed countries, and *diabetic neuropathy* (DPN) as reported (IDF, 2009). As we already mentioned, chronic hyperglycemia represents the key determinant in the development of T1DM chronic microvascular complications. Meanwhile, considerable biochemical and clinical evidence (Hadi & Suwaidi, 2007) indicated that endothelial dysfunction is a critical part of the pathogenesis of vascular

**2. Type 1 diabetes mellitus** 

nitrosamines, etc. (Akerblom et al., 2002).

genes are still not elucidated.

**3. Chronic complications in T1DM** 

complications both in T1DM and T2DM.

autoimmunity.

DN in T1DM can be defined by the presence of increased urinary albumin excretion rate (UAER) on at least two distinct occasions separated by 3–6 months (Mogensen, 2000). DN is usually accompanied by hypertension, progressive rise in proteinuria, and decline in renal function. According to several guidelines, normal UAER is defined as an excretion rate below 30 mg/24 h; microalbuminuria represents an UAER between 30-300 mg/24 h while more than 300 mg/24 h defines overt proteinuria. In T1DM, five DN stages have been proposed (Mogensen, 2000). Stage 1 is characterized by renal hypertrophy and hyperfiltration, being frequently reversible with good metabolic control. Stage 2 is typically asymptomatic and lasts for an average of 10 years. Typical histological abnormalities include diffuse thickening of the glomerular and tubular basement membranes as well as glomerular hypertrophy. About 30% of subjects will progress towards microalbuminuria. Stage 3 (incipient DN) develops 10 years after the onset of diabetes. Microalbuminuria, the earliest clinically detectable sign, is well correlated with histological findings of nodular glomerulosclerosis. About 80% of subjects will progress to overt proteinuria. This proportion may decrease with tight glycemic control, hypoproteic diet and early treatment with angiotensin I-converting enzyme (ACE) inhibitors or angiotensin II receptor (Ang II R) blockers. Stage 4 (clinical or late DN) occurs on average 15–20 years after diabetes onset and is characterized by macroalbuminuria. The glomerular filtration rate (GFR) declines progressively, UAER increases usually to more than 500 mg/day and blood pressure starts

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 7

DR is one of the most severe diabetes complications, potentially leading to severe sight decrease or even blindness. The first clinical signs of DR (incipient, non-proliferative DN) are retinal microaneurysms, dot intraretinal hemorrhages and hard exudates (Frank, 2004). The most severe stage, proliferative DR, is characterized by retinal haemorrhages from fragile neo-vessels and, in advanced eye disease by vitreous hemorrhages and tractional detachments of the retina, both resulting in visual loss. Histologically, DR is characterized by a selective loss of pericytes from the retinal capillaries followed by the loss of capillary endothelial cells (Frank, 2004). DR pathogenesis is complex, involving both metabolic and haemodynamic factors. The most important DR specific pathways seem to be the local production of several polypeptide growth factors, including VEGF, pigment-epithelium– derived factor (PEDF), growth hormone and insulin-like growth factor-1 , as well as cytokines and inflammatory mediators such as TNFα, TNFβ, TGFβ and NO (Frank, 2004). Genetic factors appear also to have an important role in generating the DR risk in T1DM subjects with similar degrees of metabolic control and disease duration (Keenan et al., 2007). The most often studied DR candidate genes include blood pressure regulators (RAS), metabolism factors (AKR1B1, AGER, GLUT1), growth factors (VEGF, PEDF), NOS2A, NOS3, TNFα, TGFβ, ET-1 and its receptors, etc. (Cimponeriu et al., 2010; Ng, 2010). As for all diabetes chronic complications, the studies of candidate genes were more often underpowered to detect true associations, and most often the results were not reconfirmed by additional, independent studies. However, published meta-analyses suggest a real role in

DR for at least four genes (Abhary et al., 2009a; Cimponeriu et al., 2010; Ng, 2010).

Similar with DN, *ACE* gene was the most studied for DR in T1DM and data regarding its involvement will be presented further (see subchapter 6.1). *VEGFA* gene on chromosome 6p12-p21 was also intensively studied and a recent large study (including both T1DM and T2DM cases) suggested a possible effect of two gene variants (*rs699946* and *rs833068*) on DR risk in T1DM subjects (Abhary et al., 2009b). Among the candidate genes from the oxidative stress/increased ROS pathway, *NOS3* gene was intensively studied in DR and data will be presented in subchapter 4.3. Finally, maybe the strongest evidence for a role in the genetic risk for DR is provided by the analysis of *AKR1B1* gene on chromosome 7q35, encoding the rate-limiting enzyme of the polyol pathway. The most intensively studied polymorphism was the *(AC)n* microsatellite located at 2.1 kb upstream of the transcription start site (*Z-2*, *Z*  and *Z+2* alleles). A recent meta-analysis (Abhary et al., 2009a) showed that the *Z-2* allele of the *(CA)n* microsatellite is significantly associated with DR risk in both T1DM and T2DM subjects. In addition, the *T* allele of *rs759853* in the *AKR1B1* promoter seems to be protective. To our best knowledge, no attempts for both GWL and GWA for identification of

DPN is a chronic microvascular complication affecting both somatic and autonomic peripheral nerves. It may be defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in people with diabetes, after the exclusion of other causes of neuropathy. Many neuropathic patients have signs of neurological dysfunction upon clinical examination, but have no symptoms at all (negative symptoms neuropathy). On the contrary, some patients have positive symptoms (burning, itching, freezing, sometimes intense pain and often with nocturnal exacerbations), usually with distal onset and proximal

**3.2 Diabetic retinopathy** 

DR genes in T1DM were reported so far.

**3.3 Diabetic polyneuropathy** 

to rise. Histologically, mesangial expansion develops, renal fibrosis becomes more evident and leads to diffuse and nodular glomerulosclerosis. Stage 5 (end-stage renal disease) occurs on average 7 years after the development of persistent proteinuria. GFR decreases below 40 ml/min and an advanced destruction of all renal structures is observed.

DN pathogenesis is very complex and comprises both metabolic and haemodynamic factors in the renal microcirculation (Stehouwer, 2000). The glucose dependent pathways were presented briefly above. Haemodynamic factors mediate renal injury via effects on systemic hypertension, intraglomerular haemodynamics or via direct effects on renal production of cytokines, such as TGFβ and vascular endothelial growth factor (VEGF), or hormones such as angiotensin II or endothelin (ET) as reported (Schrijvers et al., 2004). In addition to the diabetes duration reflected by the level of glycated hemoglobin (HbA1c), the specific risk factors for DN are the blood pressure, older age, male sex, smoking status, and ethnic background.

Despite clear evidence for the role of genetic factors in DN, success in identifying the responsible genetic variants has been limited due to both objective and subjective difficulties, the main being represented by the small size of the DNA collections available to individual research groups (Pezzolesi et al., 2009). Strategies for the genetic investigation of DN included the analysis of candidate gene polymorphisms in case-control settings (hypothesis driven approach) as well as the GWL or GWA strategies with DN (hypothesis free approach). Numerous candidate genes were tested explaining the complexity of the diabetic renal disease pathogenesis (Cimponeriu et al., 2010; Mooyaart et al., 2011) but just few of them were reconfirmed in multiple, independent, studies. We give a list of the stronger associations in Table 1.


Table 1. Gene variants associated with DN in T1DM subjects. Identified by candidate gene study and confirmed after meta-analysis of at least 2 studies (adapted from a recent report, Mooyaart et al., 2011). I-converting *ACE*, angiotensin I-converting enzyme; *AKR1B1*, aldose reductase; *APOC1*, apoprotein C1; *APOE*, apoprotein E; *EPO*, erythropoietin; *GREM1*, gremlin 1 homolog; *HSPG2*, heparan sulfate proteoglycan; *NOS3*, endothelial nitric oxide synthase; *UNC13B*, presynaptic protein; *VEGFA*, vascular endotehlial growth factor A.

#### **3.2 Diabetic retinopathy**

6 Type 1 Diabetes Complications

to rise. Histologically, mesangial expansion develops, renal fibrosis becomes more evident and leads to diffuse and nodular glomerulosclerosis. Stage 5 (end-stage renal disease) occurs on average 7 years after the development of persistent proteinuria. GFR decreases below 40

DN pathogenesis is very complex and comprises both metabolic and haemodynamic factors in the renal microcirculation (Stehouwer, 2000). The glucose dependent pathways were presented briefly above. Haemodynamic factors mediate renal injury via effects on systemic hypertension, intraglomerular haemodynamics or via direct effects on renal production of cytokines, such as TGFβ and vascular endothelial growth factor (VEGF), or hormones such as angiotensin II or endothelin (ET) as reported (Schrijvers et al., 2004). In addition to the diabetes duration reflected by the level of glycated hemoglobin (HbA1c), the specific risk factors for DN

Despite clear evidence for the role of genetic factors in DN, success in identifying the responsible genetic variants has been limited due to both objective and subjective difficulties, the main being represented by the small size of the DNA collections available to individual research groups (Pezzolesi et al., 2009). Strategies for the genetic investigation of DN included the analysis of candidate gene polymorphisms in case-control settings (hypothesis driven approach) as well as the GWL or GWA strategies with DN (hypothesis free approach). Numerous candidate genes were tested explaining the complexity of the diabetic renal disease pathogenesis (Cimponeriu et al., 2010; Mooyaart et al., 2011) but just few of them were reconfirmed in multiple, independent, studies. We give a list of the

SNP Allele No.

*ACE* 17q23 *rs179975 D* 14 2215/2685 1.13

*APOC1* 19q13.2 *rs4420638 G* 2 857/935 1.54 *APOE* 19q13.2 *E2/E3/E4 E2* 6 889/803 1.48 *EPO* 7q21 *rs1617640 T* 2 1244/715 0.67 *GREM1* 15q13-q15 *rs1129456 T* 2 859/940 1.53 *HSPG2* 1p36.1 *rs3767140 G* 2 417/240 0.64

*UNC13B* 9 *rs13293564 T* 4 1572/1910 1.23 *VEGFA* 6p12 *rs833061 C* 2 242/301 0.48 Table 1. Gene variants associated with DN in T1DM subjects. Identified by candidate gene study and confirmed after meta-analysis of at least 2 studies (adapted from a recent report, Mooyaart et al., 2011). I-converting *ACE*, angiotensin I-converting enzyme; *AKR1B1*, aldose reductase; *APOC1*, apoprotein C1; *APOE*, apoprotein E; *EPO*, erythropoietin; *GREM1*, gremlin 1 homolog; *HSPG2*, heparan sulfate proteoglycan; *NOS3*, endothelial nitric oxide synthase; *UNC13B*, presynaptic protein; *VEGFA*, vascular endotehlial growth factor A.

studies

393 bp 3 679/657 1.45

*(AC)n repeat Z-2* 10 1380/1308 1.12 *(AC)n repeat Z+2* 10 1380/1308 0.79 *rs759853 T* 4 636/537 1.58

*rs2070744 C* 2 273/450 1.39

Case/

Control OR

ml/min and an advanced destruction of all renal structures is observed.

stronger associations in Table 1.

Gene Chromo

*AKR1B1* 7q35

some

*NOS3* 7q36 *rs3138808 a*-del

are the blood pressure, older age, male sex, smoking status, and ethnic background.

DR is one of the most severe diabetes complications, potentially leading to severe sight decrease or even blindness. The first clinical signs of DR (incipient, non-proliferative DN) are retinal microaneurysms, dot intraretinal hemorrhages and hard exudates (Frank, 2004). The most severe stage, proliferative DR, is characterized by retinal haemorrhages from fragile neo-vessels and, in advanced eye disease by vitreous hemorrhages and tractional detachments of the retina, both resulting in visual loss. Histologically, DR is characterized by a selective loss of pericytes from the retinal capillaries followed by the loss of capillary endothelial cells (Frank, 2004). DR pathogenesis is complex, involving both metabolic and haemodynamic factors. The most important DR specific pathways seem to be the local production of several polypeptide growth factors, including VEGF, pigment-epithelium– derived factor (PEDF), growth hormone and insulin-like growth factor-1 , as well as cytokines and inflammatory mediators such as TNFα, TNFβ, TGFβ and NO (Frank, 2004). Genetic factors appear also to have an important role in generating the DR risk in T1DM subjects with similar degrees of metabolic control and disease duration (Keenan et al., 2007). The most often studied DR candidate genes include blood pressure regulators (RAS), metabolism factors (AKR1B1, AGER, GLUT1), growth factors (VEGF, PEDF), NOS2A, NOS3, TNFα, TGFβ, ET-1 and its receptors, etc. (Cimponeriu et al., 2010; Ng, 2010). As for all diabetes chronic complications, the studies of candidate genes were more often underpowered to detect true associations, and most often the results were not reconfirmed by additional, independent studies. However, published meta-analyses suggest a real role in DR for at least four genes (Abhary et al., 2009a; Cimponeriu et al., 2010; Ng, 2010).

Similar with DN, *ACE* gene was the most studied for DR in T1DM and data regarding its involvement will be presented further (see subchapter 6.1). *VEGFA* gene on chromosome 6p12-p21 was also intensively studied and a recent large study (including both T1DM and T2DM cases) suggested a possible effect of two gene variants (*rs699946* and *rs833068*) on DR risk in T1DM subjects (Abhary et al., 2009b). Among the candidate genes from the oxidative stress/increased ROS pathway, *NOS3* gene was intensively studied in DR and data will be presented in subchapter 4.3. Finally, maybe the strongest evidence for a role in the genetic risk for DR is provided by the analysis of *AKR1B1* gene on chromosome 7q35, encoding the rate-limiting enzyme of the polyol pathway. The most intensively studied polymorphism was the *(AC)n* microsatellite located at 2.1 kb upstream of the transcription start site (*Z-2*, *Z*  and *Z+2* alleles). A recent meta-analysis (Abhary et al., 2009a) showed that the *Z-2* allele of the *(CA)n* microsatellite is significantly associated with DR risk in both T1DM and T2DM subjects. In addition, the *T* allele of *rs759853* in the *AKR1B1* promoter seems to be protective. To our best knowledge, no attempts for both GWL and GWA for identification of DR genes in T1DM were reported so far.

#### **3.3 Diabetic polyneuropathy**

DPN is a chronic microvascular complication affecting both somatic and autonomic peripheral nerves. It may be defined as the presence of symptoms and/or signs of peripheral nerve dysfunction in people with diabetes, after the exclusion of other causes of neuropathy. Many neuropathic patients have signs of neurological dysfunction upon clinical examination, but have no symptoms at all (negative symptoms neuropathy). On the contrary, some patients have positive symptoms (burning, itching, freezing, sometimes intense pain and often with nocturnal exacerbations), usually with distal onset and proximal

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 9

induce the renal hyperfiltration and hyperperfusion and by its perturbing effect contributes to the DN appearance (Bazzaz et al., 2010). In early diabetes, the retinal circulation devoided of any extrinsic innervation and depending entirely on endothelium-mediated autoregulation, is dramatically affected by the ECs dysfunction due to the lack of the local

In active progressive DR, aqueous NO levels are significantly high, while plasma NO levels remained at the level of diabetics without DR (Yilmaz et al., 2000). Raised plasma NO levels in T1DM patients were reported (Heltianu et al., 2008) indicating that pathogenesis of diabetic-associated vascular complications is connected with a generalized increased synthesis of NO throughout the body. This phenomenon occurs early in the natural course of diabetes and independently of the presence of microvascular complications. So, we suggest that the high NO levels found in diabetic patients (including those without any clinically manifested microangiopaties) might represent an overproduction of NO that is

There is a family of NOS enzymes which produces NO. The two constitutive isoforms NOS1 (neuronal) and NOS3 (endothelial) as well as the inducible isoform NOS2 have similar enzymatic mechanisms but are encoded on separate chromosomes by different genes. The *NOS1* gene is located on chromosome 12q24.2-24.31, has 29 exons, spaning a region greater than 240 kb and encodes a protein of ~161 kDa. The *NOS2A* gene is on chromosome 17q11.2–12 having 27 exons, spaning 37 kb and encodes a protein of ~131 kDA (Li et al., 2007). *NOS3* gene is on chromosome 7q35-36, includes 26 exons, having a genomic size of 21 kb, and encodes a protein of ~133 kDa (Chen et al., 2007; http://www.genecards.org,

The NOS1-derived NO is implicated in local regulation of vascular tone and blood flow using different mechanisms. This process appears to be independent of central NOS1 action on autonomic function (Melikian et al., 2009). In the early stages of diabetes, the NOS1 expression in the nitrergic axons decreases and its level in the cell bodies is unaffected probably due to a defect in axonal transport. Insulin treatment is able to reverse NOS1 decrease. With the progression of diabetes, NOS1 accumulates in the cell bodies due to an affected transport down to the axons, and the degenerative changes become irreversible without any response to insulin treatment (Cellek, 2004). The *NOS1* gene has 12 different potential rst exons (*1A*–*1L*) and as consequence the NOS1 protein is expressed as a very complex enzyme (Wang et al., 1999). The NOS1B is expressed in renal microvasculature (Freedman et al., 2000). To our knowledge, there are only two reports in which *NOS1* polymorphisms were analyzed for the relationship with diabetic microvascular disorders. Microsatellite markers in *NOS1B* were assessed in T2DM and an association with ESDR for alleles 7 and 9 was reported (Freedman et al., 2000). The *CA* repeat in the 3'-UTR region

*NOS2A* gene has the transcription start site in exon 2 and the stop codon in exon 27. This gene encodes NOS2 protein which has two different functional catalytic enzyme domains, the oxygenase domain encoded by 1 to 13 exons, and reductase domain by 14 to 27 exons. The NOS2 differs from the constitutive forms (NOS1 and NOS3) being Ca2+ independent.

(exon 29) of *NOS1* was found not to be a risk for DPN (Zotova et al., 2005).

associated with diffuse endothelial dysfunction (Heltianu et al., 2008).

NO, a state seen in DR (Qidwai & Jamal, 2010).

version 3).

**4.1** *NOS1* **gene** 

**4.2** *NOS2A* **gene** 

progression. This form is designated as painful DPN. Correct estimates regarding the prevalence of DPN are hard to obtain since the diagnosis of the "negative symptom patients" can be made only by active screening, usually with complex investigations such as nerve conduction velocity.

It is generally accepted that DPN results from the micro-angiopathy damage of the *vasa nervorum* (responsible for the microcirculation of neural tissue) associated with the direct damage of neuronal components induced by various metabolic factors, the most important being chronic hyperglycemia (Kempler, 2002). The vascular and metabolic mechanisms act simultaneously and have an additive effect. The most important links between the two are represented by the local NO depletion and failure of antioxidant protection, both resulting in increased oxidative stress. Apart the unquestionable role of chronic hyperglycemia (diabetes duration and level of metabolic control), other risk factors for DPN are increasing age, cigarette smoking, alcohol or other drug abuse, hypertension and hypercholesterolemia.

Data regarding the genetic background of DPN are rather scarce. To our best knowledge, no GWL or GWA were performed for the identification of DPN genes in T1DM. Several data regarding the effect of some candidate genes were published, but these included usually only small number of patients/controls and few were replicated in other, independent datasets. Maybe the most significant effect on DPN genetic risk in T1DM is conferred by variants of the *AKR1B1* gene. Other significant but not reconfirmed associations with the risk for DPN were reported for variants of *PARP-1*, *NOS2A, NOS3*, uncoupling protein *UCP2* and *UCP3,* genes encoding the antioxidant proteins, catalase and the superoxide dismutase, and the gene encoding the neuronal Na+/K+-ATPase (Cimponeriu et al., 2010).

In conclusion, during the past two decades we have witnessed an explosion of studies regarding the genetic background of diabetes microvascular complications, both in T1DM and T2DM. These efforts, mainly focusing on candidate genes and often using study groups underpowered to detect genuine associations, have contributed to the identification of a few credible predisposing gene variants (Doria, 2010). In order to make significant progresses in elucidating the genetics of microvascular complications, there is an urgent need for assembling large population collections of different backgrounds for both GWA scanning and candidate gene association studies.

#### **4. Nitric oxide synthase genes**

NO is one of the vasodilatory substances released by the endothelium and has the crucial role in vascular physiopathology including regulation of vascular tone and blood pressure, hemostasis of fibrinolysis, and proliferation of vascular smooth muscle cells (SMC). In T1DM, NO has an increased stimulatory effect on the released insulin from β cells, mostly to the early phase of the effect of glucose upon insulin secretion. Abnormality in its production and action can cause endothelial dysfunction leading to increased susceptibility to hypertension, hypercholesterolemia, diabetes mellitus, thrombosis and cerebrovascular disease. Serum nitrite and nitrate (NOx) concentrations assessed as an index of NO production was used as a marker for endothelial function.

In DN, the NO production was significantly higher. A strong link between circulating NO, glomerular hyperfiltration, and microalbuminuria in young T1DM patients with early nephropathy was reported (Chiarelli et al., 2000). It has been postulated that in diabetic kidney there is increased NO synthase (NOS) activity, and the excessive NO production can induce the renal hyperfiltration and hyperperfusion and by its perturbing effect contributes to the DN appearance (Bazzaz et al., 2010). In early diabetes, the retinal circulation devoided of any extrinsic innervation and depending entirely on endothelium-mediated autoregulation, is dramatically affected by the ECs dysfunction due to the lack of the local NO, a state seen in DR (Qidwai & Jamal, 2010).

In active progressive DR, aqueous NO levels are significantly high, while plasma NO levels remained at the level of diabetics without DR (Yilmaz et al., 2000). Raised plasma NO levels in T1DM patients were reported (Heltianu et al., 2008) indicating that pathogenesis of diabetic-associated vascular complications is connected with a generalized increased synthesis of NO throughout the body. This phenomenon occurs early in the natural course of diabetes and independently of the presence of microvascular complications. So, we suggest that the high NO levels found in diabetic patients (including those without any clinically manifested microangiopaties) might represent an overproduction of NO that is associated with diffuse endothelial dysfunction (Heltianu et al., 2008).

There is a family of NOS enzymes which produces NO. The two constitutive isoforms NOS1 (neuronal) and NOS3 (endothelial) as well as the inducible isoform NOS2 have similar enzymatic mechanisms but are encoded on separate chromosomes by different genes. The *NOS1* gene is located on chromosome 12q24.2-24.31, has 29 exons, spaning a region greater than 240 kb and encodes a protein of ~161 kDa. The *NOS2A* gene is on chromosome 17q11.2–12 having 27 exons, spaning 37 kb and encodes a protein of ~131 kDA (Li et al., 2007). *NOS3* gene is on chromosome 7q35-36, includes 26 exons, having a genomic size of 21 kb, and encodes a protein of ~133 kDa (Chen et al., 2007; http://www.genecards.org, version 3).

#### **4.1** *NOS1* **gene**

8 Type 1 Diabetes Complications

progression. This form is designated as painful DPN. Correct estimates regarding the prevalence of DPN are hard to obtain since the diagnosis of the "negative symptom patients" can be made only by active screening, usually with complex investigations such as

It is generally accepted that DPN results from the micro-angiopathy damage of the *vasa nervorum* (responsible for the microcirculation of neural tissue) associated with the direct damage of neuronal components induced by various metabolic factors, the most important being chronic hyperglycemia (Kempler, 2002). The vascular and metabolic mechanisms act simultaneously and have an additive effect. The most important links between the two are represented by the local NO depletion and failure of antioxidant protection, both resulting in increased oxidative stress. Apart the unquestionable role of chronic hyperglycemia (diabetes duration and level of metabolic control), other risk factors for DPN are increasing age, cigarette smoking, alcohol or other drug abuse, hypertension and

Data regarding the genetic background of DPN are rather scarce. To our best knowledge, no GWL or GWA were performed for the identification of DPN genes in T1DM. Several data regarding the effect of some candidate genes were published, but these included usually only small number of patients/controls and few were replicated in other, independent datasets. Maybe the most significant effect on DPN genetic risk in T1DM is conferred by variants of the *AKR1B1* gene. Other significant but not reconfirmed associations with the risk for DPN were reported for variants of *PARP-1*, *NOS2A, NOS3*, uncoupling protein *UCP2* and *UCP3,* genes encoding the antioxidant proteins, catalase and the superoxide dismutase, and the gene encoding the neuronal Na+/K+-ATPase (Cimponeriu et al., 2010). In conclusion, during the past two decades we have witnessed an explosion of studies regarding the genetic background of diabetes microvascular complications, both in T1DM and T2DM. These efforts, mainly focusing on candidate genes and often using study groups underpowered to detect genuine associations, have contributed to the identification of a few credible predisposing gene variants (Doria, 2010). In order to make significant progresses in elucidating the genetics of microvascular complications, there is an urgent need for assembling large population collections of different backgrounds for both GWA scanning

NO is one of the vasodilatory substances released by the endothelium and has the crucial role in vascular physiopathology including regulation of vascular tone and blood pressure, hemostasis of fibrinolysis, and proliferation of vascular smooth muscle cells (SMC). In T1DM, NO has an increased stimulatory effect on the released insulin from β cells, mostly to the early phase of the effect of glucose upon insulin secretion. Abnormality in its production and action can cause endothelial dysfunction leading to increased susceptibility to hypertension, hypercholesterolemia, diabetes mellitus, thrombosis and cerebrovascular disease. Serum nitrite and nitrate (NOx) concentrations assessed as an index of NO

In DN, the NO production was significantly higher. A strong link between circulating NO, glomerular hyperfiltration, and microalbuminuria in young T1DM patients with early nephropathy was reported (Chiarelli et al., 2000). It has been postulated that in diabetic kidney there is increased NO synthase (NOS) activity, and the excessive NO production can

nerve conduction velocity.

hypercholesterolemia.

and candidate gene association studies.

production was used as a marker for endothelial function.

**4. Nitric oxide synthase genes** 

The NOS1-derived NO is implicated in local regulation of vascular tone and blood flow using different mechanisms. This process appears to be independent of central NOS1 action on autonomic function (Melikian et al., 2009). In the early stages of diabetes, the NOS1 expression in the nitrergic axons decreases and its level in the cell bodies is unaffected probably due to a defect in axonal transport. Insulin treatment is able to reverse NOS1 decrease. With the progression of diabetes, NOS1 accumulates in the cell bodies due to an affected transport down to the axons, and the degenerative changes become irreversible without any response to insulin treatment (Cellek, 2004). The *NOS1* gene has 12 different potential rst exons (*1A*–*1L*) and as consequence the NOS1 protein is expressed as a very complex enzyme (Wang et al., 1999). The NOS1B is expressed in renal microvasculature (Freedman et al., 2000). To our knowledge, there are only two reports in which *NOS1* polymorphisms were analyzed for the relationship with diabetic microvascular disorders. Microsatellite markers in *NOS1B* were assessed in T2DM and an association with ESDR for alleles 7 and 9 was reported (Freedman et al., 2000). The *CA* repeat in the 3'-UTR region (exon 29) of *NOS1* was found not to be a risk for DPN (Zotova et al., 2005).

#### **4.2** *NOS2A* **gene**

*NOS2A* gene has the transcription start site in exon 2 and the stop codon in exon 27. This gene encodes NOS2 protein which has two different functional catalytic enzyme domains, the oxygenase domain encoded by 1 to 13 exons, and reductase domain by 14 to 27 exons. The NOS2 differs from the constitutive forms (NOS1 and NOS3) being Ca2+ independent.

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 11

NO, in DN, induces the renal hyperfiltration and hyperperfusion and contributes to the vascular disorder. More often, reduced NO production or availability was reported in other vascular pathologies. The effect of NO on the endothelial modulation is influenced by the duration of diabetes; so, at early stages of diabetes the endothelial function is enhanced, and with the progression of diabetic duration the endothelial dysfunction is accelerated (Bazzaz

Several polymorphisms have been reported in NOS3 promoter, exon and intron regions (Table 2). The most studied variant from the promoter region was the single nucleotide polymorphism at position -786 where there is a base substitution from *T* to *C* (*rs2070744*). In previous studies it was shown that individuals with *−786C* allele had a reduced activity of the *NOS3* gene promoter (Taverna et al., 2005), explained by the fact that DNA binding protein (replication protein A1) has the ability to bind only to the *−786C* allele resulting a ~50% reduced *NOS3* transcription, with the subsequent decrease in both protein expression and serum NOx levels (Erbs et al., 2003). The interrelationships among *rs2070744* genotypes, NOS3 (mRNA, protein levels, and enzymatic activity), and plasma NOx levels have never

*NOS3* polymorphism in intron 4 (*4a/4b*) is based on a variable 27-base pair tandem repeat four (allele *4a*), five (allele *4b*) or six (allele *4c*) repeats. Previous studies have suggested that deletion of one of the five nucleotide repeats in intron 4 could affect the rates of *NOS3*  transcription and processing rate, thus resulting the modulation of NOS3 enzymatic activity and, apparently, affecting the plasma NOx concentrations (Zanchi et al., 2000), with the potentiality of this genotype to have an effect on microangiopathy later on in diabetic life

 intron 4 *4b/4ba*  150707488 intron *A/G*  150696111 exon *G/T*  150689397 intron *T/A*  150690079 the 5' promoter region *T/C*  150345745 the 3' region *G/A*  150312143 the 5' promoter region *C/T*  Del/Ins 393 bp *D/I*  150702781 intron *A/C/T*  150314562 the 5' region *G/T*  150690106 intron *C/T*  Table 2. *NOS3* gene polymorphisms. Source, http://www.genecards.org; version 3

Carriers of the *4a* allele were found exhibiting ~20% lower NOx levels that appearing in *4b/4b* homozygous subjects. The regulation of *NOS3* expression is more complicated considering the strong linked of *4a/4b* variant with *rs2373961* and *rs2070744* when the *b/b* genotype might acts independently and in coordination with the other variants (Chen et al., 2007; Zintzaras et al., 2009). Among polymorphisms found in exons of *NOS3*, the *G* to *T* polymorphism at position 894 in exon 7 (*rs1799983*) was most studied. It was reported that

position Location type Alleles

et al., 2010; Chen et al., 2007; Mamoulakis et al., 2009).

been linear.

(Mamoulakis et al., 2009).

SNP ID Chromosome

Due to strong binding of calmodulin to NOS2, this is insensitive to changes in calcium ion concentrations (Jonannesen et al., 2001; Qidwai & Jamal, 2010). Under normal conditions, NOS2 is not expressed. Exposure to high ambient glucose or cytokines, the upregulation of NOS2 occurs in a variety of cell type and tissues. As a consequence, a sudden burst of NO synthesis occurs leading to severe vasodilation and circulatory collapse. In diabetic milieu as long as NOS3 expression is low, the induction of NOS2 expression may occur in an attempt to achieve homeostasis, being crucial in preventing or delaying pathological alterations in the microcirculation (Warpeha & Chakravarthy 2003). In studies of diabetic complications, as DR and DN, influenced by vascular functional disturbances, the increased NO formation via NOS2 expression has been reported (Johannesen et al., 2000a).

In human *NOS2A* gene has been identified a large number of polymorphisms. In the promoter region there are single nucleotide polymorphisms (*-954G/C*, *-1173CT*, -*1659 AT*) and two microsatellite repeats, the biallelic *(TAAA)*n, and the *(CCTTT)*n with nine alleles, which might affect the *NOS2* transcription. Explanations for this modulation was proposed for *–1173C/T* polymorphism, when the *C* to *T* change predicts the formation of a new sequence recognition site for the GATA-1 or GATA-2 transcription factors, which further bind to specific DNA sequences and potentially increase the degree of mRNA transcription (Qidwai & Jamal, 2010). The gene variants in the coding region might alter the activity of *NOS2* with subsequence variability in the NO levels which might be responsible for the susceptibility orand severity of the disease. Other polymorphisms in exons and introns were reported, *rs16966563* (exon 4, *Pro68Pro*), *rs1137933* (exon 10, *Asp385Asp*), *rs2297518*  (exon 16, *Leu608Ser*), *rs3794763* (intron 5, *G>A*), *rs17718148* (intron 11, *C>T*), *rs2314809* (intron 17, *T>C*), and *rs2297512* (intron 20, *A>G*).

In T1DM, the *NOS2A* polymorphisms in the promoter region [-*954G/C*, (*TAAA*)n and (*CCTTT*)n] and exons (*Asp346Asp* and *Leu608Ser*) were analyzed and the results showed that none of them has a role in the development of the disease (Johannesen et al., 2000a). Using the transmission disequilibrium test, it was found in Caucasian population that there is an increased risk for T1DM among *HLA DR3/4*-positive individuals with a *T* in position 150 in exon 16 (*Leu608Ser*) of *NOS2A*. This finding suggests an interaction between the *NOS2A* locus and the HLA region and a role for *NOS2A* in the pathogenesis of human T1DM (Johannesen et al., 2001).

Assessement of polymorphisms in T1DM for the prevalence of DR showed that the *14-repeat*  allele of (*CCTTT*)*n repeat* polymorphism in *NOS2A* was significantly associated with the absence of the disease. A person with diabetes carrying this allele has 0.21-fold chance of developing retinopathy as compared to those not carrying the allele, suggesting that the carriage of the *14-repeat* allele is not a feature of diabetes itself, but is specific to DR development (Warpeha et al., 1999; Warpeha & Chakravarthy, 2003). In addition, the same *NOS2A* variant, the *14-repeat* allele, was found to represent a low risk for DN (Johannesen et al., 2000b), and other report indicated that carriers of this allele have the low risk of DPN in T1DM (Nosikov, 2004; Zotova et al., 2005).

#### **4.3** *NOS3* **gene**

NOS3 is the most relevant and frequent isoform studied to assess the role of genetic issues in the development of angiopathic disease in T1DM. This enzyme is a constitutively expressed in vascular endothelial cells, and the protein expression depends on Ca2+ and calmodulin. It was suggested a possible dual functionality of NO. Excessive production of

Due to strong binding of calmodulin to NOS2, this is insensitive to changes in calcium ion concentrations (Jonannesen et al., 2001; Qidwai & Jamal, 2010). Under normal conditions, NOS2 is not expressed. Exposure to high ambient glucose or cytokines, the upregulation of NOS2 occurs in a variety of cell type and tissues. As a consequence, a sudden burst of NO synthesis occurs leading to severe vasodilation and circulatory collapse. In diabetic milieu as long as NOS3 expression is low, the induction of NOS2 expression may occur in an attempt to achieve homeostasis, being crucial in preventing or delaying pathological alterations in the microcirculation (Warpeha & Chakravarthy 2003). In studies of diabetic complications, as DR and DN, influenced by vascular functional disturbances, the increased NO formation

In human *NOS2A* gene has been identified a large number of polymorphisms. In the promoter region there are single nucleotide polymorphisms (*-954G/C*, *-1173CT*, -*1659 AT*) and two microsatellite repeats, the biallelic *(TAAA)*n, and the *(CCTTT)*n with nine alleles, which might affect the *NOS2* transcription. Explanations for this modulation was proposed for *–1173C/T* polymorphism, when the *C* to *T* change predicts the formation of a new sequence recognition site for the GATA-1 or GATA-2 transcription factors, which further bind to specific DNA sequences and potentially increase the degree of mRNA transcription (Qidwai & Jamal, 2010). The gene variants in the coding region might alter the activity of *NOS2* with subsequence variability in the NO levels which might be responsible for the susceptibility orand severity of the disease. Other polymorphisms in exons and introns were reported, *rs16966563* (exon 4, *Pro68Pro*), *rs1137933* (exon 10, *Asp385Asp*), *rs2297518*  (exon 16, *Leu608Ser*), *rs3794763* (intron 5, *G>A*), *rs17718148* (intron 11, *C>T*), *rs2314809*

In T1DM, the *NOS2A* polymorphisms in the promoter region [-*954G/C*, (*TAAA*)n and (*CCTTT*)n] and exons (*Asp346Asp* and *Leu608Ser*) were analyzed and the results showed that none of them has a role in the development of the disease (Johannesen et al., 2000a). Using the transmission disequilibrium test, it was found in Caucasian population that there is an increased risk for T1DM among *HLA DR3/4*-positive individuals with a *T* in position 150 in exon 16 (*Leu608Ser*) of *NOS2A*. This finding suggests an interaction between the *NOS2A* locus and the HLA region and a role for *NOS2A* in the pathogenesis of human

Assessement of polymorphisms in T1DM for the prevalence of DR showed that the *14-repeat*  allele of (*CCTTT*)*n repeat* polymorphism in *NOS2A* was significantly associated with the absence of the disease. A person with diabetes carrying this allele has 0.21-fold chance of developing retinopathy as compared to those not carrying the allele, suggesting that the carriage of the *14-repeat* allele is not a feature of diabetes itself, but is specific to DR development (Warpeha et al., 1999; Warpeha & Chakravarthy, 2003). In addition, the same *NOS2A* variant, the *14-repeat* allele, was found to represent a low risk for DN (Johannesen et al., 2000b), and other report indicated that carriers of this allele have the low risk of DPN in

NOS3 is the most relevant and frequent isoform studied to assess the role of genetic issues in the development of angiopathic disease in T1DM. This enzyme is a constitutively expressed in vascular endothelial cells, and the protein expression depends on Ca2+ and calmodulin. It was suggested a possible dual functionality of NO. Excessive production of

via NOS2 expression has been reported (Johannesen et al., 2000a).

(intron 17, *T>C*), and *rs2297512* (intron 20, *A>G*).

T1DM (Johannesen et al., 2001).

**4.3** *NOS3* **gene** 

T1DM (Nosikov, 2004; Zotova et al., 2005).

NO, in DN, induces the renal hyperfiltration and hyperperfusion and contributes to the vascular disorder. More often, reduced NO production or availability was reported in other vascular pathologies. The effect of NO on the endothelial modulation is influenced by the duration of diabetes; so, at early stages of diabetes the endothelial function is enhanced, and with the progression of diabetic duration the endothelial dysfunction is accelerated (Bazzaz et al., 2010; Chen et al., 2007; Mamoulakis et al., 2009).

Several polymorphisms have been reported in NOS3 promoter, exon and intron regions (Table 2). The most studied variant from the promoter region was the single nucleotide polymorphism at position -786 where there is a base substitution from *T* to *C* (*rs2070744*). In previous studies it was shown that individuals with *−786C* allele had a reduced activity of the *NOS3* gene promoter (Taverna et al., 2005), explained by the fact that DNA binding protein (replication protein A1) has the ability to bind only to the *−786C* allele resulting a ~50% reduced *NOS3* transcription, with the subsequent decrease in both protein expression and serum NOx levels (Erbs et al., 2003). The interrelationships among *rs2070744* genotypes, NOS3 (mRNA, protein levels, and enzymatic activity), and plasma NOx levels have never been linear.

*NOS3* polymorphism in intron 4 (*4a/4b*) is based on a variable 27-base pair tandem repeat four (allele *4a*), five (allele *4b*) or six (allele *4c*) repeats. Previous studies have suggested that deletion of one of the five nucleotide repeats in intron 4 could affect the rates of *NOS3*  transcription and processing rate, thus resulting the modulation of NOS3 enzymatic activity and, apparently, affecting the plasma NOx concentrations (Zanchi et al., 2000), with the potentiality of this genotype to have an effect on microangiopathy later on in diabetic life (Mamoulakis et al., 2009).


Table 2. *NOS3* gene polymorphisms. Source, http://www.genecards.org; version 3

Carriers of the *4a* allele were found exhibiting ~20% lower NOx levels that appearing in *4b/4b* homozygous subjects. The regulation of *NOS3* expression is more complicated considering the strong linked of *4a/4b* variant with *rs2373961* and *rs2070744* when the *b/b* genotype might acts independently and in coordination with the other variants (Chen et al., 2007; Zintzaras et al., 2009). Among polymorphisms found in exons of *NOS3*, the *G* to *T* polymorphism at position 894 in exon 7 (*rs1799983*) was most studied. It was reported that

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 13

albuminuria, suggesting a strong implication of this gene in the susceptibility to kidney damage (Ned et al., 2010). The *rs3138808* variant of *NOS3* was also analyzed in a meta-

There are only few reports which analyze the influence of *NOS3* polymorphisms on DPN in T1DM. Data from Caucasian patients genotyped for *rs1799983* and *4b/4a* variants showed that both polymorphisms were not associated with DPN (Nosikov, 2004; Zotova et al., 2005**).**  Our findings showed that only *NOS3 4b/4a* was not associated with DPN (Heltianu et al., 2009). In T1DM subjects with the lowest incidence of confirmed DPN, it was reported that the *894G* carriers of *rs1799983* variant had fivefold increased risk for DPN, suggesting that despite low risk for the disease in these individuals, there is a genetic predisposition to develop diabetes-related complication (Costacou et al., 2006). In agreement with this report we found a prevalence of DPN among the *894GG* as compared with *894TT* homozygotes in diabetic patients with normal kidney function, suggesting that *894GG* genotype might be a risk factor for T1DM-related microvascular disease. This subgroup of DPN patients with *894GG* had over 42% DR as an additional vascular complication, and the presence or absence of DR did not modify the significance of the relationship between the *rs1799983*  polymorphism and DPN. In addition, these subjects were recorded with high systolic blood pressure and raised levels of NOx, indicating a possible endothelial dysfunction, as well as with high levels of triglycerides, suggesting that additional high risk lipid profile contribute to the aggravation of the microvascular disorder. We presume that the rare-type *894T* allele might have a protective role against the development of DPN and a tendency to counterbalance increased NO production due to both chronic hyperglycemia and hypoxic effect at the microvascular level, by a not yet elucidated, compensatory-type mechanism

Taken together, these results indicate that in T1DM, from various *NOS3* polymorphisms the most studied were *rs2070744*, *rs1799983* and *4b/4a* variants. Even in Caucasians there are differences among populations for the effects of gene polymorphisms on the microvascular complications. Diverse factors contribute to the variations between studies, analysis of early or late microvascular complication, incidence of the studied disorder in subjects with other confirmed disease, small sample size, the lack of haplotype analysis. Further studies on larger numbers of samples and on different populations are required to confirm these

The family of endothelins (ET) is represented by three peptides (1 to 3) and two receptors (ETRA and ETRB), which are widely distributed, in different proportions, being mostly abundant in vascular endothelial cells (EC). Their ET-1 and ET-2 are strong vasoconstrictors, whereas ET-3 is a potentially weaker vasoconstrictor compared to the other two isoforms. The ET-1 which is the most potent vasoconstrictor peptide acts as a paracrine or autocrine factor and its effects are ~10 times higher that of angiotensin II. The ET-1 has a variety of functions including its significant contribution to the maintenance of basal vascular tone, modulation of vascular permeability for proinflammatory mediators and proliferation of SMC. Having a long half-life, only a slight activation of its receptors into the signaling pathways might contribute to progressive disturbances, as hypertension and diabetic microvascular disorders (Cimponeriu et al., 2010). From the two receptors, the ETRA, expressed in SMC, has the highest affinity for ET-1, and is involved in the short term

analysis and was found to be associated with DN (Mooyaart et al., 2011).

(Heltianu et al., 2009).

**5. Endothelin genes** 

results.

this variant changes the NOS3 protein sequence, probable resulting an alteration of enzyme activity (Costacou et al., 2006), and control the NOS3 intracellular distribution interacting with proteins of degradating process (Brouet et al., 2001).

From many polymorphisms of the *NOS3* gene some of them are associated with the development of diabetic microvascular complications while others indicated their protective role (Freedman et al., 2007; Heltianu et al., 2009). A recent study of *rs2070744* in Caucasian T1DM reported a positive association with diabetes *per se* as well as DR and two possible explanations were found; either *NOS3* is a candidate gene for the microvascular disease, or there is a linkage disequilibrium between *NOS3* and the neighbouring genes. It is known that in the same position (7q35) to *NOS3* gene the AKR1B1 and T-cell receptor beta-chain (TCRBC) genes in the 7q34 position are located (Bazzaz et al., 2010). In a hyperglycaemic milieu, the retinal NO bioavailability due to the presence of C-786 mutant allele of rs2070744 is decreased, and therefore the lack of NO stimulates aldose reductase, known to be implicated in the development of diabetes complications (Chandra et al., 2002). Other report showed that the onset pattern of severe DR in longstanding C-peptide-negative T1DM is affected by *NOS3 rs2070744* and *C774T* polymorphisms (Taverna et al., 2005). In the case of *C774T NOS3* polymorphism, the association with severe DR was related to the influence of the DN presence, which is a well-known strong risk factor for DR (Cimponeriu et al., 2010). Oppose, the rare allele *4a* of *4b/4a* variant of *NOS3* was found to be related to absent or nonsevere DR in T1DM Caucasians patients, suggesting a protective role. Although the *4b* allele was more frequent among patients with severe DR, a modest effect on the microvascular disorder was evaluated from the broad confidence interval (Cheng et al., 2007). Recent reports and our studies showed that there were no relationships between *4b/4a* variant of NOS3 and DR or other microangiopathic complications. Similar results for *rs179998*3 in relation with DR were also reported (Heltianu et al., 2009; Mamulakis et al., 2009). In a metaanalysis of genetic association studies for DR in T1DM, from the three *NOS3* polymorphisms (*rs1799983*, *rs3138808* and *rs413220*52) included in the sub analysis for Caucasian subjects, none of them were found to be significantly associated with any form of DN (Abhary et al., 2009a)

The progression of renal disease was associated with the *NOS3 rs2070744* variant (Freedman et al., 2007; Zanchi et al., 2000), a result confirm recently by meta-analysis (Mooyaart et al., 2011; Ned et al., 2010). Contradictory results were obtained for the relationship of *NOS3 4b/4a* polymorphism with DN. Some reports showed no association (Degen et al., 2001; Heltianu et al., 2009) and others indicated that the *4a* allele represents an excess risk for advanced DN (Nosikov, 2004; Zanchi et al., 2000; Zinzaras et al., 2009). It was hypothesized that the *NOS3 4b/4a* itself plays a role in tissue-specific regulation of *NOS3* expression, a mechanism related to the importance of intron structure in the splicing of immature to mature RNA or to the presence of enhancer sequences within the intron 4. On the other hand, both *rs2070744* and *4b/4a* polymorphisms were specifically associated with advanced DN, and the *-786C/4a* haplotype was reported to be transmitted from heterozygous parents to siblings with advanced DN, suggesting that the *4a* allele is coupled almost exclusively with the *-786C* allele of *rs2070744* (Zanchi et al., 2000). The *NOS3 rs1799983* was analyzed in T1DM Caucazians from different countries and some reports showed no association with DN (Heltianu et al., 2009; Möllsten et al., 2009; Nosikov, 2004) and others found a marginal relationship (Ned et al., 2010) or strong association with increased risk of DN (Zintzaras et al., 2009). The *-786C/894T* haplotype of *NOS3* was found to be significantly associated with albuminuria, suggesting a strong implication of this gene in the susceptibility to kidney damage (Ned et al., 2010). The *rs3138808* variant of *NOS3* was also analyzed in a metaanalysis and was found to be associated with DN (Mooyaart et al., 2011).

There are only few reports which analyze the influence of *NOS3* polymorphisms on DPN in T1DM. Data from Caucasian patients genotyped for *rs1799983* and *4b/4a* variants showed that both polymorphisms were not associated with DPN (Nosikov, 2004; Zotova et al., 2005**).**  Our findings showed that only *NOS3 4b/4a* was not associated with DPN (Heltianu et al., 2009). In T1DM subjects with the lowest incidence of confirmed DPN, it was reported that the *894G* carriers of *rs1799983* variant had fivefold increased risk for DPN, suggesting that despite low risk for the disease in these individuals, there is a genetic predisposition to develop diabetes-related complication (Costacou et al., 2006). In agreement with this report we found a prevalence of DPN among the *894GG* as compared with *894TT* homozygotes in diabetic patients with normal kidney function, suggesting that *894GG* genotype might be a risk factor for T1DM-related microvascular disease. This subgroup of DPN patients with *894GG* had over 42% DR as an additional vascular complication, and the presence or absence of DR did not modify the significance of the relationship between the *rs1799983*  polymorphism and DPN. In addition, these subjects were recorded with high systolic blood pressure and raised levels of NOx, indicating a possible endothelial dysfunction, as well as with high levels of triglycerides, suggesting that additional high risk lipid profile contribute to the aggravation of the microvascular disorder. We presume that the rare-type *894T* allele might have a protective role against the development of DPN and a tendency to counterbalance increased NO production due to both chronic hyperglycemia and hypoxic effect at the microvascular level, by a not yet elucidated, compensatory-type mechanism (Heltianu et al., 2009).

Taken together, these results indicate that in T1DM, from various *NOS3* polymorphisms the most studied were *rs2070744*, *rs1799983* and *4b/4a* variants. Even in Caucasians there are differences among populations for the effects of gene polymorphisms on the microvascular complications. Diverse factors contribute to the variations between studies, analysis of early or late microvascular complication, incidence of the studied disorder in subjects with other confirmed disease, small sample size, the lack of haplotype analysis. Further studies on larger numbers of samples and on different populations are required to confirm these results.

#### **5. Endothelin genes**

12 Type 1 Diabetes Complications

this variant changes the NOS3 protein sequence, probable resulting an alteration of enzyme activity (Costacou et al., 2006), and control the NOS3 intracellular distribution interacting

From many polymorphisms of the *NOS3* gene some of them are associated with the development of diabetic microvascular complications while others indicated their protective role (Freedman et al., 2007; Heltianu et al., 2009). A recent study of *rs2070744* in Caucasian T1DM reported a positive association with diabetes *per se* as well as DR and two possible explanations were found; either *NOS3* is a candidate gene for the microvascular disease, or there is a linkage disequilibrium between *NOS3* and the neighbouring genes. It is known that in the same position (7q35) to *NOS3* gene the AKR1B1 and T-cell receptor beta-chain (TCRBC) genes in the 7q34 position are located (Bazzaz et al., 2010). In a hyperglycaemic milieu, the retinal NO bioavailability due to the presence of C-786 mutant allele of rs2070744 is decreased, and therefore the lack of NO stimulates aldose reductase, known to be implicated in the development of diabetes complications (Chandra et al., 2002). Other report showed that the onset pattern of severe DR in longstanding C-peptide-negative T1DM is affected by *NOS3 rs2070744* and *C774T* polymorphisms (Taverna et al., 2005). In the case of *C774T NOS3* polymorphism, the association with severe DR was related to the influence of the DN presence, which is a well-known strong risk factor for DR (Cimponeriu et al., 2010). Oppose, the rare allele *4a* of *4b/4a* variant of *NOS3* was found to be related to absent or nonsevere DR in T1DM Caucasians patients, suggesting a protective role. Although the *4b* allele was more frequent among patients with severe DR, a modest effect on the microvascular disorder was evaluated from the broad confidence interval (Cheng et al., 2007). Recent reports and our studies showed that there were no relationships between *4b/4a* variant of NOS3 and DR or other microangiopathic complications. Similar results for *rs179998*3 in relation with DR were also reported (Heltianu et al., 2009; Mamulakis et al., 2009). In a metaanalysis of genetic association studies for DR in T1DM, from the three *NOS3* polymorphisms (*rs1799983*, *rs3138808* and *rs413220*52) included in the sub analysis for Caucasian subjects, none of them were found to be significantly associated with any form of

The progression of renal disease was associated with the *NOS3 rs2070744* variant (Freedman et al., 2007; Zanchi et al., 2000), a result confirm recently by meta-analysis (Mooyaart et al., 2011; Ned et al., 2010). Contradictory results were obtained for the relationship of *NOS3 4b/4a* polymorphism with DN. Some reports showed no association (Degen et al., 2001; Heltianu et al., 2009) and others indicated that the *4a* allele represents an excess risk for advanced DN (Nosikov, 2004; Zanchi et al., 2000; Zinzaras et al., 2009). It was hypothesized that the *NOS3 4b/4a* itself plays a role in tissue-specific regulation of *NOS3* expression, a mechanism related to the importance of intron structure in the splicing of immature to mature RNA or to the presence of enhancer sequences within the intron 4. On the other hand, both *rs2070744* and *4b/4a* polymorphisms were specifically associated with advanced DN, and the *-786C/4a* haplotype was reported to be transmitted from heterozygous parents to siblings with advanced DN, suggesting that the *4a* allele is coupled almost exclusively with the *-786C* allele of *rs2070744* (Zanchi et al., 2000). The *NOS3 rs1799983* was analyzed in T1DM Caucazians from different countries and some reports showed no association with DN (Heltianu et al., 2009; Möllsten et al., 2009; Nosikov, 2004) and others found a marginal relationship (Ned et al., 2010) or strong association with increased risk of DN (Zintzaras et al., 2009). The *-786C/894T* haplotype of *NOS3* was found to be significantly associated with

with proteins of degradating process (Brouet et al., 2001).

DN (Abhary et al., 2009a)

The family of endothelins (ET) is represented by three peptides (1 to 3) and two receptors (ETRA and ETRB), which are widely distributed, in different proportions, being mostly abundant in vascular endothelial cells (EC). Their ET-1 and ET-2 are strong vasoconstrictors, whereas ET-3 is a potentially weaker vasoconstrictor compared to the other two isoforms. The ET-1 which is the most potent vasoconstrictor peptide acts as a paracrine or autocrine factor and its effects are ~10 times higher that of angiotensin II. The ET-1 has a variety of functions including its significant contribution to the maintenance of basal vascular tone, modulation of vascular permeability for proinflammatory mediators and proliferation of SMC. Having a long half-life, only a slight activation of its receptors into the signaling pathways might contribute to progressive disturbances, as hypertension and diabetic microvascular disorders (Cimponeriu et al., 2010). From the two receptors, the ETRA, expressed in SMC, has the highest affinity for ET-1, and is involved in the short term

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 15

variants are not risk factors, but plasma ET-1 level influences more the disease severity

Insufficient data exists regarding the influence of *EDN1* polymorphisms on the development of microvascular disorders. In a previous review was shown that *EDN1* gene was directly involved in hypertension and polymorphisms in *EDNRA* were associated with essential hypertension testifying the necessity of a balance within the endothelin system for normal functioning in vascular tissues. Although the importance of ET-1 expression in retinal microvasculature in high glucose was incontrovertible, it appears to be a lack of association between *EDN1* and *ECE1* polymorphisms and DR (Warpeha & Chakravarthy, 2003). Interestigly, in T2DM the *TT* genotype of *EDN1 G/T* polymorphism was associated with

Renin-angiotensin system plays a central role in blood pressure regulation and fluid electrolyte balance, being a modulator of vascular tone and structure. RAS components are produced by different organs and are delivered to their site of action by the bloodstream. Angiotensinogen (ANGT) is synthesized primarily by the liver and the released hormone precursor is cleaved by renin enzyme and aspartyl proteinase, to generate angiotensin I (Ang I). The key enzyme of RAS is angiotensin I-converting enzyme (ACE) which converts Ang I to angiotensin II (Ang II) by the release of the terminal His-Leu, when an increase of the vasoconstrictor activity of angiotensin occurs. The Ang II acts through two main receptors, the type 1 Ang II receptor and the type 2 Ang II receptor (Table 4). It is generally believed that type I Ang II receptor is the dominant one in the cardiovascular system, being expressed in different organs including the brain, kidney, heart, skeletal muscle (Abdollahi

Name Location Name Size Subcellular location

*ACE* 17q23.3 ACE 1306 149.72 secreted and

*ACE2* Xp22 ACE2 805 92.46 secreted and

Table 4. Renin-angiotensin system. Source, http://www.genecards.org; a. a., amino acids;

The RAS effects are primarily mediated by Ang II, a trophic hormone, which acts either directly on tissues, including vascular remodeling and inflammation or indirectly on NO bioavailability and its consequences (Chung et al., 2010; Ringel et al., 1997). In distinct local organs (brain, kidney, eye, vessel wall, heart) RAS regulatory mechanisms and function are

*AGT* 1q42-q43 ANGT 485 53.15 secreted

receptor

receptor

a.a. kDa

cell membrane

cell membrane

359 41.06 cell membrane

363 41.18 cell membrane

(Spinarová et al., 2008).

reduce risk of DN (Li et al., 2008).

Gene Protein

*AGTR1* 3q24 Type 1 Ang II

*AGTR2* Xq22-q23 Type 2 Ang II

et al., 2005).

kDa, kiloDalton

**6. Genes of renin – Angiotensin system** 

regulation of SMC and in the long term control of cell growth, adhesion and migration in the vasculature. The ETRB, expressed on both EC and SMC, has a dual function and can cause both vasoconstriction on SMC and vasodilation by the release of endothelial NO (Kalani, 2008; Potenza et al., 2009). The components of ET family are encoded by different genes (Table 3) with a generic name *EDN* (*EDN1*, *EDN2*, *EDN3*, *EDNRA,* and *EDNRB*). All three *EDN* genes (1 to 3) translate a respective amino acid prepropeptide, which is cleaved by one or more dibasic pair-specific endopeptidases to yield big ET. For ET-1, the large precursor is then converted into the mature and active ET-1 by a putative converting enzyme (ECE-1) encoded by the *ECE1* gene.

In diabetes, the secreted ET-1 by kidney cells activates its receptors and leads to constriction of renal vessels, inhibition of salt and water reabsorption, and enhanced glomerular proliferation. Correlations between plasma or urinary levels of ET-1 and signs of DN at different stages, as well as a close association between systemic endothelial dysfunction and microalbuminuria have been reported. Elevated ET-1 levels are present before the onset of microalbuminuria in T1DM, and worsen in association with it. In DR, the increased ET-1 levels strongly correlate with the enhanced endothelial permeability and loss of endothelial-mediated vasodilation in the retinal microvasculature (Kalani, 2008; Kankova et al., 2001). In DNP, the ET-1 is a potent vasoconstrictor of vasa nervorum and contributes to the EC abnormalities, when the balance of vasodilatation and vasoconstriction is in the favor of the latter. Moreover, ETA receptors contribute to the development of peripheral neuropathy, while ETB receptors have a protective role (Kalani, 2008; Lam, 2001). Most of the reported findings were for T2DM. A difference in the ET-1 involvement in the development of microvascular disorders in T1DM can not be excluded, knowing that differences in the pathogenesis of microangiopathy between type 1 and type 2 diabetes might exist.


Table 3. The endothelin family. Source, http://www.genecards.org; a.a., amino acids; kDa, kiloDalton

The *EDN1* gene has different polymorphisms including the *-3A/-4A*, a *−138* insertion/deletion and the *CA/CT* dinucleotide repeat in promoter, the *C8002T* or TaqI variant in intron 4, and the *Lys198Asn*, a *G/T* polymorphism in exon 5. In *EDNRA* gene were reported the *−231 A/G* and *C1363T* variants, while in *EDNRB* gene the *A30G* polymorphism. Assessments of relationship between variability of plasma concentrations of ET-1 (and big ET-1) and *EDN1* polymorphisms (*G8002A* and *−3A/−4A*) in patients with chronic heart failure indicated that there was no significant association, suggesting that the genetic variants are not risk factors, but plasma ET-1 level influences more the disease severity (Spinarová et al., 2008).

Insufficient data exists regarding the influence of *EDN1* polymorphisms on the development of microvascular disorders. In a previous review was shown that *EDN1* gene was directly involved in hypertension and polymorphisms in *EDNRA* were associated with essential hypertension testifying the necessity of a balance within the endothelin system for normal functioning in vascular tissues. Although the importance of ET-1 expression in retinal microvasculature in high glucose was incontrovertible, it appears to be a lack of association between *EDN1* and *ECE1* polymorphisms and DR (Warpeha & Chakravarthy, 2003). Interestigly, in T2DM the *TT* genotype of *EDN1 G/T* polymorphism was associated with reduce risk of DN (Li et al., 2008).

#### **6. Genes of renin – Angiotensin system**

14 Type 1 Diabetes Complications

regulation of SMC and in the long term control of cell growth, adhesion and migration in the vasculature. The ETRB, expressed on both EC and SMC, has a dual function and can cause both vasoconstriction on SMC and vasodilation by the release of endothelial NO (Kalani, 2008; Potenza et al., 2009). The components of ET family are encoded by different genes (Table 3) with a generic name *EDN* (*EDN1*, *EDN2*, *EDN3*, *EDNRA,* and *EDNRB*). All three *EDN* genes (1 to 3) translate a respective amino acid prepropeptide, which is cleaved by one or more dibasic pair-specific endopeptidases to yield big ET. For ET-1, the large precursor is then converted into the mature and active ET-1 by a putative converting

In diabetes, the secreted ET-1 by kidney cells activates its receptors and leads to constriction of renal vessels, inhibition of salt and water reabsorption, and enhanced glomerular proliferation. Correlations between plasma or urinary levels of ET-1 and signs of DN at different stages, as well as a close association between systemic endothelial dysfunction and microalbuminuria have been reported. Elevated ET-1 levels are present before the onset of microalbuminuria in T1DM, and worsen in association with it. In DR, the increased ET-1 levels strongly correlate with the enhanced endothelial permeability and loss of endothelial-mediated vasodilation in the retinal microvasculature (Kalani, 2008; Kankova et al., 2001). In DNP, the ET-1 is a potent vasoconstrictor of vasa nervorum and contributes to the EC abnormalities, when the balance of vasodilatation and vasoconstriction is in the favor of the latter. Moreover, ETA receptors contribute to the development of peripheral neuropathy, while ETB receptors have a protective role (Kalani, 2008; Lam, 2001). Most of the reported findings were for T2DM. A difference in the ET-1 involvement in the development of microvascular disorders in T1DM can not be excluded, knowing that differences in the pathogenesis of microangiopathy between type

Gene Protein

Name Location Name Size Subcellular

*EDN1* 6p24.1 ET-1 212 24.43 secreted *EDN2* 1p34.2 ET-2 178 19.96 secreted *EDN3* 20q13.2-13.3 ET-3 238 25.45 secreted *ECE1* 1p36.1 ECE1 770 87.16 cell membrane *EDNRA* 4q31.22 ETA receptor 427 48.72 cell membrane *EDNRB* 13q22 ETB receptor 442 49.64 cell membrane Table 3. The endothelin family. Source, http://www.genecards.org; a.a., amino acids; kDa,

The *EDN1* gene has different polymorphisms including the *-3A/-4A*, a *−138* insertion/deletion and the *CA/CT* dinucleotide repeat in promoter, the *C8002T* or TaqI variant in intron 4, and the *Lys198Asn*, a *G/T* polymorphism in exon 5. In *EDNRA* gene were reported the *−231 A/G* and *C1363T* variants, while in *EDNRB* gene the *A30G* polymorphism. Assessments of relationship between variability of plasma concentrations of ET-1 (and big ET-1) and *EDN1* polymorphisms (*G8002A* and *−3A/−4A*) in patients with chronic heart failure indicated that there was no significant association, suggesting that the genetic

a.a. kDa location

enzyme (ECE-1) encoded by the *ECE1* gene.

1 and type 2 diabetes might exist.

kiloDalton

Renin-angiotensin system plays a central role in blood pressure regulation and fluid electrolyte balance, being a modulator of vascular tone and structure. RAS components are produced by different organs and are delivered to their site of action by the bloodstream. Angiotensinogen (ANGT) is synthesized primarily by the liver and the released hormone precursor is cleaved by renin enzyme and aspartyl proteinase, to generate angiotensin I (Ang I). The key enzyme of RAS is angiotensin I-converting enzyme (ACE) which converts Ang I to angiotensin II (Ang II) by the release of the terminal His-Leu, when an increase of the vasoconstrictor activity of angiotensin occurs. The Ang II acts through two main receptors, the type 1 Ang II receptor and the type 2 Ang II receptor (Table 4). It is generally believed that type I Ang II receptor is the dominant one in the cardiovascular system, being expressed in different organs including the brain, kidney, heart, skeletal muscle (Abdollahi et al., 2005).


Table 4. Renin-angiotensin system. Source, http://www.genecards.org; a. a., amino acids; kDa, kiloDalton

The RAS effects are primarily mediated by Ang II, a trophic hormone, which acts either directly on tissues, including vascular remodeling and inflammation or indirectly on NO bioavailability and its consequences (Chung et al., 2010; Ringel et al., 1997). In distinct local organs (brain, kidney, eye, vessel wall, heart) RAS regulatory mechanisms and function are

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 17

subjects with *DD* genotype. Other report showed that about 24% of the variance in the ACE activity was attributed to other *ACE* polymorphisms *rs4343*, *rs495828* and *rs8176746*

Some reports indicated in Caucasian T1DM patients that *ACE I/D* was not associated with the development of persistent microalbuminuria, or overt DN (Möllsten et al., 2008; Ringel et al., 1997; Tarnow et al., 2000). A protective role to the homozygosity for the insertion (*I/I*) of *ACE*  gene in the DN development was attributed. With the increase of duration of diabetes it seems that the *ACE I/I* genotype is associated with longevity and survival in T1DM patients but not particularly in DN subjects (Boright et al., 2005). Other reports indicated that the *ACE D/D*  genotype was more frequent in patients with DN and the presence of the *ACE D/D* or *I/D* genotypes was associated with a faster rate of the decline of the renal function, suggesting that the *ACE D* allele represents an increased risk for both the onset and the progression of DN

From other *ACE* polymorphisms studied for the association with DN, it was reported that the *G7831A* (Nosikov, 2004), *rs4293* and *rs4309* (Currie et al., 2010) were not associate, while *rs1800764* and *rs9896208* (Boright et al., 2005) were associate with the disease. Regarding the *rs1800764* (*T/C*) variant, patients who carried the wild-type *T* allele were at lower risk for persistent microalbuminuria or severe DN, while heterozygous patients *(T/C)* had a higher risk for severe nephropathy, suggesting a genotype rather than an allele effect (Hadjadj et al., 2007). The reported haplotypic structure of *ACE* was considered to contain four polymorphisms *rs4311, rs4366, rs1244978* and *rs1800764 (*Hadjadj et al., 2007). Interestingly, the homozygosity for the common haplotype that carries the *ACE I* allele*,* as *TIC* haplotype, corresponding to the wild type alleles of *rs1800764*, *I/D* and *rs9896208*, respectively was associated with lower risk for development of severe DN. This finding provides a strong evidence that genetic variation at the *ACE* gene is associated with the development of DN (Boright et al., 2005). On the other hand it was reported that a haplotype containing the rare allele the *D* of *I/D* variant, *G* of *rs4366* and *G* of *rs12449782* was associated with a higher risk

Diabetic nephropathy is rarely diagnosed using invasive kidney biopsies and generally in genetic studies DN patients were those who presented albuminuria. Conflicting results of the gene association with the disease might occur, in addition, from the fact that a substantial number of subjects were classified as having DN but actually have nondiabetic kidney disease instead. Certain investigators have proposed that DN cases should be required to have diabetic retinopathy as well. From 1994 up to 2006 there were numbers of reports analyzing T1DM patients with DN having in addition various proportion of DR (Ng et al., 2008). The relationship between *ACE* polymorphisms and DR was less studied. Most reports found no association of *ACE I/D* with the development of any form of DR in adult or younger Type 1 diabetic patients (Abhary et al., 2009a; Zhou & Yang, 2010). Other data showed that in patients with DR, the severity of DR was associated with *ACE I/D* polymorphism (Marre et al., 1997). While nearly all T1DM individuals can develop DR or DPN, only a fraction of the subjects develops DN. So, it is hard to determine whether any observed association between *ACE I/D* and DN or DR or combined DN/DR truly exists. Including DR in the identification of potential genetic factors for the microvascular disorders might help, considering that some

Taken together, these data indicate that the *I/D* variant of *ACE* gene, considered a "reference" polymorphism, responsible, at least in part, for the interindividual variability of plasma ACE levels, is associated with the faster rate decline of renal function, particularly in patients with a less than 10 years of diabetes duration, and the *ACE D* allele represents an

(Costacou et al., 2006; Gumprecht et al., 2000; Ng et al., 2005; Nosikov, 2004).

patients manifest a joint retinal-renal phenotype (Ng et al., 2008).

(Chung et al., 2010).

for DN (Hadjadj et al., 2007).

different, so the Ang II actions may be modulated by a specific physiological process of a given tissue system. A variety of stimuli, including hyperglycemia, hypertension, sodium intake, inflammation modulate the expression of the tissue RAS components in pathophysiological states, and chronic production of Ang II may proceed remodeling and restructuring in various cardiovascular organs (Conen et al., 2008).

Discovery of ACE homologue, angiotensin I-converting enzyme 2 (ACE2) increased the complexity of RAS. ACE2 is predominantly expressed in endothelium of different tissues (i.e. kidney), although its distribution is much less widespread than ACE. The enzyme hydrolyses different peptides, including Ang I and Ang II, and is implicated in hypertension, diabetic nephropathy, and cardiovascular disease (Fröjdö et al., 2005). ACE2 seems to act as a negative regulator of the RAS, counterbalancing the function of ACE thus promoting vasodilation (Giunti et al., 2006).

RAS is a causative factor in diabetic microvascular complications inducing a variety of tissue responses including vasoconstriction, inflammation, oxidative stress, cell hypertrophy and proliferation, angiogenesis and fibrosis. Most of previous reports showed the RAS role in the initiation and progression of diabetic nephropathy. In the kidney, Ang II affects renal hemodynamics, tubular transport and stimulates growth and proto-oncogenes in various renal cell types. Increased production of angiotensin II within nephrons and their vasculature could participate in the local renal injury through both hemodynamic and nonhemodynamic actions and is a well-established factor promoting renal damage (Gumprecht et al., 2000). Because the low conversion of Ang I in the kidney, it has been proposed that the plasma ACE circulating through the kidney is an important contributor but yet a limiting factor in angiotensin II production within the renal circulation (Marre et al., 1997).

On the other hand, ACE2 which has a similar distribution to ACE, being largely localized in renal tubules, when is downregulated, as in diabetes-associated kidney disease, leads to an increase of tubular Ang II, which, in turn, may promote tubulointerstitial fibrosis. In early phases of diabetes in the absence of renal injury, it was suggested that ACE2 expression is increased, and in compensation the ACE was inhibited preventing the diabetes associated renal disease. These findings suggest that ACE inhibition may confer a renoprotective effect (Giunti et al., 2006). In diabetes, damage to the retina occurs in the vasculature, neurons and glia resulting in pathological angiogenesis, vascular leakage and a loss in retinal function. All components of RAS have been identified in the retina and iris and it is likely that the local rather than systemic RAS is involved in ocular neovascularization. It was reported that the RAS components were upregulated in DR.

#### **6.1** *ACE* **gene**

ACE, the main enzyme of RAS, is encoding by the *ACE* gene, composed of 26 exons, and span a total of 21 kb (Table 4). The genetic structure is made up of three ancestral regions, and two intragenic ancestral recombination breakpoints flank the gene region (Boright et al., 2005). Several polymorphisms have been reported in *ACE* gene. The two biallelic SNPs within and flanking the gene are in strong linkage disequilibrium with each other (Boright et al., 2005). The most extensively studied polymorphism was insertion/deletion of a *287 bp Alu* repeat in intron 16 (*rs179975; Ins/Del; I/D)* being considered a "reference" polymorphism (Hadjadj et al., 2007; Mooyaart et al., 2011). ACE activity is significantly connected with genetic variations at the *ACE* gene. The *rs179975* accounts for 44% of the interindividual variability of plasma ACE levels, and high ACE values were found among

different, so the Ang II actions may be modulated by a specific physiological process of a given tissue system. A variety of stimuli, including hyperglycemia, hypertension, sodium intake, inflammation modulate the expression of the tissue RAS components in pathophysiological states, and chronic production of Ang II may proceed remodeling and

Discovery of ACE homologue, angiotensin I-converting enzyme 2 (ACE2) increased the complexity of RAS. ACE2 is predominantly expressed in endothelium of different tissues (i.e. kidney), although its distribution is much less widespread than ACE. The enzyme hydrolyses different peptides, including Ang I and Ang II, and is implicated in hypertension, diabetic nephropathy, and cardiovascular disease (Fröjdö et al., 2005). ACE2 seems to act as a negative regulator of the RAS, counterbalancing the function of ACE thus

RAS is a causative factor in diabetic microvascular complications inducing a variety of tissue responses including vasoconstriction, inflammation, oxidative stress, cell hypertrophy and proliferation, angiogenesis and fibrosis. Most of previous reports showed the RAS role in the initiation and progression of diabetic nephropathy. In the kidney, Ang II affects renal hemodynamics, tubular transport and stimulates growth and proto-oncogenes in various renal cell types. Increased production of angiotensin II within nephrons and their vasculature could participate in the local renal injury through both hemodynamic and nonhemodynamic actions and is a well-established factor promoting renal damage (Gumprecht et al., 2000). Because the low conversion of Ang I in the kidney, it has been proposed that the plasma ACE circulating through the kidney is an important contributor but yet a limiting factor in angiotensin II production within the renal circulation (Marre et

On the other hand, ACE2 which has a similar distribution to ACE, being largely localized in renal tubules, when is downregulated, as in diabetes-associated kidney disease, leads to an increase of tubular Ang II, which, in turn, may promote tubulointerstitial fibrosis. In early phases of diabetes in the absence of renal injury, it was suggested that ACE2 expression is increased, and in compensation the ACE was inhibited preventing the diabetes associated renal disease. These findings suggest that ACE inhibition may confer a renoprotective effect (Giunti et al., 2006). In diabetes, damage to the retina occurs in the vasculature, neurons and glia resulting in pathological angiogenesis, vascular leakage and a loss in retinal function. All components of RAS have been identified in the retina and iris and it is likely that the local rather than systemic RAS is involved in ocular neovascularization. It was reported that

ACE, the main enzyme of RAS, is encoding by the *ACE* gene, composed of 26 exons, and span a total of 21 kb (Table 4). The genetic structure is made up of three ancestral regions, and two intragenic ancestral recombination breakpoints flank the gene region (Boright et al., 2005). Several polymorphisms have been reported in *ACE* gene. The two biallelic SNPs within and flanking the gene are in strong linkage disequilibrium with each other (Boright et al., 2005). The most extensively studied polymorphism was insertion/deletion of a *287 bp Alu* repeat in intron 16 (*rs179975; Ins/Del; I/D)* being considered a "reference" polymorphism (Hadjadj et al., 2007; Mooyaart et al., 2011). ACE activity is significantly connected with genetic variations at the *ACE* gene. The *rs179975* accounts for 44% of the interindividual variability of plasma ACE levels, and high ACE values were found among

restructuring in various cardiovascular organs (Conen et al., 2008).

promoting vasodilation (Giunti et al., 2006).

the RAS components were upregulated in DR.

al., 1997).

**6.1** *ACE* **gene** 

subjects with *DD* genotype. Other report showed that about 24% of the variance in the ACE activity was attributed to other *ACE* polymorphisms *rs4343*, *rs495828* and *rs8176746* (Chung et al., 2010).

Some reports indicated in Caucasian T1DM patients that *ACE I/D* was not associated with the development of persistent microalbuminuria, or overt DN (Möllsten et al., 2008; Ringel et al., 1997; Tarnow et al., 2000). A protective role to the homozygosity for the insertion (*I/I*) of *ACE*  gene in the DN development was attributed. With the increase of duration of diabetes it seems that the *ACE I/I* genotype is associated with longevity and survival in T1DM patients but not particularly in DN subjects (Boright et al., 2005). Other reports indicated that the *ACE D/D*  genotype was more frequent in patients with DN and the presence of the *ACE D/D* or *I/D* genotypes was associated with a faster rate of the decline of the renal function, suggesting that the *ACE D* allele represents an increased risk for both the onset and the progression of DN (Costacou et al., 2006; Gumprecht et al., 2000; Ng et al., 2005; Nosikov, 2004).

From other *ACE* polymorphisms studied for the association with DN, it was reported that the *G7831A* (Nosikov, 2004), *rs4293* and *rs4309* (Currie et al., 2010) were not associate, while *rs1800764* and *rs9896208* (Boright et al., 2005) were associate with the disease. Regarding the *rs1800764* (*T/C*) variant, patients who carried the wild-type *T* allele were at lower risk for persistent microalbuminuria or severe DN, while heterozygous patients *(T/C)* had a higher risk for severe nephropathy, suggesting a genotype rather than an allele effect (Hadjadj et al., 2007). The reported haplotypic structure of *ACE* was considered to contain four polymorphisms *rs4311, rs4366, rs1244978* and *rs1800764 (*Hadjadj et al., 2007). Interestingly, the homozygosity for the common haplotype that carries the *ACE I* allele*,* as *TIC* haplotype, corresponding to the wild type alleles of *rs1800764*, *I/D* and *rs9896208*, respectively was associated with lower risk for development of severe DN. This finding provides a strong evidence that genetic variation at the *ACE* gene is associated with the development of DN (Boright et al., 2005). On the other hand it was reported that a haplotype containing the rare allele the *D* of *I/D* variant, *G* of *rs4366* and *G* of *rs12449782* was associated with a higher risk for DN (Hadjadj et al., 2007).

Diabetic nephropathy is rarely diagnosed using invasive kidney biopsies and generally in genetic studies DN patients were those who presented albuminuria. Conflicting results of the gene association with the disease might occur, in addition, from the fact that a substantial number of subjects were classified as having DN but actually have nondiabetic kidney disease instead. Certain investigators have proposed that DN cases should be required to have diabetic retinopathy as well. From 1994 up to 2006 there were numbers of reports analyzing T1DM patients with DN having in addition various proportion of DR (Ng et al., 2008). The relationship between *ACE* polymorphisms and DR was less studied. Most reports found no association of *ACE I/D* with the development of any form of DR in adult or younger Type 1 diabetic patients (Abhary et al., 2009a; Zhou & Yang, 2010). Other data showed that in patients with DR, the severity of DR was associated with *ACE I/D* polymorphism (Marre et al., 1997). While nearly all T1DM individuals can develop DR or DPN, only a fraction of the subjects develops DN. So, it is hard to determine whether any observed association between *ACE I/D* and DN or DR or combined DN/DR truly exists. Including DR in the identification of potential genetic factors for the microvascular disorders might help, considering that some patients manifest a joint retinal-renal phenotype (Ng et al., 2008).

Taken together, these data indicate that the *I/D* variant of *ACE* gene, considered a "reference" polymorphism, responsible, at least in part, for the interindividual variability of plasma ACE levels, is associated with the faster rate decline of renal function, particularly in patients with a less than 10 years of diabetes duration, and the *ACE D* allele represents an

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 19

compared to *AC* and *CC* genotypes (Möllsten et al., 2008). The treatment with renoprotective antihypertensive (losartan) for slowing down the progression of diabetic glomerulopathy reduced significantly albuminuria, systolic and diastolic blood pressure in the *A* allele vs *C* allele carriers of *rs5186* polymorphism (Dragović et al., 2010). Oppose, the rs5186 of *AGTR1* was found not associated with DN in other reports (Gallego et al., 2008; Nosikov, 2004; Tarnow et al., 2000). There are no data on the influence of *AGTR1* gene polymorphism on the development of DR or DPN. For *AGTR2* gene have been identified few variants, *U20860* (*T3786C*; *rs5192*; *Ala/Ala*), *G1675A* (*rs1403543*), *G4297T* (*rs5193*), *A4303G* (*rs5194*) but there are no reports showing the involvement of one polymorphism with T1DM microvascular

Although the prognosis of patients with DN has improved, the decline in the GFR still varies among T1DM patients. The nongenetic risk factors (elevated blood pressure, albuminuria, and HbA1c) for excessive loss of GFR, explain only approximately 30 to 50% of the decrease, and the epistatic interactions between *ACE*, *ACE2*, *AGT*, *AGTR1* or *AGTR2* polymorphisms in the RAS, a concept previously suggested (Jacobsen et al., 2003) might represent a risk factor for DN. It was reported that despite the non-significant effects of a single-gene on DN progression, a combined genetic variable including the potential "bad" alleles (*D* of *ACE I/D*, *M* of *AGT rs699*, and *A* of *AGTR1 rs5186*) represent a risk factor for the disease (Jacobsen et al., 2003). These data suggest that in some conditions a single gene variant may cause appreciable phenotypic changes only upon combination with other polymorphisms, having additional or synergistic effects on the same metabolic pathways (Ruggenenti et al., 2008). Oppose, other data showed that DN was not influenced by the epistatic interactions between the polymorphisms of the RAS genes. (Gallego et al., 2008;

All over the world, the incidence of type 1 diabetes continue to be much higher than in general population. Despite major progresses done in the recent years to identify candidate genes involved in the development of diabetic microvascular complications, there are still controversial results and insufficient knowledge in the literature, although a variety of genomic strategies were applied. While the degree of metabolic control remains the main risk factor for the development of diabetic chronic complications, the genetic risk factors, common for retinopathy, neuropathy, and renal disease, or specific for each of them, are important contributors to the disease severity. Discrepancies between reported data are due to differences in the genetic background between studied populations, small sample sizes, insufficient phenotype description, genotyping procedures, individual gene polymorphism assessment, few numbers of loci included in the studies, and requirement of interaction analysis between gene-gene variants. Genetic prediction and use of individual aetiological processes, as well as the translation of recent molecular knowledge into potential therapeutic agents will contribute selectively to the preventive and therapeutic interventions

This work was financially supported by grants from the Romanian Academy, Ministry of

Education and Research and by an EFSD New Horizons Grant.

disorders.

Tarnow et al., 2000)

in this complex disease.

**8. Acknowledgment** 

**7. Conclusion** 

increased risk for both the onset and the progression of DN. These findings were confirmed by multiple, independent studies. This potential genetic factor for DN development might be correlated also with DR, suggesting its involvement in the diabetic complex phenotype.

#### **6.2 Other RAS genes**

ACE2 has approximately 40% homology with ACE sharing 42% identity with the catalytic domain of somatic ACE, and promotes vasodilatation counterbalancing the ACE effect. The *ACE2* gene consists of 18 exons is stable and conserved, indicating, that the genetic effect is small, and it intertwines and functions in concert with many other genes, suggesting the presence of epistatic effects (Fröjdö et al., 2005; Zhou & Yang, 2010). Data of genes for other RAS components are presented in Table 4. From the variants (*rs714205; rs879922; rs1978124; rs2023802; rs2048684; rs2074192; rs2285666; rs4646188; rs5978731*) reported few studies implied genomic analyses of diabetes microvascular disorders. None of the studied polymorphisms were associated with DR (Currie et al., 2010; Fröjdö et al., 2005). An increased in ACE2 expression in early phases of diabetes in the absence of renal injury was reported (Giunti et al., 2006).

Angiotensinogen gene has more than 30 genetic polymorphisms as reported in different studies, *M24686*, *C1015T* (*T174M; rs4762; Thr/Met*), *T1198C (M235T*; *rs699*; *Met/Thr), A1237G (Tyr/Cys), A1204C* (*A-20C; rs5050), G1218A* (*G-6A; rs5051*). The most studied polymorphism in relation with diabetic microvascular disorders was *rs699* in exon 2 when a *T* to *C* base substitution at position 702 take place, with the consequent replacement of methionine 235 with threonine. A relationship between the *T* allele of *rs699* and increased plasma Ang II levels was reported only in male subjects and may account for no more than 5% of ANGT variability (Marre et al., 1997, Ruggenenti et al., 2008). Reports on the association of *AGT rs699* and the development of DN in adults with T1DM showed conflicting results; some finding indicated no association (Chowdhury et al., 1996; Currie et al., 2010; Hadjadj et al., 2001; Möllsten et al., 2008; Nosikov, 2004; Ringel et al., 1997; Tarnow et al., 2000), and others suggested that this variant contribute to the increased risk for chronic renal failure (Gumprecht et al., 2000). In young T1DM subjects, the *TT* genotype of *AGT rs699* had a fourfold increased risk for persistent microalbuminuria, suggesting that this variant is a strong predictor for early stage of DN (Gallego et al., 2008). One report analyzed *AGT T174M* in relation with DN, and the findings indicated no association with the disease (Nosikov, 2004). There were no reports indicated a significant relationship between *AGT rs699* and DR, but in patients with incipient diabetic renal failure the *T* allele of *AGT rs699* was associated with DR. In these patients interaction between the *D* allele of *ACE I/D* and the *T* allele of *AGT rs699* tended towards protection against DN (Van Ittersum et al., 2000). Oppose, other study indicated that the same interaction increases risk for DN in patients cu DR (Marre et al., 1997). All these data suggest that extensive studies has to be done in large number of T1DM patients with combined DN and DR vs only one microvascular disorder for the epistatic interactions between *ACE* and *AGT* polymorphisms and their relationship of the disease.

In *AGTR1* gene were identify a variety of polymorphisms *AF245699*, *A49954G* (*rs5183*; *A1878G*; *Pro/Pro*), *A50058C* (*rs5186*; *A1166C*), *T4955A* (*rs275651*), *T5052G* (*rs275652*), *C5245T*  (*rs1492078*)*, A1062G*, *T573C*, *G1517T (*Ruggenenti et al., 2008). Reports on the relationship of *AGTR1* polymorphisms with DN showed that the *AA* genotype of *rs5186* was independently associated with overt DN, being with a threefold increase in the risk for the disease compared to *AC* and *CC* genotypes (Möllsten et al., 2008). The treatment with renoprotective antihypertensive (losartan) for slowing down the progression of diabetic glomerulopathy reduced significantly albuminuria, systolic and diastolic blood pressure in the *A* allele vs *C* allele carriers of *rs5186* polymorphism (Dragović et al., 2010). Oppose, the rs5186 of *AGTR1* was found not associated with DN in other reports (Gallego et al., 2008; Nosikov, 2004; Tarnow et al., 2000). There are no data on the influence of *AGTR1* gene polymorphism on the development of DR or DPN. For *AGTR2* gene have been identified few variants, *U20860* (*T3786C*; *rs5192*; *Ala/Ala*), *G1675A* (*rs1403543*), *G4297T* (*rs5193*), *A4303G* (*rs5194*) but there are no reports showing the involvement of one polymorphism with T1DM microvascular disorders.

Although the prognosis of patients with DN has improved, the decline in the GFR still varies among T1DM patients. The nongenetic risk factors (elevated blood pressure, albuminuria, and HbA1c) for excessive loss of GFR, explain only approximately 30 to 50% of the decrease, and the epistatic interactions between *ACE*, *ACE2*, *AGT*, *AGTR1* or *AGTR2* polymorphisms in the RAS, a concept previously suggested (Jacobsen et al., 2003) might represent a risk factor for DN. It was reported that despite the non-significant effects of a single-gene on DN progression, a combined genetic variable including the potential "bad" alleles (*D* of *ACE I/D*, *M* of *AGT rs699*, and *A* of *AGTR1 rs5186*) represent a risk factor for the disease (Jacobsen et al., 2003). These data suggest that in some conditions a single gene variant may cause appreciable phenotypic changes only upon combination with other polymorphisms, having additional or synergistic effects on the same metabolic pathways (Ruggenenti et al., 2008). Oppose, other data showed that DN was not influenced by the epistatic interactions between the polymorphisms of the RAS genes. (Gallego et al., 2008; Tarnow et al., 2000)

#### **7. Conclusion**

18 Type 1 Diabetes Complications

increased risk for both the onset and the progression of DN. These findings were confirmed by multiple, independent studies. This potential genetic factor for DN development might be correlated also with DR, suggesting its involvement in the diabetic complex phenotype.

ACE2 has approximately 40% homology with ACE sharing 42% identity with the catalytic domain of somatic ACE, and promotes vasodilatation counterbalancing the ACE effect. The *ACE2* gene consists of 18 exons is stable and conserved, indicating, that the genetic effect is small, and it intertwines and functions in concert with many other genes, suggesting the presence of epistatic effects (Fröjdö et al., 2005; Zhou & Yang, 2010). Data of genes for other RAS components are presented in Table 4. From the variants (*rs714205; rs879922; rs1978124; rs2023802; rs2048684; rs2074192; rs2285666; rs4646188; rs5978731*) reported few studies implied genomic analyses of diabetes microvascular disorders. None of the studied polymorphisms were associated with DR (Currie et al., 2010; Fröjdö et al., 2005). An increased in ACE2 expression in early phases of diabetes in the absence of renal injury was

Angiotensinogen gene has more than 30 genetic polymorphisms as reported in different studies, *M24686*, *C1015T* (*T174M; rs4762; Thr/Met*), *T1198C (M235T*; *rs699*; *Met/Thr), A1237G (Tyr/Cys), A1204C* (*A-20C; rs5050), G1218A* (*G-6A; rs5051*). The most studied polymorphism in relation with diabetic microvascular disorders was *rs699* in exon 2 when a *T* to *C* base substitution at position 702 take place, with the consequent replacement of methionine 235 with threonine. A relationship between the *T* allele of *rs699* and increased plasma Ang II levels was reported only in male subjects and may account for no more than 5% of ANGT variability (Marre et al., 1997, Ruggenenti et al., 2008). Reports on the association of *AGT rs699* and the development of DN in adults with T1DM showed conflicting results; some finding indicated no association (Chowdhury et al., 1996; Currie et al., 2010; Hadjadj et al., 2001; Möllsten et al., 2008; Nosikov, 2004; Ringel et al., 1997; Tarnow et al., 2000), and others suggested that this variant contribute to the increased risk for chronic renal failure (Gumprecht et al., 2000). In young T1DM subjects, the *TT* genotype of *AGT rs699* had a fourfold increased risk for persistent microalbuminuria, suggesting that this variant is a strong predictor for early stage of DN (Gallego et al., 2008). One report analyzed *AGT T174M* in relation with DN, and the findings indicated no association with the disease (Nosikov, 2004). There were no reports indicated a significant relationship between *AGT rs699* and DR, but in patients with incipient diabetic renal failure the *T* allele of *AGT rs699* was associated with DR. In these patients interaction between the *D* allele of *ACE I/D* and the *T* allele of *AGT rs699* tended towards protection against DN (Van Ittersum et al., 2000). Oppose, other study indicated that the same interaction increases risk for DN in patients cu DR (Marre et al., 1997). All these data suggest that extensive studies has to be done in large number of T1DM patients with combined DN and DR vs only one microvascular disorder for the epistatic interactions between *ACE* and *AGT* polymorphisms and their relationship

In *AGTR1* gene were identify a variety of polymorphisms *AF245699*, *A49954G* (*rs5183*; *A1878G*; *Pro/Pro*), *A50058C* (*rs5186*; *A1166C*), *T4955A* (*rs275651*), *T5052G* (*rs275652*), *C5245T*  (*rs1492078*)*, A1062G*, *T573C*, *G1517T (*Ruggenenti et al., 2008). Reports on the relationship of *AGTR1* polymorphisms with DN showed that the *AA* genotype of *rs5186* was independently associated with overt DN, being with a threefold increase in the risk for the disease

**6.2 Other RAS genes** 

reported (Giunti et al., 2006).

of the disease.

All over the world, the incidence of type 1 diabetes continue to be much higher than in general population. Despite major progresses done in the recent years to identify candidate genes involved in the development of diabetic microvascular complications, there are still controversial results and insufficient knowledge in the literature, although a variety of genomic strategies were applied. While the degree of metabolic control remains the main risk factor for the development of diabetic chronic complications, the genetic risk factors, common for retinopathy, neuropathy, and renal disease, or specific for each of them, are important contributors to the disease severity. Discrepancies between reported data are due to differences in the genetic background between studied populations, small sample sizes, insufficient phenotype description, genotyping procedures, individual gene polymorphism assessment, few numbers of loci included in the studies, and requirement of interaction analysis between gene-gene variants. Genetic prediction and use of individual aetiological processes, as well as the translation of recent molecular knowledge into potential therapeutic agents will contribute selectively to the preventive and therapeutic interventions in this complex disease.

#### **8. Acknowledgment**

This work was financially supported by grants from the Romanian Academy, Ministry of Education and Research and by an EFSD New Horizons Grant.

Genetic Determinants of Microvascular Complications in Type 1 Diabetes 21

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**2** 

*Spain* 

**Early and Late Onset Type 1 Diabetes: One and** 

Type 1 diabetes is a complex autoimmune disease in which genetic and environmental factors add up to induce an autoimmune destruction of the insulin-producing pancreatic cells. Although type 1 diabetes is popularly associated to an onset in infancy or adolescence, it can begin at any age. The reasons behind this temporal difference in the onset of the disease are probably a mixture of genetic and environmental factors, just as the induction of the disease itself. Despite the great progress that the study of the genetics of type 1 diabetes has experienced in the last years, the genetic factors that could modify the age at diagnosis of type 1 diabetes have not been analyzed so deeply. This knowledge would be interesting to discover new routes to delay the disease onset and preserve the cell mass as long as possible. In this chapter, we will review the characteristics of adult-onset type 1 diabetes patients and afterwards we will focus on the studies in type 1 diabetes genetics and the reported associations of genetics and age at onset of the disease. Finally, we will present an analysis of ten genetic associations in a group of Spanish patients with early and late onset

**2. Diagnostic criteria for diabetes and further classification of the disease** 

Type 1 diabetes is the most prevalent chronic disease in childhood and it is also the most frequent form of diabetes in subjects diagnosed before age 19 (Duncan, 2006). Adults can also suffer from type 1 diabetes, given that the prevalence rate does not vary greatly with age, but the diagnosis of the disease in adult age is complicated by the higher prevalence of type 2 diabetes, which is the most frequent form of diabetes in adulthood (American Diabetes Association [ADA], 2010). Classical diabetes classifications used to categorize patients by their age at diagnosis or insulin requirement. Thus, type 1 diabetes was termed juvenile diabetes or insulin-dependent diabetes mellitus (IDDM) and type 2 diabetes could be diabetes of the adult or non-insulin dependent diabetes mellitus (NIDDM). However, type 2 diabetes can begin as an insulin-dependent condition or at an early age, type 1 diabetes can begin at any age, and a certain form of adult onset autoimmune diabetes termed latent autoimmune diabetes of the adult (LADA) is non-insulin-dependent by

**1. Introduction** 

of type 1 diabetes.

**the Same or Two Distinct Genetic Entities?** 

Laura Espino-Paisan, Elena Urcelay,

Emilio Gómez de la Concha and Jose Luis Santiago *Clinical Immunology Department, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria San Carlos (IdISSC),* 


### **Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities?**

Laura Espino-Paisan, Elena Urcelay, Emilio Gómez de la Concha and Jose Luis Santiago *Clinical Immunology Department, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria San Carlos (IdISSC), Spain* 

#### **1. Introduction**

28 Type 1 Diabetes Complications

Wang, Y., Newton, DC. & Marsden, PA. (1999) Neuronal NOS: gene structure, mRNA

Warpeha, KM., Xu, W., Liu, L., Charles, IG., Patterson, CC., Ah-Fat, F., Harding, S., Hart,

Warpeha, KM. & Chakravarthy, U. (2003) Molecular genetics of microvascular disease in

*Journal*, Vol. 13, No. 13, (October), pp. 1825-1832, ISSN 0892-6638.

21-43, ISSN 0892-0915.

222X.

diversity, and functional relevance. *Critique Review Neurobiology*, Vol. 13, No. 1, pp.

PM., Chakravarthy, U. & Hughes, AE. (1999) Genotyping and functional analysis of a polymorphic (CCTTT)(n) repeat of NOS2A in diabetic retinopathy. *FASEB*

diabetic retinopathy. *Eye (London)*, Vol. 17, No. 3, (April), pp. 305-311, ISSN 0950-

Type 1 diabetes is a complex autoimmune disease in which genetic and environmental factors add up to induce an autoimmune destruction of the insulin-producing pancreatic cells. Although type 1 diabetes is popularly associated to an onset in infancy or adolescence, it can begin at any age. The reasons behind this temporal difference in the onset of the disease are probably a mixture of genetic and environmental factors, just as the induction of the disease itself. Despite the great progress that the study of the genetics of type 1 diabetes has experienced in the last years, the genetic factors that could modify the age at diagnosis of type 1 diabetes have not been analyzed so deeply. This knowledge would be interesting to discover new routes to delay the disease onset and preserve the cell mass as long as possible. In this chapter, we will review the characteristics of adult-onset type 1 diabetes patients and afterwards we will focus on the studies in type 1 diabetes genetics and the reported associations of genetics and age at onset of the disease. Finally, we will present an analysis of ten genetic associations in a group of Spanish patients with early and late onset of type 1 diabetes.

#### **2. Diagnostic criteria for diabetes and further classification of the disease**

Type 1 diabetes is the most prevalent chronic disease in childhood and it is also the most frequent form of diabetes in subjects diagnosed before age 19 (Duncan, 2006). Adults can also suffer from type 1 diabetes, given that the prevalence rate does not vary greatly with age, but the diagnosis of the disease in adult age is complicated by the higher prevalence of type 2 diabetes, which is the most frequent form of diabetes in adulthood (American Diabetes Association [ADA], 2010). Classical diabetes classifications used to categorize patients by their age at diagnosis or insulin requirement. Thus, type 1 diabetes was termed juvenile diabetes or insulin-dependent diabetes mellitus (IDDM) and type 2 diabetes could be diabetes of the adult or non-insulin dependent diabetes mellitus (NIDDM). However, type 2 diabetes can begin as an insulin-dependent condition or at an early age, type 1 diabetes can begin at any age, and a certain form of adult onset autoimmune diabetes termed latent autoimmune diabetes of the adult (LADA) is non-insulin-dependent by

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 31

Type 2 diabetes includes 90-95% of the total cases of diabetes and accounts for 80-85% of the cases diagnosed in adulthood (ADA, 2010). The disease is a result of a combination of peripheral insulin resistance and relative insulin deficiency that develops into hyperglycemia. The hyperglycemia in type 2 diabetes appears gradually, usually with absence of the classic symptoms (polyuria, polydipsia) and can go undetected for long time before diagnosis. The etiologic factors of type 2 diabetes are unknown and probably this trait, more than type 1 diabetes, is composed of several different diseases with the common clinical manifestation of hyperglycemia. However, there is no proof of an implication of an autoimmune response, and thus autoantibodies against pancreatic antigens are always negative (ADA, 2010). Patients are usually obese and non-insulin dependent, although insulin can become a necessary therapy for a good control of hyperglycemia in some cases. However, insulin treatment in type 2 diabetes is not required for survival. C-peptide levels can be lower than in healthy controls, reflecting the relative insulin deficiency, but they are higher than in type 1 diabetes patients and decrease more gradually with time (Hosszufalusi et al, 2003). Diabetic ketoacidosis is quite rare and tends to develop due to underlying conditions, such as an infection (ADA,

Early after the discovery of antibodies against pancreatic antigens in the serum of type 1 diabetic subjects, it was noticed by clinicians that 10% of patients first diagnosed with type 2 diabetes tested positive for type 1 diabetes antibodies (mainly GADA and ICA) (Palmer et al, 2005). Those patients slowly but relentlessly progressed to insulin-dependency and showed signs of pancreatic islet dysfunction such as a progressive decrease of C-peptide levels. These characteristics defined a new category in the diabetes spectra called the latent autoimmune diabetes of the adult, and abbreviated LADA. Diagnostic criteria for these patients are age at diagnosis over 30 years, presence of antibodies against pancreatic islets, with a higher frequency of single positivity and GADA or ICA antibodies than type 1 diabetes patients, and no requirement of insulin for at least six months since diagnosis (Palmer et al, 2005; Leslie et al, 2006). There seems to exist a similar but milder genetic background to that of type 1 diabetic patients (Hosszufalusi et al, 2003; Palmer et al, 2005), and some researchers think of LADA as a slower and less aggressive form of type 1 diabetes, to the point that this condition is usually termed type 1.5 diabetes. Debate exists over LADA being an entity of its own or just a less aggressive form of type 1 diabetes at an older age (Hosszufalusi et al, 2003; Palmer et al, 2005; Leslie et al, 2006; Steck & Eisenbarth, 2008). Anyway, this group of patients poses a very interesting subset for testing cell preserving therapies in an autoimmune form of diabetes due to its slow progression to

When recruiting patients for genetic studies, a careful evaluation of adult-onset patients must be carried out to avoid misclassification. In most cases, the three more common forms of diabetes in adults can be distinguished with a test for antibodies against pancreatic

antigens at diagnosis and the requirement for insulin therapy (see table 2).

**2.2 Type 2 diabetes** 

2010).

insulin-dependency.

**2.3 Latent autoimmune diabetes of the adult (LADA)** 

**2.4 The diagnosis of an adult-onset diabetic patient** 

definition at the time of diagnosis, nevertheless it is an autoimmune form of diabetes. Therefore, cataloging the different forms of diabetes is not so simple and classifications based in age at diagnosis or insulin requirement are no longer employed. Hence, we will review the diagnostic criteria for diabetes and define what can be considered adult-onset type 1 diabetes.

According to the most recent classification of the American Diabetes Association (ADA, 2010), the diagnostic criteria for diabetes are 1) levels of glycosilated haemoglobin over 6.5%, 2) fasting plasma glucose levels over 126 mg/dl, defining fasting state as no caloric intake for at least eight hours, 3) plasma glucose over 200 mg/dl at two hours during an oral glucose tolerance test (OGTT) or 4) random plasma glucose over 200 mg/dl in a patient with classic symptoms of hyperglycemia (poliuria, polidypsia or glucosuria). Patients with type 1, type 2 diabetes or LADA must feet these criteria. Then, further classification of the patient should be considered.

#### **2.1 Type 1 diabetes**

Type 1 diabetes accounts for 5-10% of the total cases of diabetes and it is the 90% of the cases of diabetes diagnosed in children (ADA, 2010). The disease is an autoimmune condition characterized by the destruction of the pancreatic cells by autoreactive T lymphocytes. Hyperglicemia manifests when 60-90% of the cell mass has been lost. As a result of the autoimmune insult, antibodies against pancreatic islets are synthesized and can be detectable in serum (see table 1). These antibodies precede in several years the clinical symptoms. They are not pathogenic (Wong et al, 2010), but its detection helps in the classification of the patient as type 1 diabetes, especially when the disease is diagnosed in adulthood. Antibodies against pancreatic antigens are positive at diagnosis in 90% of type 1 diabetes patients. Obesity is quite uncommon in these patients, but not incompatible with the disease. Patients are frequently insulin-dependent since diagnosis and insulinreplacement therapy is ultimately necessary for survival. Also, C-peptide levels (a measure of cell activity) are usually low or undetectable. When untreated, type 1 diabetes leads to diabetic ketoacidosis, a life-threatening condition derived from the use of fat deposits (ADA, 2010).

Adult-onset type 1 diabetes patients tend to have a softer disease onset, with a lower frequency of diabetic ketoacidosis and a slower loss of insulin secretion capacity (Hosszufalusi et al, 2003; Leslie et al, 2006). These characteristics lead to think that a slower autoimmune reaction is taking place in the patient with adult onset.


Table 1. Autoantibodies against pancreatic antigens in type 1 diabetes.

#### **2.2 Type 2 diabetes**

30 Type 1 Diabetes Complications

definition at the time of diagnosis, nevertheless it is an autoimmune form of diabetes. Therefore, cataloging the different forms of diabetes is not so simple and classifications based in age at diagnosis or insulin requirement are no longer employed. Hence, we will review the diagnostic criteria for diabetes and define what can be considered adult-onset

According to the most recent classification of the American Diabetes Association (ADA, 2010), the diagnostic criteria for diabetes are 1) levels of glycosilated haemoglobin over 6.5%, 2) fasting plasma glucose levels over 126 mg/dl, defining fasting state as no caloric intake for at least eight hours, 3) plasma glucose over 200 mg/dl at two hours during an oral glucose tolerance test (OGTT) or 4) random plasma glucose over 200 mg/dl in a patient with classic symptoms of hyperglycemia (poliuria, polidypsia or glucosuria). Patients with type 1, type 2 diabetes or LADA must feet these criteria. Then, further classification of the patient

Type 1 diabetes accounts for 5-10% of the total cases of diabetes and it is the 90% of the cases of diabetes diagnosed in children (ADA, 2010). The disease is an autoimmune condition characterized by the destruction of the pancreatic cells by autoreactive T lymphocytes. Hyperglicemia manifests when 60-90% of the cell mass has been lost. As a result of the autoimmune insult, antibodies against pancreatic islets are synthesized and can be detectable in serum (see table 1). These antibodies precede in several years the clinical symptoms. They are not pathogenic (Wong et al, 2010), but its detection helps in the classification of the patient as type 1 diabetes, especially when the disease is diagnosed in adulthood. Antibodies against pancreatic antigens are positive at diagnosis in 90% of type 1 diabetes patients. Obesity is quite uncommon in these patients, but not incompatible with the disease. Patients are frequently insulin-dependent since diagnosis and insulinreplacement therapy is ultimately necessary for survival. Also, C-peptide levels (a measure of cell activity) are usually low or undetectable. When untreated, type 1 diabetes leads to diabetic ketoacidosis, a life-threatening condition derived from the use of fat deposits (ADA,

Adult-onset type 1 diabetes patients tend to have a softer disease onset, with a lower frequency of diabetic ketoacidosis and a slower loss of insulin secretion capacity (Hosszufalusi et al, 2003; Leslie et al, 2006). These characteristics lead to think that a slower

**IAA** Anti-insulin antibodies Pancreas

**IA2-A** Anti-insulinoma associated 2 antibodies Pancreas **ICA** Anti-islet cell antibodies (several antigens) Pancreas **SCL38A** Antibodies against the zinc channel ZnT8 Pancreas

**GADA** Anti-glutamate decarboxilase antibodies Pancreas/nervous system

**Autoantibody Antigen expression** 

autoimmune reaction is taking place in the patient with adult onset.

Table 1. Autoantibodies against pancreatic antigens in type 1 diabetes.

type 1 diabetes.

should be considered.

**2.1 Type 1 diabetes** 

2010).

Type 2 diabetes includes 90-95% of the total cases of diabetes and accounts for 80-85% of the cases diagnosed in adulthood (ADA, 2010). The disease is a result of a combination of peripheral insulin resistance and relative insulin deficiency that develops into hyperglycemia. The hyperglycemia in type 2 diabetes appears gradually, usually with absence of the classic symptoms (polyuria, polydipsia) and can go undetected for long time before diagnosis. The etiologic factors of type 2 diabetes are unknown and probably this trait, more than type 1 diabetes, is composed of several different diseases with the common clinical manifestation of hyperglycemia. However, there is no proof of an implication of an autoimmune response, and thus autoantibodies against pancreatic antigens are always negative (ADA, 2010). Patients are usually obese and non-insulin dependent, although insulin can become a necessary therapy for a good control of hyperglycemia in some cases. However, insulin treatment in type 2 diabetes is not required for survival. C-peptide levels can be lower than in healthy controls, reflecting the relative insulin deficiency, but they are higher than in type 1 diabetes patients and decrease more gradually with time (Hosszufalusi et al, 2003). Diabetic ketoacidosis is quite rare and tends to develop due to underlying conditions, such as an infection (ADA, 2010).

#### **2.3 Latent autoimmune diabetes of the adult (LADA)**

Early after the discovery of antibodies against pancreatic antigens in the serum of type 1 diabetic subjects, it was noticed by clinicians that 10% of patients first diagnosed with type 2 diabetes tested positive for type 1 diabetes antibodies (mainly GADA and ICA) (Palmer et al, 2005). Those patients slowly but relentlessly progressed to insulin-dependency and showed signs of pancreatic islet dysfunction such as a progressive decrease of C-peptide levels. These characteristics defined a new category in the diabetes spectra called the latent autoimmune diabetes of the adult, and abbreviated LADA. Diagnostic criteria for these patients are age at diagnosis over 30 years, presence of antibodies against pancreatic islets, with a higher frequency of single positivity and GADA or ICA antibodies than type 1 diabetes patients, and no requirement of insulin for at least six months since diagnosis (Palmer et al, 2005; Leslie et al, 2006). There seems to exist a similar but milder genetic background to that of type 1 diabetic patients (Hosszufalusi et al, 2003; Palmer et al, 2005), and some researchers think of LADA as a slower and less aggressive form of type 1 diabetes, to the point that this condition is usually termed type 1.5 diabetes. Debate exists over LADA being an entity of its own or just a less aggressive form of type 1 diabetes at an older age (Hosszufalusi et al, 2003; Palmer et al, 2005; Leslie et al, 2006; Steck & Eisenbarth, 2008). Anyway, this group of patients poses a very interesting subset for testing cell preserving therapies in an autoimmune form of diabetes due to its slow progression to insulin-dependency.

#### **2.4 The diagnosis of an adult-onset diabetic patient**

When recruiting patients for genetic studies, a careful evaluation of adult-onset patients must be carried out to avoid misclassification. In most cases, the three more common forms of diabetes in adults can be distinguished with a test for antibodies against pancreatic antigens at diagnosis and the requirement for insulin therapy (see table 2).

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 33

environmental factors (Redondo et al, 2001a; Pociot et al, 2010). Half of this genetic contribution is due to alleles and haplotypic combinations in the HLA region (Erlich et al, 2008), which is the strongest genetic modifier of type 1 diabetes risk. The rest of the genetic load is composed of genes with smaller effects, some of which have been unveiled in the last thirty years through different approaches developed as the technology for DNA study evolved. These studies ranged from the association studies of candidate genes to the complex hypothesis-free genome-wide association studies. In this section, we will briefly review the methodology employed in these studies and its importance in the

The association studies are suited for the detection of variants with moderate or low effects on the disease, as long as the studied variants are relatively frequent in the population of study (minor allele frequency over 5%) (Pociot et al, 2010; Steck & Rewers, 2011). Association can be measured in a case-control design (study of the differences between a set of unrelated patients and healthy controls) or a family design (analysis of deviations in the theoretical 50% transmission of the variant from healthy parents to patients). Previous to the massive knowledge that the Human Genome Project provided to genetic studies in humans, association studies had to focus on the selection of candidate genes, which limited these studies to genes with known function and biased the selection by what was known (or believed) about the pathogenesis of the disease at the moment. Nevertheless, this approach discovered the five classical regions associated with

(Nerup et al, 1974) HLA class II Antigen presentation in antigen-presenting cells.

(Bell et al, 1984) *INS* Expression levels in the thymus regulate the

Table 3. Classical type 1 diabetes associated genes discovered through association studies of

presence of insulin-reactive T cells.

Modulator of inactivation of the immune response. Constitutively expressed in regulatory T cells, a lymphocyte subset specialized in the suppression of autoimmunity.

Suppressor of signals through the TCR. Susceptibility variant is believed to favor the survival of auto-reactive T cells in the thymus.

Alpha subunit of the high affinity IL2 receptor. Constitutively expressed in regulatory T cells, it is essential for the maintenance of this cell subset.

**First reported Gene Function** 

unraveling of the type 1 diabetes genetic component.

**3.1 Association studies of candidate genes** 

type 1 diabetes and detailed in table 3.

1970-1980

1984

1996

2004

2005

(Nistico et al, 1996) *CTLA4* 

(Bottini et al, 2004) *PTPN22* 

(Vella et al, 2005) *IL2RA* 

candidate genes. TCR: T cell receptor.


Table 2. Summary of the characteristics of the three more common forms of diabetes in adulthood: adult-onset type 1 diabetes, type 2 diabetes and LADA (Leslie et al, 2006; ADA, 2010).

The subgroup of adult-onset type 1 diabetic patients is relatively easy to separate from the other two clinical manifestations of diabetes in adulthood: patients should be insulindependent since diagnosis, positive for at least two type 1 diabetes autoantibodies and usually of lean body type. However, many genetic studies in the last years have excluded the adult subset of type 1 diabetic patients as a precaution to avoid contamination with type 2 diabetic patients. This conservative measure has excluded a group of patients that could give us important information about the genetics of the disease: the existence of genes that modify the progression of the disease, together with unknown environmental factors, which would be the causatives of the fast immune cell destruction in a child and the slower destruction in an adult patient.

From now on, we will focus on the genetics of type 1 diabetes and the study of the influence of genetics in age at disease onset.

#### **3. Genetic studies in type 1 diabetes**

The existence of a genetic basis that influences the development of type 1 diabetes is known since the first studies associated alleles and haplotypes in the Human Leukocyte Antigen (HLA) complex with type 1 diabetes risk in the late 70s (Nerup et al, 1974). Familial studies have been able to quantify the genetic basis of the disease in a range between 30-70% of the total contributing factors, being the remainder due to environmental factors (Redondo et al, 2001a; Pociot et al, 2010). Half of this genetic contribution is due to alleles and haplotypic combinations in the HLA region (Erlich et al, 2008), which is the strongest genetic modifier of type 1 diabetes risk. The rest of the genetic load is composed of genes with smaller effects, some of which have been unveiled in the last thirty years through different approaches developed as the technology for DNA study evolved. These studies ranged from the association studies of candidate genes to the complex hypothesis-free genome-wide association studies. In this section, we will briefly review the methodology employed in these studies and its importance in the unraveling of the type 1 diabetes genetic component.

#### **3.1 Association studies of candidate genes**

32 Type 1 Diabetes Complications

Corporal phenotype Usually lean Usually obese Variable

Diabetic ketoacidosis Present Rare Rare

pancreatic islet antigens Positive Negative Positive

% of total adult diabetes 5-10% 80-85% 5-10%

Table 2. Summary of the characteristics of the three more common forms of diabetes in adulthood: adult-onset type 1 diabetes, type 2 diabetes and LADA (Leslie et al, 2006; ADA,

susceptibility Present Absent Present

The subgroup of adult-onset type 1 diabetic patients is relatively easy to separate from the other two clinical manifestations of diabetes in adulthood: patients should be insulindependent since diagnosis, positive for at least two type 1 diabetes autoantibodies and usually of lean body type. However, many genetic studies in the last years have excluded the adult subset of type 1 diabetic patients as a precaution to avoid contamination with type 2 diabetic patients. This conservative measure has excluded a group of patients that could give us important information about the genetics of the disease: the existence of genes that modify the progression of the disease, together with unknown environmental factors, which would be the causatives of the fast immune cell destruction in a child and the slower

From now on, we will focus on the genetics of type 1 diabetes and the study of the influence

The existence of a genetic basis that influences the development of type 1 diabetes is known since the first studies associated alleles and haplotypes in the Human Leukocyte Antigen (HLA) complex with type 1 diabetes risk in the late 70s (Nerup et al, 1974). Familial studies have been able to quantify the genetic basis of the disease in a range between 30-70% of the total contributing factors, being the remainder due to

**1 diabetes Type 2 diabetes LADA** 

Useful for improved glycemic control in some cases

Not for the first 6 months after diagnosis. Patients eventually evolve to insulindependency

At least 1 positive High frequency of GADA+ and/or ICA+

decreased Low

**Adult-onset type** 

Ultimately needed for survival

Antibodies 2 or more positive Negative

C-peptide levels Low or absent Normal or slightly

Insulin requirement

T-cell response against

Type 1 diabetes HLA

destruction in an adult patient.

of genetics in age at disease onset.

**3. Genetic studies in type 1 diabetes** 

2010).

The association studies are suited for the detection of variants with moderate or low effects on the disease, as long as the studied variants are relatively frequent in the population of study (minor allele frequency over 5%) (Pociot et al, 2010; Steck & Rewers, 2011). Association can be measured in a case-control design (study of the differences between a set of unrelated patients and healthy controls) or a family design (analysis of deviations in the theoretical 50% transmission of the variant from healthy parents to patients). Previous to the massive knowledge that the Human Genome Project provided to genetic studies in humans, association studies had to focus on the selection of candidate genes, which limited these studies to genes with known function and biased the selection by what was known (or believed) about the pathogenesis of the disease at the moment. Nevertheless, this approach discovered the five classical regions associated with type 1 diabetes and detailed in table 3.


Table 3. Classical type 1 diabetes associated genes discovered through association studies of candidate genes. TCR: T cell receptor.

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 35

**Chromosome Candidate gene OR Reference study Published age-at-**

1p13.2 *PTPN22* 2.05 (Smyth et al. 2008) Yes

2q32.2 *STAT4* 1.10 (Fung et al. 2009) Yes

10p15.1 *IL2RA* 0.62 (Smyth et al. 2008) Yes

11p15.5 *INS* 0.42 (Smyth et al. 2008) Yes

12q13.2 *ERBB3* 1.31 (Barrett et al. 2009) Yes 12q24.12 *SH2B3* 1.28 (Smyth et al. 2008) Yes

16p13.13 *CLEC16A* 0.81 (Smyth et al. 2008) Yes

Table 4. Summary of the 50 chromosomal regions currently associated with type 1 diabetes (continues in next page). The odds ratio in the table has been extracted from the reference study. Data come from the on-line database www.t1dbase.org, belonging to the Type 1 Diabetes Genetics Consortium (T1DGC). The reference study does not correspond with the published age-at-onset study. Genes *CLEC16A* and *SH2B3* were analyzed in Todd et al (2007) and where not associated with age at onset. The rest of the age-at-onset associations

(Ounissi-Benkalha and Polychronakos 2008)

1q31.2 *RGS1* 0.89 (Smyth et al. 2008) 1q32.1 *IL20-IL10-IL19* 0.84 (Barrett et al. 2009) 2q11.2 Several -- (Barrett et al. 2009) 2q24.2 *IFIH1* 0.86 (Smyth et al. 2008)

2q33.2 *CTLA4* 0.82 (Smyth et al. 2008) 3p21.31 *CCR5* 0.85 (Smyth et al. 2008) 4p15.2 *AC111003.1* 1.09 (Barrett et al. 2009) 4q27 *IL2-IL21* 1.13 (Barrett et al. 2009)

6q15 *BACH2* 1.13 (Cooper et al. 2008) 6q22.32 *CENPW* 1.17 (Barrett et al. 2009) 6q23.3 *TNFAIP3* 0.90 (Fung et al. 2009) 6q25.3 *TAGAP* 0.92 (Smyth et al. 2008) 7p15.2 Several 0.88 (Barrett et al. 2009) 7p12.1 *COBL* 0.77 (Barrett et al. 2009) 9p24.2 *GLIS3* 0.88 (Barrett et al. 2009)

10p15.1 *PRKCQ* 0.69 (Lowe et al. 2007) 10q22.3 *ZMIZ1* -- (Barrett et al. 2009) 10q23.31 *RNLS* 0.75 (Barrett et al. 2009)

12p13.31 *CLEC2D-CD69* 1.09 (Barrett et al. 2009) 12q13.3 *CYP27B1* 1.22 (Bailey et al. 2007)

13.32.3 *GRP183* 1.15 (Heinig et al. 2010) 14q24.1 Several 0.86 (Barrett et al. 2009) 14q32.2 Several 1.09 (Barrett et al. 2009) 14q32.2 Several 0.90 (Wallace et al. 2009) 15q14 *RASGRP1* 1.21 (Qu et al. 2009) 15q25.1 *CTSH* 0.86 (Smyth et al. 2008)

16p11.2 *IL27* 0.86 (Barrett et al. 2009) 16q23.1 Several 1.28 (Barrett et al. 2009) 17q12 Several 0.87 (Barrett et al. 2009) 17q21.2 *SMARCE1* 0.95 (Barrett et al. 2009)

are reviewed in section 4.

6p21 *HLA* 0.02-49.2

**onset analysis** 

Yes

#### **3.2 The genome wide association studies**

The first genome-wide association study was published in 2007 (The Wellcome Trust Case-Control Consortium [WTCCC], 2007). Samples from seven diseases (among them type 1 and type 2 diabetes) were collected, recruiting 2000 cases of each disease and a common subset of 3000 healthy controls. This single study described four new regions strongly associated with type 1 diabetes, almost the same number of regions that the previous association studies had taken three decades to discover.

Genome-wide association studies were possible thanks to the great development in the knowledge of human genetics and in the techniques to study DNA, derived from initiatives such as the Human Genome Project. The design of a genome-wide study is based on the analysis of over 500.000 single nucleotide polymorphisms (SNPs) throughout the genome in each subject recruited. The study design is usually a case-control approach, but both casecontrol and family studies are commonly used to replicate the stronger associations in the recent genome-wide studies (Hakonarson et al, 2007; Barrett et al, 2009). Since their object of analysis is the whole genome, these kind of studies are hypothesis-free and are able to detect associations in regions and genes that a candidate gene study would have never considered, such as regions with genes of unknown function and genes in routes not classically considered to take part in the pathogenesis of the disease. Thus, from the four regions associated with type 1 diabetes and described in the WTCCC study (WTCCC, 2007), one (16p13) covered a gene of unknown function, and two (12q13 and 12q24) pointed to regions with several candidate genes.

Despite the advantage that poses the hypothesis-free design, the genome-wide association studies have a great disadvantage in return: the high number of polymorphisms studied implies an elevated number of statistical comparisons and an increased probability of obtaining false-positive associations. Therefore, these studies are subject to a strong statistical correction (WTCCC, 2007), and, depending on the number of markers analyzed, p values should be as low as 10-7 to be considered statistically significant (Todd et al, 2007). Moreover, the results obtained (especially those that are borderline significant) should be replicated in independent populations to assure that the result is not a false positive and it is not influenced by population variability (McCarthy et al, 2008). At this stage is where follow-up studies take place. Follow-up studies select associations from genome-wide studies for replication purposes, being the more interesting those that are borderline significant.

Genome-wide association studies and their follow-up have been successful in uncovering associations of high to moderate effect (odds ratio over 1.15) in variants with a minor allele frequency over 5%. Now, the remainder of the genetic component in type 1 diabetes is proposed to reside in rare variants with high effect and common variants with low effect on the disease (Pociot et al, 2010). Due to the stringent statistical correction required, genomewide studies are not suitable for detecting these associations and new approaches will be necessary. Despite their limitations, in just five years genome-wide studies have revealed ten times more genetic regions than the older approaches did in thirty years, providing fifty genetic regions associated with type 1 diabetes. A brief summary of these regions can be consulted in table 4.

#### **3.3 The problem with age at diagnosis**

Although type 1 diabetes has a similar prevalence in all ages, the restriction to pediatric patients has been a popular criterion for recruitment of patients in studies on the genetics of the disease, as it is shown in table 5. Its purpose is to avoid the inclusion of misdiagnosed

The first genome-wide association study was published in 2007 (The Wellcome Trust Case-Control Consortium [WTCCC], 2007). Samples from seven diseases (among them type 1 and type 2 diabetes) were collected, recruiting 2000 cases of each disease and a common subset of 3000 healthy controls. This single study described four new regions strongly associated with type 1 diabetes, almost the same number of regions that the previous association

Genome-wide association studies were possible thanks to the great development in the knowledge of human genetics and in the techniques to study DNA, derived from initiatives such as the Human Genome Project. The design of a genome-wide study is based on the analysis of over 500.000 single nucleotide polymorphisms (SNPs) throughout the genome in each subject recruited. The study design is usually a case-control approach, but both casecontrol and family studies are commonly used to replicate the stronger associations in the recent genome-wide studies (Hakonarson et al, 2007; Barrett et al, 2009). Since their object of analysis is the whole genome, these kind of studies are hypothesis-free and are able to detect associations in regions and genes that a candidate gene study would have never considered, such as regions with genes of unknown function and genes in routes not classically considered to take part in the pathogenesis of the disease. Thus, from the four regions associated with type 1 diabetes and described in the WTCCC study (WTCCC, 2007), one (16p13) covered a gene of unknown function, and two (12q13 and 12q24) pointed to regions

Despite the advantage that poses the hypothesis-free design, the genome-wide association studies have a great disadvantage in return: the high number of polymorphisms studied implies an elevated number of statistical comparisons and an increased probability of obtaining false-positive associations. Therefore, these studies are subject to a strong statistical correction (WTCCC, 2007), and, depending on the number of markers analyzed, p values should be as low as 10-7 to be considered statistically significant (Todd et al, 2007). Moreover, the results obtained (especially those that are borderline significant) should be replicated in independent populations to assure that the result is not a false positive and it is not influenced by population variability (McCarthy et al, 2008). At this stage is where follow-up studies take place. Follow-up studies select associations from genome-wide studies for replication

Genome-wide association studies and their follow-up have been successful in uncovering associations of high to moderate effect (odds ratio over 1.15) in variants with a minor allele frequency over 5%. Now, the remainder of the genetic component in type 1 diabetes is proposed to reside in rare variants with high effect and common variants with low effect on the disease (Pociot et al, 2010). Due to the stringent statistical correction required, genomewide studies are not suitable for detecting these associations and new approaches will be necessary. Despite their limitations, in just five years genome-wide studies have revealed ten times more genetic regions than the older approaches did in thirty years, providing fifty genetic regions associated with type 1 diabetes. A brief summary of these regions can be

Although type 1 diabetes has a similar prevalence in all ages, the restriction to pediatric patients has been a popular criterion for recruitment of patients in studies on the genetics of the disease, as it is shown in table 5. Its purpose is to avoid the inclusion of misdiagnosed

purposes, being the more interesting those that are borderline significant.

**3.2 The genome wide association studies** 

studies had taken three decades to discover.

with several candidate genes.

consulted in table 4.

**3.3 The problem with age at diagnosis** 


Table 4. Summary of the 50 chromosomal regions currently associated with type 1 diabetes (continues in next page). The odds ratio in the table has been extracted from the reference study. Data come from the on-line database www.t1dbase.org, belonging to the Type 1 Diabetes Genetics Consortium (T1DGC). The reference study does not correspond with the published age-at-onset study. Genes *CLEC16A* and *SH2B3* were analyzed in Todd et al (2007) and where not associated with age at onset. The rest of the age-at-onset associations are reviewed in section 4.

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 37

4000 cases and 2997 British families

563 cases and 1422 families from Britain, the US and Australia

1785 US cases

8064 cases and 3064 families from US, Finland, Ireland, Norway and Romania

T1DGC population 3983 cases and 2319 families from Britain, the US and Australia

Table 5. Genome-wide and major genetic studies performed in type 1 diabetes. Studies 2, 4 and 6 performed metanalysis with the WTCCC data. Also, the last genome-wide carried out by Barrett et al (study 6) performed a meta-analysis of the three larger genome-wide studies

The T1DGC (Type 1 Diabetes Genetics Consortium) is an initiative constituted in 2002 with the aim of providing resources to the research in type 1 diabetes. Since 2007, the consortium has published several studies on type 1 diabetes genetics (Erlich et al, 2008; Hakonarson et al, 2008; Howson et al, 2009; Qu et al, 2009) and, although the genome-wide study in which the T1DGC population was genotyped did not include an age-at-onset analysis, this group is lately including age-at-onset analyses in their publications and some of their studies have provided the first evidences of influence of genetics in age at onset of the post-genome wide era (Hakonarson et al, 2008). The selection criteria for adult patients are year at diagnosis under 35 years and uninterrupted insulin treatment for at least 6 months (Hakonarson et al, 2007). However, despite the wider limit in age at onset in this population, the majority of the patients included are pediatric, as reflected in the mean age at onset (around ten years)

The influence of genetics in age at onset of type 1 diabetes has been analyzed in some studies. However, initiatives to replicate these first studies or to establish a protocol to analyze the genetics or early and late onset are lacking, and therefore there is disparity in the definition and selection of late-onset type 1 diabetes patients, and in the statistical methods employed to analyze the associations. In this section, we will review some selected

found in the published studies (Hakonarson et al, 2008; Howson et al, 2009).

**4. Reported genetic associations of genes and age-at-diagnosis** 

studies on the influence of genetics in age at diagnosis of type 1 diabetes.

**Age limit for selection of participants** 

years

Diagnosis before 17 years

Most diagnosed

Diagnosis before 31 years

Diagnosis before 17 years

Diagnosis before 35 years

before 18 years No

**Study of genetics and age at diagnosis** 

No

Yes

No

No

No

**publication Population** 

(1) WTCCC 2007 2000 cases, British Diagnosis before 17

**Study Year of** 

(3) Hakonarson et al 2007

(6) Barrett et al 2009

(1, 4 and 6) performed in type 1 diabetes.

2007 Follow-up from WTCCC

(4) Cooper et al 2008 GoKinD population

2008 Major genetic study

(2) Todd et al

(5) Smyth et al


Table 4 (continuation). Summary of the 50 chromosome regions currently associated with type 1 diabetes.

patients (type 2 diabetics or LADA patients) within adult-onset diabetic patients. However, enough criteria exist to discriminate type 1 diabetic patients from the remainder of diabetic adults, and the exclusion of adult type 1 diabetes patients limits the knowledge of the genetics of the disease only to its early onset. Adult-onset patients show signs of a slower immune reaction to cells. The factors that cause a rapid destruction of cells in a child but a slower degeneration in an adult-onset patient are unknown, nevertheless they are probably a mixture of genetic and environmental factors. Some hypotheses may explain the different speed in clinical manifestations: the genetic load of adult-onset diabetes could be composed of a lower number of associated genes than in the early-onset patients, or could be the same genes but with less effect in the adult disease, or maybe the adult-onset population has genes associated that are exclusive of adult-onset. Besides, the simple replication in adult-onset patients of associations found in pediatric type 1 diabetes is interesting to prove that, from a genetic perspective, adult-onset patients are as much type 1 diabetes as the pediatric-onset ones.

Four genome-wide studies and a series of follow-up have been published in type 1 diabetes in the last five years (table 5). Two included systematically some adult-onset patients in their populations; however most of these studies lacked an analysis of the influence of genetics in the age of diagnosis. The characteristics of the four genome-wide and some selected followup and major genetic studies, and the populations included in them, can be consulted in table 5.

The two populations that recruit late-onset patients deserve a more detailed commentary. The GoKinD (Genetics of kidneys in diabetes) population, included in Cooper et al in 2008, belongs to a United States project that aims at studying the genetics of kidney diseases in type 1 diabetes. Selection criteria for type 1 diabetes were age at diagnosis before 31 years, insulin therapy needed within the first year of diagnosis and not interrupted for any reason ever since. Patients had a minimum disease duration of 10 years. Analysis of the influence of genetics in age at diagnosis was not carried out in this genome-wide study.

**Chromosome Candidate gene OR Reference study Published age-at-**

18p11.21 *PTPN2* 1.28 (Smyth et al. 2008) Yes

Table 4 (continuation). Summary of the 50 chromosome regions currently associated with

patients (type 2 diabetics or LADA patients) within adult-onset diabetic patients. However, enough criteria exist to discriminate type 1 diabetic patients from the remainder of diabetic adults, and the exclusion of adult type 1 diabetes patients limits the knowledge of the genetics of the disease only to its early onset. Adult-onset patients show signs of a slower immune reaction to cells. The factors that cause a rapid destruction of cells in a child but a slower degeneration in an adult-onset patient are unknown, nevertheless they are probably a mixture of genetic and environmental factors. Some hypotheses may explain the different speed in clinical manifestations: the genetic load of adult-onset diabetes could be composed of a lower number of associated genes than in the early-onset patients, or could be the same genes but with less effect in the adult disease, or maybe the adult-onset population has genes associated that are exclusive of adult-onset. Besides, the simple replication in adult-onset patients of associations found in pediatric type 1 diabetes is interesting to prove that, from a genetic perspective, adult-onset patients are as much type 1

Four genome-wide studies and a series of follow-up have been published in type 1 diabetes in the last five years (table 5). Two included systematically some adult-onset patients in their populations; however most of these studies lacked an analysis of the influence of genetics in the age of diagnosis. The characteristics of the four genome-wide and some selected followup and major genetic studies, and the populations included in them, can be consulted in

The two populations that recruit late-onset patients deserve a more detailed commentary. The GoKinD (Genetics of kidneys in diabetes) population, included in Cooper et al in 2008, belongs to a United States project that aims at studying the genetics of kidney diseases in type 1 diabetes. Selection criteria for type 1 diabetes were age at diagnosis before 31 years, insulin therapy needed within the first year of diagnosis and not interrupted for any reason ever since. Patients had a minimum disease duration of 10 years. Analysis of the influence of

genetics in age at diagnosis was not carried out in this genome-wide study.

type 1 diabetes.

table 5.

diabetes as the pediatric-onset ones.

18q22.2 *CD226* 1.16 (Smyth et al. 2008) 19p13.2 *TYK2* 0.86 (Wallace et al. 2009) 19q13.32 Several 0.86 (Barrett et al. 2009) 19q13.4 *FUT2* -- (Barrett et al. 2009) 20p13 Several 0.90 (Barrett et al. 2009) 21q22.3 *UBASH3A* 1.13 (Smyth et al. 2008) 22q12.2 Several 1.10 (Barrett et al. 2009) 22q12.3 *IL2RB* -- (Barrett et al. 2009) 22q13.1 *C1QTNF6* 1.11 (Cooper et al. 2008) Xp22.2 *TLR8* 0.84 (Barrett et al. 2009) Xq28 Several 1.16 (Barrett et al. 2009)

**onset analysis** 


Table 5. Genome-wide and major genetic studies performed in type 1 diabetes. Studies 2, 4 and 6 performed metanalysis with the WTCCC data. Also, the last genome-wide carried out by Barrett et al (study 6) performed a meta-analysis of the three larger genome-wide studies (1, 4 and 6) performed in type 1 diabetes.

The T1DGC (Type 1 Diabetes Genetics Consortium) is an initiative constituted in 2002 with the aim of providing resources to the research in type 1 diabetes. Since 2007, the consortium has published several studies on type 1 diabetes genetics (Erlich et al, 2008; Hakonarson et al, 2008; Howson et al, 2009; Qu et al, 2009) and, although the genome-wide study in which the T1DGC population was genotyped did not include an age-at-onset analysis, this group is lately including age-at-onset analyses in their publications and some of their studies have provided the first evidences of influence of genetics in age at onset of the post-genome wide era (Hakonarson et al, 2008). The selection criteria for adult patients are year at diagnosis under 35 years and uninterrupted insulin treatment for at least 6 months (Hakonarson et al, 2007). However, despite the wider limit in age at onset in this population, the majority of the patients included are pediatric, as reflected in the mean age at onset (around ten years) found in the published studies (Hakonarson et al, 2008; Howson et al, 2009).

#### **4. Reported genetic associations of genes and age-at-diagnosis**

The influence of genetics in age at onset of type 1 diabetes has been analyzed in some studies. However, initiatives to replicate these first studies or to establish a protocol to analyze the genetics or early and late onset are lacking, and therefore there is disparity in the definition and selection of late-onset type 1 diabetes patients, and in the statistical methods employed to analyze the associations. In this section, we will review some selected studies on the influence of genetics in age at diagnosis of type 1 diabetes.

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 39

 Insulin gene: the T1DGC group analyzed several classical type 1 diabetes genes (Howson et al, 2009) and found association of the susceptibility variant in the insulin gene to an onset of type 1 diabetes two years earlier than the protective allele. However, this effect was not replicated in one of the cohorts included in the study. We will

 *IL2RA*: a Finnish study analyzed several classical type 1 diabetes genes in a group with late-onset of the disease (Klinker et al, 2010). They found associations of the insulin, *PTPN22*, *IFIH1* and *CTLA4* genes with late-onset patients, and replicated the age-atonset effect of the *DRB1\*03-DRB1\*04-DQB1\*03:02* heterozygote. They also found that *IL2RA* was associated with an earlier disease onset. However, the T1DGC studied the *IL2RA* gene and did not find any effect in age at onset, although they did not include the stronger association in the gene that the Finnish study did analyze (Howson et al, 2009). Our group conducted a replication study in polymorphisms in the *IL2RA* gene and we found them associated to both early and late disease onset (Espino-Paisan et al,

 *PTPN22*: a German group studied the C1858T polymorphism in the *PTPN22* gene and found that the susceptibility polymorphism was associated to an earlier onset of the disease in a group of pediatric-onset patients (Kordonouri et al, 2010). Patients with the susceptibility polymorphism had an onset of the disease two years earlier than homozygotes for the protective allele. However, this observation was not replicated in the T1DGC study (Howson et al, 2009). We will study this polymorphism in our group

 *PTPN2*: our group studied the influence in age at disease onset of two polymorphisms in the *PTPN2* gene that had been previously associated with type 1 diabetes (Todd et al, 2007; WTCCC, 2007). We found that one of the studied polymorphisms was associated with an earlier disease onset, with carriers of the susceptibility allele having a disease onset almost three years earlier than homozygotes for the protective allele (Espino-

We have selected ten chromosome regions (five classical genes and five genome-wide discoveries) previously studied in type 1 diabetes to test their association with pediatric and late-onset patients. We will briefly review their role in the pathogenesis of the disease and

A total of 444 type 1 diabetes patients (47% female) were included in this study. All patients were recruited from the Madrid area (Spain), all where Caucasoid and diagnosed according to the criteria of the American Diabetes Association (ADA, 2010). Age at diagnosis was available for 415 patients and ranged from 1 to 65 years. Mean age at onset of the population was 18.611.1 years and median age at onset was 16 years. All patients were insulin-dependent since diagnosis and had been on uninterrupted insulin treatment for at least 6 months. Adult patients diagnosed over 35 years were included on the basis of positivity to autoantibodies, lean body type and insulin-dependency status. Also, a maximum of 888 ethnically matched controls

(53.7% female) with no history of type 1 diabetes in first degree relatives were recruited.

**5. A practical study: Genetic analysis of a population with early and** 

analyze the same polymorphism in our study in section 5.

of pediatric and adult patients in section 5.

2011c).

Paisan et al, 2011a).

**late-onset type 1 diabetes patients** 

**5.1 Population of study and methods** 

compare our results with the previous reported associations.


 Familial studies: analysis in monozygotic twins with one member affected with type 1 diabetes have shown that the probability of developing the disease in the non-affected twin is considerably higher (38%) when the affected twin developed type 1 diabetes at an early age (under 24 years) than when the affected twin developed the disease after 25 years of age (6% risk for the non-affected twin) (Redondo et al, 2001b). This observation might suggest that early-onset type 1 diabetes has a stronger genetic component (responsible of the higher concordance rate) than the same disease with a late onset. HLA associations to age at onset: several studies (Redondo et al, 2001a; Leslie et al, 2006; Klinker et al, 2010) have pointed out to the higher risk and earlier onset of type 1 diabetes in patients that are heterozygote for the HLA class II risk haplotypes *DRB1\*03* and *DRB1\*04-DQB1\*03:02*. On the other hand, the influence of HLA class I alleles in age at diagnosis has been thoroughly studied, and alleles *B\*39* and *A\*24* have been consistently associated to an earlier onset of the disease (Valdes et al, 2005; Nejentsev et al, 2007). The *B\*39* allele, for example, precipitates the age of diagnosis in four years

 *IL12B*: the gene *IL12B* codes for the p40 subunit of the interleukin 12, also shared with interleukin 23. A 2004 study (Windsor et al, 2004) carried out in an Australian cohort including early and late onset patients found association of a polymorphism in position +1188 of the gene with late-onset of the disease (over 25 years). Neither associations on the *IL12B* gene with type 1 diabetes nor the described age-at-onset association have

 *CAPSL-IL7R*: this region was first associated with type 1 diabetes in a study of nonsynonymous polymorphisms, finding a marker in the *CAPSL* gene that was highly associated with the disease (Smyth et al, 2006). Another polymorphism in the *IL7R* gene has been associated to type 1 diabetes and multiple sclerosis (Hafler et al, 2007; Todd et al, 2007). Our group undertook a replication study on both polymorphisms briefly after the discovery of the first signal (Santiago et al, 2008). We found association with type 1 diabetes in both polymorphisms and also described that both markers were associated

 Region 12q13 (*ERBB3* gene): in a replication study of borderline significant signals from a previous genome-wide (Hakonarson et al, 2007), the T1DGC found evidence of the association of three 12q13 polymorphisms with age at diagnosis (Hakonarson et al, 2008). The influence of this region on age at onset of type 1 diabetes has been subsequently analyzed in two independent studies (Awata et al, 2009; Wang et al, 2010) that, with different statistical methodology, did not replicate the effect seen in the first study. Finally, we have studied several signals in this region and found an age-atdiagnosis effect stronger than the previously described, with homozygotes for the susceptibility allele having an age at onset five years earlier than carriers of protective

 Region 2q32 (*STAT4* gene): a study in 2008 described an association of polymorphisms in the *STAT4* gene with type 1 diabetes patients with an onset earlier than 8 years (Lee et al, 2008). Among the polymorphisms studied was rs7574865, which we will include in our study on age at onset in section 5. This study was carried out in a pediatric Korean population, therefore population differences have to be taken in consideration, given that the genetics of Asian and Caucasian type 1 diabetes patients present some

with an earlier onset, an effect more noticeable in the *IL7R* polymorphism.

(Valdes et al, 2005).

been replicated in recent studies.

alleles (Espino-Paisan et al, 2011b).

important differences (Ikegami et al, 2007).


#### **5. A practical study: Genetic analysis of a population with early and late-onset type 1 diabetes patients**

We have selected ten chromosome regions (five classical genes and five genome-wide discoveries) previously studied in type 1 diabetes to test their association with pediatric and late-onset patients. We will briefly review their role in the pathogenesis of the disease and compare our results with the previous reported associations.

#### **5.1 Population of study and methods**

A total of 444 type 1 diabetes patients (47% female) were included in this study. All patients were recruited from the Madrid area (Spain), all where Caucasoid and diagnosed according to the criteria of the American Diabetes Association (ADA, 2010). Age at diagnosis was available for 415 patients and ranged from 1 to 65 years. Mean age at onset of the population was 18.611.1 years and median age at onset was 16 years. All patients were insulin-dependent since diagnosis and had been on uninterrupted insulin treatment for at least 6 months. Adult patients diagnosed over 35 years were included on the basis of positivity to autoantibodies, lean body type and insulin-dependency status. Also, a maximum of 888 ethnically matched controls (53.7% female) with no history of type 1 diabetes in first degree relatives were recruited.

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 41

**Class II HLA alleles (chromosome 6p21)**: class II HLA binds extra-cellular antigens processed by antigen presenting cells and presents them to CD4+ helper T cells. The proposed mechanism in the pathogenesis of type 1 diabetes takes place at the negative selection process during lymphocyte thymic maturation (Redondo et al, 2001a; Ounissi-Benkalha and Polychronakos, 2008). Negative selection occurs when a T cell with an autoreactive T cell receptor (TCR) binds a HLA molecule loaded with an autoantigen. This union sends a strong activation signal through the TCR that is deleterious to the autoreactive T cell. Theoretically, susceptibility HLA alleles bind pancreatic antigens less efficiently, lowering the activation signal to non-deleterious levels and allowing the

The HLA associations detected in our group in the case-control analysis and the study on age at onset can be consulted in table 7. Due to the high number of alleles and haplotypic combinations in this region, and the low frequency of some of them, a stratified analysis would imply a marked loss of statistical power, so we choose to perform only the continuous analysis. We did not find evidence of an influence in age at diagnosis in any of the haplotypic combinations included, which means that our adult-onset patients have the same HLA contribution to type 1 diabetes than our pediatric patients. Of notice, there are two haplotypes with a marked difference in the mean age at diagnosis: the *DRB1\*04- DQB1\*03:02* homozygote (carriers have an onset three years earlier) and carriers of the protective haplotype *DRB1\*15:01-DQB1\*06:02* (carriers have an onset ten years later than non carriers). None of these comparisons are statistically significant, but it could be a problem of low statistical power since both genotypes are quite infrequent. A larger sample

Many studies describe the *DRB1\*03-DRB1\*04* heterozygote as associated with earlier age at diagnosis of type 1 diabetes, and also as the haplotypic combination that confers a higher risk to the disease. In our population we do not see an age effect (p=0.9). Moreover, the *DRB1\*03-DRB1\*04* heterozygote does not confer the higher risk, but the *DRB1\*04*

**Insulin gene (chromosome 11p15)**: the insulin gene is also proposed to participate in the generation of autoreactive T cells in the thymus. The polymorphism associated with type 1 diabetes is a VNTR (variable number of tandem repeats) that locates upstream of the *INS* gene and modifies its expression in the thymus (Pugliese et al, 1997; Vafiadis et al, 1997). Alleles in this VNTR range from 26 to 210 repetitions of a consensus sequence and are usually classified in three groups: short class I alleles (26 to 64 repetitions), intermediate class II alleles (64 to 139 repetitions, infrequent in Caucasian and Asian populations) and large class III alleles (140 to 210 repetitions). Large alleles are associated with protection from type 1 diabetes and are related to a higher expression of insulin in the thymus (Vafiadis et al, 1997). This is thought to favor the negative selection of insulin-reactive T cells, a theory that would be consistent with the lower levels of insulin antibodies detected in patients that carry the large class III alleles (Hermann et al, 2005). We have selected a polymorphism (rs689) in linkage disequilibrium with the two main classes of alleles that is usually employed in genetic studies (Hermann et al, 2005; Todd et al, 2007; Smyth et al,

homozygote, the *DRB1\*03* or *DRB1\*04* carrier, and the *DRB1\*03* homozygote.

**5.2 Selected genes and results** 

**5.2.1 Classical type 1 diabetes associations** 

autoreactive T cell to escape from thymic selection.

would be needed to elucidate the possible associations.

2008) as a proxy to the VNTR genotyping.

Genes in the HLA complex were genotyped by two SSOP (Sequence Specific Oligonucleotide Probe) procedures: dot-blot hybridization and Luminex technology. The remaining genes were studied through genotyping of single nucleotide polymorphisms by TaqMan Assays in a 7900HT fast real-time PCR system (Applied Biosystems Foster City, CA, USA). The call rate for each SNP was 95%. A summary of these studied polymorphisms can be consulted in table 6.


Table 6. Summary of the genotyped SNPs in each gene. MAF: minor allele frequency.

No statistically significant deviations from Hardy-Weinberg equilibrium were found in the control subset for each polymorphism. A case-control analysis was performed to asses association of the selected variants with type 1 diabetes. Differences were calculated through Chi-square and Fisher's exact tests when necessary. Analysis of age at onset was performed through a stratified and a continuous approach. For the stratified analysis, cases were classified in early-onset (age at diagnosis under 17 years) or late-onset (age at diagnosis over 16 years) and compared with Chi-square test or Fisher's exact test. Associations were estimated by the odds ratio (OR) with 95% confidence interval. All Chi-square and Fisher's exact test comparisons were calculated with Epi Info v.5 (CDC, Atlanta, USA). For the continuous analysis approach, ages at onset associated to each allele were compared with the non-parametric U Mann-Whitney test implemented in SPSS v.15.0 (Chicago, Illinois, USA).

#### **5.2 Selected genes and results**

40 Type 1 Diabetes Complications

Genes in the HLA complex were genotyped by two SSOP (Sequence Specific Oligonucleotide Probe) procedures: dot-blot hybridization and Luminex technology. The remaining genes were studied through genotyping of single nucleotide polymorphisms by TaqMan Assays in a 7900HT fast real-time PCR system (Applied Biosystems Foster City, CA, USA). The call rate for each SNP was 95%. A summary of these studied polymorphisms

**Chromosome Candidate gene SNP Assay reference Control MAF** 

1p13 *PTPN22* rs2476601 By design T (0.06)

2q24 *IFIH1* rs1990760 C\_\_\_2780299\_30 G (0.46)

2q32.3 *STAT4* rs7574865 C\_\_29882391\_10 T (0.21)

6q23.3 *TNFAIP3* rs10499194 C\_\_\_1575581\_10 T (0.32)

9q33.2 *TRAF1* rs2269059 C\_\_15875924\_10 A (0.07)

11p15.5 *INS* rs689 C\_\_\_1223317\_10 A (0.28)

12p13.31 *CLEC2D* rs11052552 C\_\_32169467\_10 G (0.49)

16p13.13 *CLEC16A* rs2903692 C\_\_15941578\_10 A (0.42)

No statistically significant deviations from Hardy-Weinberg equilibrium were found in the control subset for each polymorphism. A case-control analysis was performed to asses association of the selected variants with type 1 diabetes. Differences were calculated through Chi-square and Fisher's exact tests when necessary. Analysis of age at onset was performed through a stratified and a continuous approach. For the stratified analysis, cases were classified in early-onset (age at diagnosis under 17 years) or late-onset (age at diagnosis over 16 years) and compared with Chi-square test or Fisher's exact test. Associations were estimated by the odds ratio (OR) with 95% confidence interval. All Chi-square and Fisher's exact test comparisons were calculated with Epi Info v.5 (CDC, Atlanta, USA). For the continuous analysis approach, ages at onset associated to each allele were compared with the non-parametric U Mann-Whitney test implemented in SPSS v.15.0 (Chicago, Illinois,

Table 6. Summary of the genotyped SNPs in each gene. MAF: minor allele frequency.

rs231775 C\_\_\_2415786\_20 T (0.29)

rs3087243 C\_\_\_3296043\_10 G (0.48)

can be consulted in table 6.

2q33.2 *CTLA-4* 

USA).

#### **5.2.1 Classical type 1 diabetes associations**

**Class II HLA alleles (chromosome 6p21)**: class II HLA binds extra-cellular antigens processed by antigen presenting cells and presents them to CD4+ helper T cells. The proposed mechanism in the pathogenesis of type 1 diabetes takes place at the negative selection process during lymphocyte thymic maturation (Redondo et al, 2001a; Ounissi-Benkalha and Polychronakos, 2008). Negative selection occurs when a T cell with an autoreactive T cell receptor (TCR) binds a HLA molecule loaded with an autoantigen. This union sends a strong activation signal through the TCR that is deleterious to the autoreactive T cell. Theoretically, susceptibility HLA alleles bind pancreatic antigens less efficiently, lowering the activation signal to non-deleterious levels and allowing the autoreactive T cell to escape from thymic selection.

The HLA associations detected in our group in the case-control analysis and the study on age at onset can be consulted in table 7. Due to the high number of alleles and haplotypic combinations in this region, and the low frequency of some of them, a stratified analysis would imply a marked loss of statistical power, so we choose to perform only the continuous analysis. We did not find evidence of an influence in age at diagnosis in any of the haplotypic combinations included, which means that our adult-onset patients have the same HLA contribution to type 1 diabetes than our pediatric patients. Of notice, there are two haplotypes with a marked difference in the mean age at diagnosis: the *DRB1\*04- DQB1\*03:02* homozygote (carriers have an onset three years earlier) and carriers of the protective haplotype *DRB1\*15:01-DQB1\*06:02* (carriers have an onset ten years later than non carriers). None of these comparisons are statistically significant, but it could be a problem of low statistical power since both genotypes are quite infrequent. A larger sample would be needed to elucidate the possible associations.

Many studies describe the *DRB1\*03-DRB1\*04* heterozygote as associated with earlier age at diagnosis of type 1 diabetes, and also as the haplotypic combination that confers a higher risk to the disease. In our population we do not see an age effect (p=0.9). Moreover, the *DRB1\*03-DRB1\*04* heterozygote does not confer the higher risk, but the *DRB1\*04* homozygote, the *DRB1\*03* or *DRB1\*04* carrier, and the *DRB1\*03* homozygote.

**Insulin gene (chromosome 11p15)**: the insulin gene is also proposed to participate in the generation of autoreactive T cells in the thymus. The polymorphism associated with type 1 diabetes is a VNTR (variable number of tandem repeats) that locates upstream of the *INS* gene and modifies its expression in the thymus (Pugliese et al, 1997; Vafiadis et al, 1997). Alleles in this VNTR range from 26 to 210 repetitions of a consensus sequence and are usually classified in three groups: short class I alleles (26 to 64 repetitions), intermediate class II alleles (64 to 139 repetitions, infrequent in Caucasian and Asian populations) and large class III alleles (140 to 210 repetitions). Large alleles are associated with protection from type 1 diabetes and are related to a higher expression of insulin in the thymus (Vafiadis et al, 1997). This is thought to favor the negative selection of insulin-reactive T cells, a theory that would be consistent with the lower levels of insulin antibodies detected in patients that carry the large class III alleles (Hermann et al, 2005). We have selected a polymorphism (rs689) in linkage disequilibrium with the two main classes of alleles that is usually employed in genetic studies (Hermann et al, 2005; Todd et al, 2007; Smyth et al, 2008) as a proxy to the VNTR genotyping.

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 43

In our study, we replicate the association previously described in the *INS* gene and we do not find effects on age at onset in the stratified and continuous analysis. Results are provided in

> **CASE-CONTROL AND AGE-STRATIFIED ANALYSES**

> > **MAF p OR**

T1D-control 0.175 0.282 8.3x10-9 0.54

T1D-control 0.328 0.299 0.1 1.14

T1D-control 0.477 0.518 0.05 0.85

T1D-control 0.115 0.064 7x10-6 1.91

T1D-control 0.373 0.409 0.1 0.86

Whitney test are presented. *INS*: insulin gene. T1D: type 1 diabetes.

Table 8. Analysis of classical gene associations in type 1 diabetes. Minor allele frequency (MAF) is provided in case-control and age-stratified analyses, and comparisons were calculated with Chi-square and Fisher's exact test when necessary. In the continuous analysis, mean ages at onset associated to each allele and the p value from the U Mann-

**CTLA4 (chromosome 2q33)**: this gene encodes a negative regulator of lymphocytic activation. Its expression is induced in activated lymphocytes, but it is also constitutively expressed in regulatory T cells, a lymphocyte subpopulation specialized in the suppression of autoimmunity. Also, a soluble form of CTLA4 is secreted in the serum, and it is believed that this form contributes to the downregulation of activation in the immune system (Ueda et al, 2003). Several polymorphisms have been identified and associated with type 1 diabetes (Ueda et al, 2003; Qu et al, 2009). We have selected two functional polymorphisms: one aminoacidic change related to lower membrane expression of the protein (rs231775) and a polymorphism in the 3' end that is related to higher expression of soluble CTLA4 (rs3087243), which also is one of the strongest associations with type 1 diabetes in the gene

Pediatric-adult T1D 0.162 0.177 0.6

Pediatric-adult T1D 0.323 0.339 0.6

Pediatric-adult T1D 0.493 0.451 0.2

Pediatric-adult T1D 0.113 0.127 0.5

Pediatric-adult T1D 0.373 0.394 0.6

(Ueda et al, 2003; Qu et al, 2009).

**CONTINUOUS ANALYSIS** 

> **Minor allele**

18.0 (10.7) 0.4

18.9 (11.4) 0.6

17.8 (10.8) 0.2

19.0 (10.6) 0.4

17.2 (10.6) 0.9

**Mean age at onset P Major** 

**allele** 

19.3 (11.8)

18.4 (10.9)

18.9 (11.0)

18.4 (11.2)

17.2 (10.3)

(0.43-0.67)

(0.96-1.37)

(0.72-1.00)

(1.42-2.56)

(0.70-1.06)

table 8.

*INS* 

**Gene** 

*CTLA4* **rs231775** 

*CTLA4* **rs3087243** 

*PTPN22* 

*IFIH1* 


Table 7. Case-control analysis of selected HLA haplotypes. Comparisons were calculated with Chi-square and Fisher's exact test when necessary. HLA haplotypes have been abbreviated: DR4-DQ8 (*DRB1\*04-DQA1\*03:01-DQB1\*03:02*), DR3-DQ2 (*DRB1\*03- DQA1\*05:01-DQB1\*02:01*), DR2-DQ6 (*DRB1\*15:01-DQA1\*01:02-DQB1\*06:02*). T1D: type 1 diabetes.


Table 7 (continuation). Analysis of age at onset in selected HLA haplotypes. Comparisons were calculated with the U Mann-Whitney test. HLA haplotypes: DR4-DQ8 (*DRB1\*04- DQA1\*03:01-DQB1\*03:02*), DR3-DQ2 (*DRB1\*03-DQA1\*05:01-DQB1\*02:01*), DR2-DQ6 (*DRB1\*15:01-DQA1\*01:02-DQB1\*06:02*). T1D: type 1 diabetes.

**T1D Controls** 

**DR4-DQ8 homozygote** 0.052 0.002 6.0x10-8 33.59 (5.40-1386)

**DR3-DQ2 carrier** 0.143 0.015 3.1x10-16 11.19 (5.31-24.41)

**DR4-DQ8 – X (not DR3)** 0.256 0.123 3.7x10-8 2.43 (1.74-3.39) **DR3-DQ2 – X (not DR4)** 0.369 0.221 2.0x10-7 2.03 (1.53-2.69) **DR2-DQ6 – X** 0.011 0.186 4.5x10-19 0.05 (0.02-0.12)

Table 7. Case-control analysis of selected HLA haplotypes. Comparisons were calculated with Chi-square and Fisher's exact test when necessary. HLA haplotypes have been abbreviated: DR4-DQ8 (*DRB1\*04-DQA1\*03:01-DQB1\*03:02*), DR3-DQ2 (*DRB1\*03-*

*DQA1\*05:01-DQB1\*02:01*), DR2-DQ6 (*DRB1\*15:01-DQA1\*01:02-DQB1\*06:02*). T1D: type 1

**DR4-DQ8 homozygote** 15.9 (11.5) 18.6 (11.1) 0.2

**DR3-DQ2 carrier** 17.5 (11.2) 18.6 (11.0) 0.2

**DR4-DQ8 – X (not DR3)** 18.0 (10.5) 18.9 (11.5) 0.7 **DR3-DQ2 – X (not DR4)** 18.8 (11.9) 18.4 (11.5) 0.9

Table 7 (continuation). Analysis of age at onset in selected HLA haplotypes. Comparisons were calculated with the U Mann-Whitney test. HLA haplotypes: DR4-DQ8 (*DRB1\*04- DQA1\*03:01-DQB1\*03:02*), DR3-DQ2 (*DRB1\*03-DQA1\*05:01-DQB1\*02:01*), DR2-DQ6

**DR4-DQ8** 18.0 (10.7) 18.6 (11.2) 0.9

**DR2-DQ6 – X** 28.0 (13.7) 18.5 (11.0) 0.2

**DR4-DQ8** 0.249 0.031 1.6x10-26 10.36 (6.11-17.75)

**Case-control analysis** 

0.894 0.384 2.1x10-61 13.24 (9.27-18.96)

**Continuous analysis** 

18.3 (11.1) 19.4 (10.2) 0.4

**Carrier Non carrier** 

**Mean age at onset P** 

**Genotype frequency p OR** 

**Genotype** 

**DR3-DQ2 carrier or DR4-DQ8 carrier** 

**DR3-DQ2** 

**Genotype** 

**DR3-DQ2 carrier Or DR4-DQ8 carrier** 

**DR3-DQ2** 

(*DRB1\*15:01-DQA1\*01:02-DQB1\*06:02*). T1D: type 1 diabetes.

diabetes.

In our study, we replicate the association previously described in the *INS* gene and we do not find effects on age at onset in the stratified and continuous analysis. Results are provided in table 8.


Table 8. Analysis of classical gene associations in type 1 diabetes. Minor allele frequency (MAF) is provided in case-control and age-stratified analyses, and comparisons were calculated with Chi-square and Fisher's exact test when necessary. In the continuous analysis, mean ages at onset associated to each allele and the p value from the U Mann-Whitney test are presented. *INS*: insulin gene. T1D: type 1 diabetes.

**CTLA4 (chromosome 2q33)**: this gene encodes a negative regulator of lymphocytic activation. Its expression is induced in activated lymphocytes, but it is also constitutively expressed in regulatory T cells, a lymphocyte subpopulation specialized in the suppression of autoimmunity. Also, a soluble form of CTLA4 is secreted in the serum, and it is believed that this form contributes to the downregulation of activation in the immune system (Ueda et al, 2003). Several polymorphisms have been identified and associated with type 1 diabetes (Ueda et al, 2003; Qu et al, 2009). We have selected two functional polymorphisms: one aminoacidic change related to lower membrane expression of the protein (rs231775) and a polymorphism in the 3' end that is related to higher expression of soluble CTLA4 (rs3087243), which also is one of the strongest associations with type 1 diabetes in the gene (Ueda et al, 2003; Qu et al, 2009).

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 45

**CASE-CONTROL AND AGE-STRATIFIED ANALYSES** 

**MAF p OR** 

Table 9. Analysis of selected gene associations from genome-wide studies in type 1 diabetes. Minor allele frequency (MAF) is provided in case-control and pediatric vs adult onset analyses, and comparisons are calculated with Chi-square and Fisher's exact test when necessary. In the continuous analysis, mean ages at onset associated to each allele and the p

**Region 6q23 (TNFAIP3)**: two polymorphisms located in an intergenic space adjacent to the *TNFAIP3* gene have been associated to several autoimmune diseases, among them type 1 diabetes (Fung et al, 2009). We have selected the polymorphism that shows a stronger association with the disease. The gene *TNFAIP3* is expressed in cells and serves as an antiinflammatory mechanism by its downregulation of the NF-B activation (Liuwantara et al, 2006); therefore, it poses an interesting candidate gene in the pathogenesis of type 1 diabetes. To our knowledge, this is the first time that this region is studied in relation to its

In our study, we replicate the association previously seen in type 1 diabetes and we do not find differences in age at onset either in the stratified or continuous analyses. Results are

**Region 9q33 (TRAF1)**: this region was first associated to rheumatoid arthritis (Kurreeman et al, 2007). Our group took part in a collaborative study that analyzed this region in several autoimmune diseases and found association with type 1 diabetes, among others

T1D-control 0.240 0.192 0.01 1.33

Pediatric-adult T1D 0.237 0.250 0.7 --

T1D-control 0.282 0.321 0.05 0.83

Pediatric-adult T1D 0.297 0.271 0.4 --

Pediatric-adult T1D 0.063 0.104 0.04 --

T1D-control 0.462 0.504 0.06 0.85

Pediatric-adult T1D 0.471 0.460 0.8 --

T1D-control 0.368 0.416 0.05 0.82

Pediatric-adult T1D 0.359 0.394 0.4 --

value from the U Mann-Whitney test are presented. T1D: type 1 diabetes.

T1D-control 0.085 0.071 0.2 -- 18.4

**CONTINUOUS ANALYSIS** 

> **Minor allele**

> > 16.7 (9.4) 0.6

17.8 (10.6) 0.3

21.2 (10.5) 0.02

18.5 (11.0) 0.4

18.0 (11.5) 0.5

**Mean age at onset P Major** 

**allele** 

17.6 (10.6)

18.7 (11.1)

(11.1)

17.6 (10.7)

16.9 (9.7)

(1.05-1.68)

(0.69-1.01)

(0.71-1.01)

(0.66-1.01)

**Gene** 

**2q32 (***STAT4***)**

**6q23 (***TNFAIP3***)** 

**9q33 (***TRAF1***)** 

**12p13 (***CLEC2D***)**

**16p13 (***CLEC16A***)**

influence in age at onset.

provided in table 9.

We replicate the association described in *CTLA4* rs3087243. Differences in *CTLA4* rs231775 do not reach statistical signification, but this could be due to low statistical power to detect the previously described association (Ueda et al, 2003). We do not find effects of any of the polymorphisms on age at onset. Results can be consulted in table 8.

**PTPN22 (chromosome 1p13)**: this gene encodes a lymphoid-specific phosphatase called LYP, which is an important downregulator of T cell activation through the TCR. We selected the classical non-synonymous polymorphism C1858T that causes a substitution from arginine to tryptophan in the aminoacid 620 of the encoded protein (Bottini et al, 2004). The mutant form shows a higher phosphatase activity, and therefore it suppresses T cell activation more efficiently. Its role in type 1 diabetes is believed to be at the thymic selection process, where the mutant PTPN22 would lower the activation signal sent to the autoreactive T cell through its TCR, thus contributing to its survival (Bottini et al, 2006). It also has been proposed that the increased suppression of activation associated to the mutant form could affect negatively the activation of regulatory T cells (Bottini et al, 2006).

We replicate the association previously described in the *PTPN22* gene and we do not find effects on age at onset in the stratified and continuous analysis. Results can be consulted in table 8.

**IFIH1/MDA5 (chromosome 2q24)**: certain viral infections such as that caused by Enterovirus are more prevalent in type 1 diabetes patients than in the healthy population, and it has been proposed that they could participate in the development or acceleration of the immune response against the cell (Hober & Sauter 2010). The helicase IFIH1 recognizes viral double stranded RNA (dsRNA) and it is expressed in the cytoplasm of several cells, including cells. In the presence of a viral infection, IFIH1 binds the dsRNA and induces the synthesis of pro-inflammatory cytokines. Functional experiments show that protection from type 1 diabetes is achieved through a lower performance of the sentinel role of IFIH1 that would end up in lower activation of the immune system in response to the viral infection (Colli et al, 2010).

We detect a lower frequency of the minor allele of *IFIH1* in type 1 diabetes patients respect to controls; however this difference is not statistically significant, probably due to low statistical power of our study. We do not find effects on age at diagnosis of type 1 diabetes in the stratified and continuous analysis. Results are provided in table 8.

#### **5.2.2 Genome-wide associations**

**Region 2q32 (STAT4)**: the gene *STAT4* is an interesting candidate for type 1 diabetes. Member of a family of transcription factors, STAT4 activates the transcription of several genes including IFN- in response to interleukin-12 signaling. The pathway IL12-STAT4- IFN polarizes the immune response to a Th1 type, the kind of response that is thought to be responsible of the type 1 diabetes autoimmune reaction (Raz et al, 2005). We have selected a polymorphism that was first discovered associated with rheumatoid arthritis (Remmers et al, 2007). Our group studied this polymorphism in several autoimmune diseases and described its association with type 1 diabetes (Martinez et al, 2008), as it can be seen in table 9. Influence of this polymorphism in age at onset has been previously studied in a pediatric-onset Korean population, as we described in section 4. We performed a stratified and continuous analysis of age at onset and we did not find evidences of the influence of this polymorphism in age at onset of the disease. Results can be consulted in table 9.

We replicate the association described in *CTLA4* rs3087243. Differences in *CTLA4* rs231775 do not reach statistical signification, but this could be due to low statistical power to detect the previously described association (Ueda et al, 2003). We do not find effects of any of the

**PTPN22 (chromosome 1p13)**: this gene encodes a lymphoid-specific phosphatase called LYP, which is an important downregulator of T cell activation through the TCR. We selected the classical non-synonymous polymorphism C1858T that causes a substitution from arginine to tryptophan in the aminoacid 620 of the encoded protein (Bottini et al, 2004). The mutant form shows a higher phosphatase activity, and therefore it suppresses T cell activation more efficiently. Its role in type 1 diabetes is believed to be at the thymic selection process, where the mutant PTPN22 would lower the activation signal sent to the autoreactive T cell through its TCR, thus contributing to its survival (Bottini et al, 2006). It also has been proposed that the increased suppression of activation associated to the mutant

form could affect negatively the activation of regulatory T cells (Bottini et al, 2006).

We replicate the association previously described in the *PTPN22* gene and we do not find effects on age at onset in the stratified and continuous analysis. Results can be consulted in

**IFIH1/MDA5 (chromosome 2q24)**: certain viral infections such as that caused by Enterovirus are more prevalent in type 1 diabetes patients than in the healthy population, and it has been proposed that they could participate in the development or acceleration of the immune response against the cell (Hober & Sauter 2010). The helicase IFIH1 recognizes viral double stranded RNA (dsRNA) and it is expressed in the cytoplasm of several cells, including cells. In the presence of a viral infection, IFIH1 binds the dsRNA and induces the synthesis of pro-inflammatory cytokines. Functional experiments show that protection from type 1 diabetes is achieved through a lower performance of the sentinel role of IFIH1 that would end up in lower activation of the immune system in response to the viral infection

We detect a lower frequency of the minor allele of *IFIH1* in type 1 diabetes patients respect to controls; however this difference is not statistically significant, probably due to low statistical power of our study. We do not find effects on age at diagnosis of type 1 diabetes

**Region 2q32 (STAT4)**: the gene *STAT4* is an interesting candidate for type 1 diabetes. Member of a family of transcription factors, STAT4 activates the transcription of several genes including IFN- in response to interleukin-12 signaling. The pathway IL12-STAT4- IFN polarizes the immune response to a Th1 type, the kind of response that is thought to be responsible of the type 1 diabetes autoimmune reaction (Raz et al, 2005). We have selected a polymorphism that was first discovered associated with rheumatoid arthritis (Remmers et al, 2007). Our group studied this polymorphism in several autoimmune diseases and described its association with type 1 diabetes (Martinez et al, 2008), as it can be seen in table 9. Influence of this polymorphism in age at onset has been previously studied in a pediatric-onset Korean population, as we described in section 4. We performed a stratified and continuous analysis of age at onset and we did not find evidences of the influence of this polymorphism in age at onset of the disease. Results can

in the stratified and continuous analysis. Results are provided in table 8.

polymorphisms on age at onset. Results can be consulted in table 8.

table 8.

(Colli et al, 2010).

**5.2.2 Genome-wide associations** 

be consulted in table 9.


Table 9. Analysis of selected gene associations from genome-wide studies in type 1 diabetes. Minor allele frequency (MAF) is provided in case-control and pediatric vs adult onset analyses, and comparisons are calculated with Chi-square and Fisher's exact test when necessary. In the continuous analysis, mean ages at onset associated to each allele and the p value from the U Mann-Whitney test are presented. T1D: type 1 diabetes.

**Region 6q23 (TNFAIP3)**: two polymorphisms located in an intergenic space adjacent to the *TNFAIP3* gene have been associated to several autoimmune diseases, among them type 1 diabetes (Fung et al, 2009). We have selected the polymorphism that shows a stronger association with the disease. The gene *TNFAIP3* is expressed in cells and serves as an antiinflammatory mechanism by its downregulation of the NF-B activation (Liuwantara et al, 2006); therefore, it poses an interesting candidate gene in the pathogenesis of type 1 diabetes. To our knowledge, this is the first time that this region is studied in relation to its influence in age at onset.

In our study, we replicate the association previously seen in type 1 diabetes and we do not find differences in age at onset either in the stratified or continuous analyses. Results are provided in table 9.

**Region 9q33 (TRAF1)**: this region was first associated to rheumatoid arthritis (Kurreeman et al, 2007). Our group took part in a collaborative study that analyzed this region in several autoimmune diseases and found association with type 1 diabetes, among others

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 47

pediatric-onset type 1 diabetes. Also, a recent study from the T1DGC (Howson et al, 2009) that replicated 19 genes, including *PTPN22*, *IFIH1* and *CTLA4*, studied age at onset in each one and did not find statistical differences, supporting the idea that the classical genetic

Interestingly, in our population we do not find differences in the HLA associations with early and late onset. It has been described before that the *DRB1\*03-DRB1\*04* heterozygote is the combination that confers a higher risk and it is associated with an earlier age at onset of type 1 diabetes (Redondo et al, 2001a; Leslie et al, 2006; Klinker et al, 2010). However, in our population the heterozygote is the fourth combination in risk conferred to the disease after the *DRB1\*04-DQB1\*03:02* homozygote, the *DRB1\*03* or *DRB1\*04- DQB1\*03:02* carrier and the *DRB1\*03* homozygote, and we do not see an effect on age at diagnosis of the heterozygote. This could be due to populational differences, quite important in the HLA complex. It is well known that not all the *DRB1\*03* haplotypes confer the same susceptibility to the disease. An extended conserved haplotype marked by *B\*18-DRB1\*03-DQB1\*02:01* is described to confer higher susceptibility among the *DRB1\*03*-carrying haplotypes (Johansson et al, 2003; Urcelay et al, 2005). This haplotype is more frequent in the Mediterranean area and its frequency descends in Northern Europe. Therefore, it could be possible that the higher frequency of this high risk haplotype enhances the risk conferred by being a *DRB1\*03* homozygote in a Mediterranean

A recent study with the T1DGC family cohort (Howson et al, 2009) found a mild effect of the insulin gene in age at diagnosis of the disease, with the susceptibility allele conferring an onset two years earlier than the protective allele. We do not see an effect on age at diagnosis (continuous analysis, p=0.4). Moreover, in the aforementioned Finnish report, the authors also studied the *INS* gene and found association in late onset patients (Klinker et al, 2010). It is possible that our study lacks statistical power to detect a difference of two years in the age at diagnosis. However, the authors of the T1DGC study tried to replicate their findings in a case series of 900 patients and did not find the effect they saw in the family cohort, opening

Finally, we find that *TRAF1* is only associated to late-onset type 1 diabetes. Although our study is limited by low statistical power and would require replication in an independent late onset type 1 diabetes cohort, this is an interesting finding that would justify the study of

From the study of these ten genetic regions in our group composed of early and late onset patients, we propose that there are no major genetic differences between patients with an

Although the knowledge on the genetics of type 1 diabetes has experienced a great development in the last years, it has not provided many hints on the basis of the genetic components that could modify the age at onset of the disease. Several studies have approached the subject, but few of the reported associations have been properly replicated in independent populations. Also, the heterogeneity in the methodology of the published studies should be discussed: some studies select only paediatric patients. If the influence of a genetic region reaches a peak in the paediatric age and then decreases with

the door to the possibility that the described effect is a false positive.

the late-onset patients as a distinct set among the type 1 diabetes patients.

associations to type 1 diabetes are shared between the early and late onset patients.

population such as the Spanish.

early and a late onset of type 1 diabetes.

**6. Conclusions** 

(Kurreeman et al, 2010). As an extension of the cited study, we selected and studied new polymorphisms in the *TRAF1* gene and analyzed their influence in age at onset of the disease. We present in table 9 the results obtained in one polymorphism that has not been previously studied in type 1 diabetes. Like *TNFAIP3*, the gene *TRAF1* is expressed in cells and protects them against cytokine-mediated apoptosis in an inflammatory environment (Sarkar et al, 2009).

The polymorphism in *TRAF1* shows interesting data (table 9). We do not see statistical differences in the case-control analysis, but the age-stratified analysis shows an elevation of the minor allele frequency only in the adult-onset patients, that is statistically significant when compared to pediatric patients (p=0.04) and to controls (OR=1.52 [1.00- 2.31]; p=0.04). On the other hand, the pediatric patients are similar to controls (p=0.6). The continuous analysis confirms the difference observed in the stratified analysis, showing an age at onset associated to the minor allele (mean 21.2) that is almost three years higher than the age at onset associated to the major allele (mean 18.4). Therefore, there seems to exist an association in this gene that is exclusive of our adult-onset type 1 diabetes patients.

**Region 12p13 (CLEC2D)**: first associated with type 1 diabetes in the WTCCC study (WTCCC, 2007), it includes several genes. Polymorphisms associated with the disease (WTCCC, 2007; Barrett et al, 2009) have been identified in the surroundings of two genes with immunological function: *CLEC2D* and *CD69*. We will focus on the polymorphism near *CLEC2D* (coding the NK receptor LLT1), which was the strongest signal reported by the WTCCC (WTCCC, 2007) in this region. To date, the influence of region 12p13 on age at onset has not been studied.

In our study, we detect a trend towards association with type 1 diabetes of the studied polymorphism. We do not find differences in age at onset in the stratified and continuous analyses. Results are provided in table 9.

**Region 16p13 (CLEC16A)**: this region was one of the most strongly associated with type 1 diabetes in the WTCCC (WTCCC, 2007) and was also discovered independently in a parallel study (Hakonarson et al, 2007). It covers a gene of unknown function termed *CLEC16A*, which is expressed in antigen presenting cells and NK cells, but little else is known about this gene or its possible role in the pathogenesis of the disease. Our group replicated the association detected in the WTCCC study (Martinez et al, 2010). Here we will analyze its influence in age at onset.

We replicate the association previously seen in type 1 diabetes. We do not find differences in age at onset in the stratified and continuous analyses. Results are provided in table 9.

#### **5.3 Discussion**

In this study we analyzed ten genetic regions, five of them classical type 1 diabetes genes and another five extracted from recent genome-wide studies. The analysis of age at onset (either age-stratified or considering age as a continuum) did not provide statistical differences in nine of the ten regions studied, therefore we propose that our adult-onset patients have the same genetic background in the studied genes that our pediatric-onset patients. Hence there is no reason to exclude these late-onset patients from genetic studies on type 1 diabetes. The results we observe in the classical type 1 diabetes susceptibility genes (table 8) are concordant with a recent Finnish study (Klinker et al, 2010) that analyzes these genes in late-onset patients and finds the same associations previously described in pediatric-onset type 1 diabetes. Also, a recent study from the T1DGC (Howson et al, 2009) that replicated 19 genes, including *PTPN22*, *IFIH1* and *CTLA4*, studied age at onset in each one and did not find statistical differences, supporting the idea that the classical genetic associations to type 1 diabetes are shared between the early and late onset patients.

Interestingly, in our population we do not find differences in the HLA associations with early and late onset. It has been described before that the *DRB1\*03-DRB1\*04* heterozygote is the combination that confers a higher risk and it is associated with an earlier age at onset of type 1 diabetes (Redondo et al, 2001a; Leslie et al, 2006; Klinker et al, 2010). However, in our population the heterozygote is the fourth combination in risk conferred to the disease after the *DRB1\*04-DQB1\*03:02* homozygote, the *DRB1\*03* or *DRB1\*04- DQB1\*03:02* carrier and the *DRB1\*03* homozygote, and we do not see an effect on age at diagnosis of the heterozygote. This could be due to populational differences, quite important in the HLA complex. It is well known that not all the *DRB1\*03* haplotypes confer the same susceptibility to the disease. An extended conserved haplotype marked by *B\*18-DRB1\*03-DQB1\*02:01* is described to confer higher susceptibility among the *DRB1\*03*-carrying haplotypes (Johansson et al, 2003; Urcelay et al, 2005). This haplotype is more frequent in the Mediterranean area and its frequency descends in Northern Europe. Therefore, it could be possible that the higher frequency of this high risk haplotype enhances the risk conferred by being a *DRB1\*03* homozygote in a Mediterranean population such as the Spanish.

A recent study with the T1DGC family cohort (Howson et al, 2009) found a mild effect of the insulin gene in age at diagnosis of the disease, with the susceptibility allele conferring an onset two years earlier than the protective allele. We do not see an effect on age at diagnosis (continuous analysis, p=0.4). Moreover, in the aforementioned Finnish report, the authors also studied the *INS* gene and found association in late onset patients (Klinker et al, 2010). It is possible that our study lacks statistical power to detect a difference of two years in the age at diagnosis. However, the authors of the T1DGC study tried to replicate their findings in a case series of 900 patients and did not find the effect they saw in the family cohort, opening the door to the possibility that the described effect is a false positive.

Finally, we find that *TRAF1* is only associated to late-onset type 1 diabetes. Although our study is limited by low statistical power and would require replication in an independent late onset type 1 diabetes cohort, this is an interesting finding that would justify the study of the late-onset patients as a distinct set among the type 1 diabetes patients.

From the study of these ten genetic regions in our group composed of early and late onset patients, we propose that there are no major genetic differences between patients with an early and a late onset of type 1 diabetes.

#### **6. Conclusions**

46 Type 1 Diabetes Complications

(Kurreeman et al, 2010). As an extension of the cited study, we selected and studied new polymorphisms in the *TRAF1* gene and analyzed their influence in age at onset of the disease. We present in table 9 the results obtained in one polymorphism that has not been previously studied in type 1 diabetes. Like *TNFAIP3*, the gene *TRAF1* is expressed in cells and protects them against cytokine-mediated apoptosis in an inflammatory

The polymorphism in *TRAF1* shows interesting data (table 9). We do not see statistical differences in the case-control analysis, but the age-stratified analysis shows an elevation of the minor allele frequency only in the adult-onset patients, that is statistically significant when compared to pediatric patients (p=0.04) and to controls (OR=1.52 [1.00- 2.31]; p=0.04). On the other hand, the pediatric patients are similar to controls (p=0.6). The continuous analysis confirms the difference observed in the stratified analysis, showing an age at onset associated to the minor allele (mean 21.2) that is almost three years higher than the age at onset associated to the major allele (mean 18.4). Therefore, there seems to exist an association in this gene that is exclusive of our adult-onset type 1 diabetes

**Region 12p13 (CLEC2D)**: first associated with type 1 diabetes in the WTCCC study (WTCCC, 2007), it includes several genes. Polymorphisms associated with the disease (WTCCC, 2007; Barrett et al, 2009) have been identified in the surroundings of two genes with immunological function: *CLEC2D* and *CD69*. We will focus on the polymorphism near *CLEC2D* (coding the NK receptor LLT1), which was the strongest signal reported by the WTCCC (WTCCC, 2007) in this region. To date, the influence of region 12p13 on age at

In our study, we detect a trend towards association with type 1 diabetes of the studied polymorphism. We do not find differences in age at onset in the stratified and continuous

**Region 16p13 (CLEC16A)**: this region was one of the most strongly associated with type 1 diabetes in the WTCCC (WTCCC, 2007) and was also discovered independently in a parallel study (Hakonarson et al, 2007). It covers a gene of unknown function termed *CLEC16A*, which is expressed in antigen presenting cells and NK cells, but little else is known about this gene or its possible role in the pathogenesis of the disease. Our group replicated the association detected in the WTCCC study (Martinez et al, 2010). Here we will analyze its

We replicate the association previously seen in type 1 diabetes. We do not find differences in

In this study we analyzed ten genetic regions, five of them classical type 1 diabetes genes and another five extracted from recent genome-wide studies. The analysis of age at onset (either age-stratified or considering age as a continuum) did not provide statistical differences in nine of the ten regions studied, therefore we propose that our adult-onset patients have the same genetic background in the studied genes that our pediatric-onset patients. Hence there is no reason to exclude these late-onset patients from genetic studies on type 1 diabetes. The results we observe in the classical type 1 diabetes susceptibility genes (table 8) are concordant with a recent Finnish study (Klinker et al, 2010) that analyzes these genes in late-onset patients and finds the same associations previously described in

age at onset in the stratified and continuous analyses. Results are provided in table 9.

environment (Sarkar et al, 2009).

onset has not been studied.

influence in age at onset.

**5.3 Discussion** 

analyses. Results are provided in table 9.

patients.

Although the knowledge on the genetics of type 1 diabetes has experienced a great development in the last years, it has not provided many hints on the basis of the genetic components that could modify the age at onset of the disease. Several studies have approached the subject, but few of the reported associations have been properly replicated in independent populations. Also, the heterogeneity in the methodology of the published studies should be discussed: some studies select only paediatric patients. If the influence of a genetic region reaches a peak in the paediatric age and then decreases with

Early and Late Onset Type 1 Diabetes: One and the Same or Two Distinct Genetic Entities? 49

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Dunger, D. B., Ramos-Lopez, E., Badenhoop, K., Nejentsev, S., and Todd, J. A. (2007) Association of the vitamin D metabolism gene CYP27B1 with type 1

C., Morahan, G., Nerup, J., Nierras, C., Plagnol, V., Pociot, F., Schuilenburg, H., Smyth, D. J., Stevens, H., Todd, J. A., Walker, N. M., and Rich, S. S. (2009) Genomewide association study and meta-analysis find that over 40 loci affect risk of type 1

MacMurray, J., Meloni, G. F., Lucarelli, P., Pellecchia, M., Eisenbarth, G. S., Comings, D., and Mustelin, T. (2004) A functional variant of lymphoid tyrosine

two candidate genes for type 1 diabetes, modify pancreatic beta-cell responses to

Barrett, J. C., Healy, B. C., Mychaleckyj, J. C., Warram, J. H., and Todd, J. A. (2008) Meta-analysis of genome-wide association study data identifies additional type 1

adolescents: National Health and Nutrition Examination Survey, 1999-2002. *Arch* 

C., Todd, J. A., Bonella, P., Fear, A. L., Lavant, E., Louey, A., and Moonsamy, P. (2008) HLA DR-DQ haplotypes and genotypes and type 1 diabetes risk: analysis of

G., Urcelay, E., and Santiago, J. L. (2011a) A polymorphism in PTPN2 gene is associated with an earlier onset of type 1 diabetes. *Immunogenetics 63:*255-258 Espino-Paisan, L., de la Calle, H., Fernandez-Arquero, M., Figueredo, M. A., de la Concha, E.

G., Urcelay, E., and Santiago, J. L. (2011b) Polymorphisms in chromosome region

G., Urcelay, E., and Santiago, J. L. (2011c) Study of polymorphisms in 4q27, 10p15 and 22q13 regions in autoantibodies stratified type 1 diabetes patients.

S., and Todd, J. A. (2009) Analysis of 17 autoimmune disease-associated variants in

time, a study with paediatric and adult patients would better estimate the difference than a study with only paediatric patients in which the difference may be seen, but also may be smaller than it really is. The majority of the published studies analyze the age at diagnosis of type 1 diabetes as a continuous variable, but some studies adopt a stratified analysis that implies the fragmentation of the patients in two or more groups and the individual analysis of each subgroup. This strategy defines artificial groups with limits that do not have a biological justification, and makes it more likely to produce false results in underpowered studies. We recommend the age-stratified analysis as a screening method or as a confirmation analysis, but we consider the analysis of age as a continuous variable a more accurate method to detect differences, given that it only takes into consideration the genotype studied and the ages at onset of all the patients carrying this genotype. Therefore, we propose that age at diagnosis of type 1 diabetes should be studied in groups that include paediatric and adult onset patients, and that the statistical analysis should include at least one method that considers age at diagnosis as a continuum.

Despite the improvements that can be incorporated to age-at-onset analysis, what is known to date about the influence of the genetics on the early and late onset of type 1 diabetes allows us to formulate a tentative answer to the question that gives title to this chapter: are early and late onset type 1 diabetes the same or two distinct genetic entities? Our data, presented in section 5, and the previous studies reviewed in section 4 suggest that there are no major differences in the genetic component of paediatric and adult patients. Both share the risk conferred by the main type 1 diabetes risk modifiers such as the HLA, the insulin gene or *PTPN22*. However, two points have to be taken into consideration: first, minor differences can be found between the adult and the paediatric patient, such as the elevated prevalence of the higher risk HLA alleles in subjects with early onset. Second, the genetics of adult-onset type 1 diabetes patients has frequently been studied only in relation to genes that already showed association in paediatric patients. This approach precludes the possibility of discovering genes that could be associated exclusively to late-onset type 1 diabetes. The study of these two points is of great interest given that it could point to metabolic routes that take part in the acceleration of the disease and, therefore, genes in these routes would be excellent candidates for therapeutic strategies focused on the delay of the autoimmune cell destruction.

In conclusion, we propose that type 1 diabetes, whether in its early or late-onset, is an autoimmune disease defined by a number of primary risk genes and a constellation of minor genetic modifiers that, together with environmental factors, define the pace of the autoimmune reaction that will determine the age at onset.

#### **7. References**

American Diabetes Association (2010) Diagnosis and classification of diabetes mellitus. *Diabetes Care 33 Suppl 1:*S62-69

Awata, T., Kawasaki, E., Tanaka, S., Ikegami, H., Maruyama, T., Shimada, A., Nakanishi, K., Kobayashi, T., Iizuka, H., Uga, M., Kawabata, Y., Kanazawa, Y., Kurihara, S., Osaki, M., and Katayama, S. (2009) Association of type 1 diabetes with two Loci on 12q13

time, a study with paediatric and adult patients would better estimate the difference than a study with only paediatric patients in which the difference may be seen, but also may be smaller than it really is. The majority of the published studies analyze the age at diagnosis of type 1 diabetes as a continuous variable, but some studies adopt a stratified analysis that implies the fragmentation of the patients in two or more groups and the individual analysis of each subgroup. This strategy defines artificial groups with limits that do not have a biological justification, and makes it more likely to produce false results in underpowered studies. We recommend the age-stratified analysis as a screening method or as a confirmation analysis, but we consider the analysis of age as a continuous variable a more accurate method to detect differences, given that it only takes into consideration the genotype studied and the ages at onset of all the patients carrying this genotype. Therefore, we propose that age at diagnosis of type 1 diabetes should be studied in groups that include paediatric and adult onset patients, and that the statistical analysis should include at least one method that considers age at diagnosis as a

Despite the improvements that can be incorporated to age-at-onset analysis, what is known to date about the influence of the genetics on the early and late onset of type 1 diabetes allows us to formulate a tentative answer to the question that gives title to this chapter: are early and late onset type 1 diabetes the same or two distinct genetic entities? Our data, presented in section 5, and the previous studies reviewed in section 4 suggest that there are no major differences in the genetic component of paediatric and adult patients. Both share the risk conferred by the main type 1 diabetes risk modifiers such as the HLA, the insulin gene or *PTPN22*. However, two points have to be taken into consideration: first, minor differences can be found between the adult and the paediatric patient, such as the elevated prevalence of the higher risk HLA alleles in subjects with early onset. Second, the genetics of adult-onset type 1 diabetes patients has frequently been studied only in relation to genes that already showed association in paediatric patients. This approach precludes the possibility of discovering genes that could be associated exclusively to late-onset type 1 diabetes. The study of these two points is of great interest given that it could point to metabolic routes that take part in the acceleration of the disease and, therefore, genes in these routes would be excellent candidates for therapeutic strategies focused on the delay of

In conclusion, we propose that type 1 diabetes, whether in its early or late-onset, is an autoimmune disease defined by a number of primary risk genes and a constellation of minor genetic modifiers that, together with environmental factors, define the pace of the

American Diabetes Association (2010) Diagnosis and classification of diabetes mellitus.

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continuum.

the autoimmune cell destruction.

**7. References** 

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**3** 

*Italy* 

**Islet Endothelium: Role in Type 1 Diabetes and** 

The heterogeneity of microvascular endothelial cells derived from different organs, suggests that these cells have specialised functions at different anatomical sites. The microvasculature is, in fact, a key interface between blood and tissues and participates in numerous pathophysiological processes. Pancreatic islet microcirculation exhibits distinctive features, in an interdependent physical and functional relationship with β cells, from organogenesis to adult life. The islet microendothelium behaves as an active "gatekeeper" in the control of leukocyte recruitment into the islets during autoimmune

Furthermore, microvascular endothelial cells, forming the key lining between the vascular space and organ parenchyma, have been shown to influence organ and tissue specific susceptibility to viral infection, and to modulate the pathological expression of virusinduced diseases, which potentially includes type 1 diabetes. Endothelial cells expressing appropriate receptors would fail to act as effective barrier to infections, allowing viral particles to pass through, and replicate in, the vascular endothelium. Human Enteroviruses (EV), especially those of the Coxsackievirus B (CVB) group, are associated with a wide variety of clinical syndromes and have long been considered possible culprits of inflammatory conditions and immune-mediated pathological processes, such as chronic dilated cardiomyopathy, chronic myositis and type 1 diabetes mellitus (Rose et al., 1993; Luppi et al., 1998; Hyöty & Taylor, 2002). Several mechanisms, including molecular mimicry, bystander activation of autoreactive T cells, superantigenic activity of viral proteins, not mutually exclusive, have been proposed to explain the relationship between EV infections and induction of autoimmune diseases (Varela-Calvino & Peakman, 2003; Horwitz et al., 1998; Wucherpfennig, 2001). Evidences of a link between viral infections and initiation or acceleration of pancreatic islet autoimmunity have been under investigation for almost 30 years, and EVs, especially those of the Coxsackievirus B (CVB) group, are historically the prime suspects as important aetiological determinants in type 1 diabetes (Hyöty & Taylor, 2002; Varela-Calvino & Peakman, 2003). Endothelial cells derived from different organs show distinct susceptibility to CVB infections, and the behaviour against a viral challenge of endothelial cells in large vessels and microvessels may differ (Friedman et al. 1981; Huber et al., 1990; Conaldi et al. 1997; Zanone et al.,

**1. Introduction** 

insulitis in type 1 diabetes.

2003; Saijets et al., 2003).

Enrica Favaro, Ilaria Miceli, Elisa Camussi and Maria M. Zanone

**in Coxsackievirus Infections** 

*Department of Internal Medicine, University of Turin,* 


### **Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections**

Enrica Favaro, Ilaria Miceli, Elisa Camussi and Maria M. Zanone *Department of Internal Medicine, University of Turin, Italy* 

#### **1. Introduction**

54 Type 1 Diabetes Complications

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The heterogeneity of microvascular endothelial cells derived from different organs, suggests that these cells have specialised functions at different anatomical sites. The microvasculature is, in fact, a key interface between blood and tissues and participates in numerous pathophysiological processes. Pancreatic islet microcirculation exhibits distinctive features, in an interdependent physical and functional relationship with β cells, from organogenesis to adult life. The islet microendothelium behaves as an active "gatekeeper" in the control of leukocyte recruitment into the islets during autoimmune insulitis in type 1 diabetes.

Furthermore, microvascular endothelial cells, forming the key lining between the vascular space and organ parenchyma, have been shown to influence organ and tissue specific susceptibility to viral infection, and to modulate the pathological expression of virusinduced diseases, which potentially includes type 1 diabetes. Endothelial cells expressing appropriate receptors would fail to act as effective barrier to infections, allowing viral particles to pass through, and replicate in, the vascular endothelium. Human Enteroviruses (EV), especially those of the Coxsackievirus B (CVB) group, are associated with a wide variety of clinical syndromes and have long been considered possible culprits of inflammatory conditions and immune-mediated pathological processes, such as chronic dilated cardiomyopathy, chronic myositis and type 1 diabetes mellitus (Rose et al., 1993; Luppi et al., 1998; Hyöty & Taylor, 2002). Several mechanisms, including molecular mimicry, bystander activation of autoreactive T cells, superantigenic activity of viral proteins, not mutually exclusive, have been proposed to explain the relationship between EV infections and induction of autoimmune diseases (Varela-Calvino & Peakman, 2003; Horwitz et al., 1998; Wucherpfennig, 2001). Evidences of a link between viral infections and initiation or acceleration of pancreatic islet autoimmunity have been under investigation for almost 30 years, and EVs, especially those of the Coxsackievirus B (CVB) group, are historically the prime suspects as important aetiological determinants in type 1 diabetes (Hyöty & Taylor, 2002; Varela-Calvino & Peakman, 2003). Endothelial cells derived from different organs show distinct susceptibility to CVB infections, and the behaviour against a viral challenge of endothelial cells in large vessels and microvessels may differ (Friedman et al. 1981; Huber et al., 1990; Conaldi et al. 1997; Zanone et al., 2003; Saijets et al., 2003).

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 57

Representative micrograph of scanning electron microscopy of primary islet MECs. The arrows show typical cellular fenestrations (original magnification 1500X). Inset: representative

Nephrin appears to be more than just a structural component, as it is an adhesion and signalling molecule that can activate mitogen-activated protein kinase cascades, modulating a variety of cellular programs, including proliferation, differentiation and apoptosis (Karin et al., 1997; Flickinger & Olson, 1999). It has been shown that nephrin, once phosphorylated associates with PI3K and itself stimulates the Akt-dependent signaling pathway (Huber et al., 2003) that plays a pivotal role in preventing apoptosis in a variety of settings (Datta et al., 1999). In particular, Akt activation is crucial for the ability of factors such as insulin, IGF-I and VEGF to inhibit apoptosis in cultured endothelium (Jung et al., 2000). Recent data highlight the Akt role also in insulin-mediated glucose transport and pancreatic β cell mass

Islet endothelium is crucially involved in fine-tuning blood glucose sensing and regulation (Lammert et al., 2003). Besides providing oxygen and nutrients to the endocrine cells, islet endothelium is in fact involved in the trans-endothelial rapid passage of secreted insulin into the circulation. In perfusion experiments with horseradish peroxidase it has been demonstrated that the endothelial fenestrae are sites through which proteins quickly permeate (Takahashi et al., 2002). Thus the islet fenestrae allow the fastest way for insulin to enter the circulation. Studies in mice with pancreatic VEGF-A deletion, showed that these

magnification of a cellular fenestra (original magnification 15,000X). Bar: 0.1 μm.

and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006).

Fig. 1. Cultured islet endothelial cells.

#### **2. Pancreatic islet microvasculature: Structure and specialised functions**

It is widely accepted that remarkable heterogeneity of endothelial phenotype and function exists amongst different vascular beds (Kubota et al., 1988; Charo etal., 1984; Swerlick et al., 1991; Swerlick et al., 1992; Fujimoto & Singer, 1988; Lidington et al., 1999), in particular between cells derived from large versus small vessels, supporting the notion that tissue-specific vascular beds have specialised functions. These diversities include morphology, growth requirement in vitro (Kubota et al., 1988; Charo et al., 1984; Swerlick et al., 1991; Swerlick et al., 1992; Fujimoto & Singer, 1988; Lidington et al., 1999, Folkman et al., 1979) prostaglandin secretory profile (Charo etal., 1984), immunologic phenotype (Swerlick et al., 1992) and amounts and regulation of cell adhesion molecules (Swerlick et al., 1992; Fujimoto & Singer, 1988; Petzelbauer et al., 1993; Swerlick A.R., et al., 1992; Lee et al., 1992). At a functional level, differential and sequential expression of adhesion molecules mediates trafficking of leukocytes to specific lymphoid and nonlymphoid tissues.

Endothelium heterogeneity is the result of microenvironmental signals, in particular those induced by the family of vascular endothelial growth factor (VEGF) proteins (D'Amore & Ng, 2002). VEGF is a major stimulator of neovascularisation by inducing proliferation and migration of endothelial cells and tube formation. Pancreatic islets are one of the most vascularised organs, having a blood perfusion of about 10% of that of the whole pancreas, despite representing only 1% of the gland; this reflects high exchange demand with the endocrine cells and high metabolic supply (Figure 1). Deletion studies indicate that VEGF-A is responsible for this dense islet vascularisation, being more expressed in the endocrine than the exocrine pancreas (Lammert et al., 2003). Endothelial cells migrate to the source of VEGF-A, the neighboring cells, proliferate and form blood vessels, organised in a network of sinusoidal capillaries reminiscent of those present in the renal glomerulus, with a five times higher density and ten times more fenestrations than in the exocrine tissue.

Islets receive blood from 1 to 3 arterioles and drain into collecting venules forming a network covering the islet surface, and an insulo-acinar portal system connects the islet capillaries with capillaries of the exocrine pancreas. The pattern of blood flow within the islet is still a matter of debate, with the cell-rich islet core possibly perfused before the non cells in the periphery of the islet (Brunicardi et al., 1996).

Specific markers of islet microvasculature have been identified. These include the -1 proteinase inhibitor (Api, -1 antitrypsin), a major proteinase inhibitor with immunoregulatory properties, and nephrin ( Favaro et al., 2005), a highly specific barrier protein, known to be located in the renal glomerular ultrathin filter membrane "slit diaphragm" (Ruotsalainen et al., 1999; Tryggvason & Wartiovaara, 2001) (Figure 2). The nephrin expressed in islet microendothelial cells has functional characteristics that are highly reminiscent of the same protein expressed by podocytes in the renal glomeruli, in that in both cell types treatment with TNF- acts on the cell cytoskeleton to induce a marked redistribution of nephrin expression. Nephrin is a cell adhesion transmembrane protein of the immunoglobulin superfamily, which has a pivotal role in the regulation of renal glomerular selective permeability (Henderson & Moss, 1985; Bonner-Weir, 1993; Konstantinova & Lammert, 2004; Bonner-Weir & Orci, 1982). Islet endothelial expression of this protein is consistent with the ultrastructural features of these cells in the islets, which form a microvasculature that is characterized by a glomerulus-like network of fenestrated capillaries.

It is widely accepted that remarkable heterogeneity of endothelial phenotype and function exists amongst different vascular beds (Kubota et al., 1988; Charo etal., 1984; Swerlick et al., 1991; Swerlick et al., 1992; Fujimoto & Singer, 1988; Lidington et al., 1999), in particular between cells derived from large versus small vessels, supporting the notion that tissue-specific vascular beds have specialised functions. These diversities include morphology, growth requirement in vitro (Kubota et al., 1988; Charo et al., 1984; Swerlick et al., 1991; Swerlick et al., 1992; Fujimoto & Singer, 1988; Lidington et al., 1999, Folkman et al., 1979) prostaglandin secretory profile (Charo etal., 1984), immunologic phenotype (Swerlick et al., 1992) and amounts and regulation of cell adhesion molecules (Swerlick et al., 1992; Fujimoto & Singer, 1988; Petzelbauer et al., 1993; Swerlick A.R., et al., 1992; Lee et al., 1992). At a functional level, differential and sequential expression of adhesion molecules mediates trafficking of leukocytes to specific lymphoid and non-

Endothelium heterogeneity is the result of microenvironmental signals, in particular those induced by the family of vascular endothelial growth factor (VEGF) proteins (D'Amore & Ng, 2002). VEGF is a major stimulator of neovascularisation by inducing proliferation and migration of endothelial cells and tube formation. Pancreatic islets are one of the most vascularised organs, having a blood perfusion of about 10% of that of the whole pancreas, despite representing only 1% of the gland; this reflects high exchange demand with the endocrine cells and high metabolic supply (Figure 1). Deletion studies indicate that VEGF-A is responsible for this dense islet vascularisation, being more expressed in the endocrine than the exocrine pancreas (Lammert et al., 2003). Endothelial cells migrate to the source of VEGF-A, the neighboring cells, proliferate and form blood vessels, organised in a network of sinusoidal capillaries reminiscent of those present in the renal glomerulus, with a five

times higher density and ten times more fenestrations than in the exocrine tissue.

cells in the periphery of the islet (Brunicardi et al., 1996).

Islets receive blood from 1 to 3 arterioles and drain into collecting venules forming a network covering the islet surface, and an insulo-acinar portal system connects the islet capillaries with capillaries of the exocrine pancreas. The pattern of blood flow within the islet is still a matter of debate, with the cell-rich islet core possibly perfused before the non-

Specific markers of islet microvasculature have been identified. These include the -1 proteinase inhibitor (Api, -1 antitrypsin), a major proteinase inhibitor with immunoregulatory properties, and nephrin ( Favaro et al., 2005), a highly specific barrier protein, known to be located in the renal glomerular ultrathin filter membrane "slit diaphragm" (Ruotsalainen et al., 1999; Tryggvason & Wartiovaara, 2001) (Figure 2). The nephrin expressed in islet microendothelial cells has functional characteristics that are highly reminiscent of the same protein expressed by podocytes in the renal glomeruli, in that in both cell types treatment with TNF- acts on the cell cytoskeleton to induce a marked redistribution of nephrin expression. Nephrin is a cell adhesion transmembrane protein of the immunoglobulin superfamily, which has a pivotal role in the regulation of renal glomerular selective permeability (Henderson & Moss, 1985; Bonner-Weir, 1993; Konstantinova & Lammert, 2004; Bonner-Weir & Orci, 1982). Islet endothelial expression of this protein is consistent with the ultrastructural features of these cells in the islets, which form a microvasculature that is characterized by a glomerulus-like network of fenestrated

**2. Pancreatic islet microvasculature: Structure and specialised functions** 

lymphoid tissues.

capillaries.

Representative micrograph of scanning electron microscopy of primary islet MECs. The arrows show typical cellular fenestrations (original magnification 1500X). Inset: representative magnification of a cellular fenestra (original magnification 15,000X). Bar: 0.1 μm.

Nephrin appears to be more than just a structural component, as it is an adhesion and signalling molecule that can activate mitogen-activated protein kinase cascades, modulating a variety of cellular programs, including proliferation, differentiation and apoptosis (Karin et al., 1997; Flickinger & Olson, 1999). It has been shown that nephrin, once phosphorylated associates with PI3K and itself stimulates the Akt-dependent signaling pathway (Huber et al., 2003) that plays a pivotal role in preventing apoptosis in a variety of settings (Datta et al., 1999). In particular, Akt activation is crucial for the ability of factors such as insulin, IGF-I and VEGF to inhibit apoptosis in cultured endothelium (Jung et al., 2000). Recent data highlight the Akt role also in insulin-mediated glucose transport and pancreatic β cell mass and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006).

Islet endothelium is crucially involved in fine-tuning blood glucose sensing and regulation (Lammert et al., 2003). Besides providing oxygen and nutrients to the endocrine cells, islet endothelium is in fact involved in the trans-endothelial rapid passage of secreted insulin into the circulation. In perfusion experiments with horseradish peroxidase it has been demonstrated that the endothelial fenestrae are sites through which proteins quickly permeate (Takahashi et al., 2002). Thus the islet fenestrae allow the fastest way for insulin to enter the circulation. Studies in mice with pancreatic VEGF-A deletion, showed that these

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 59

remains controversial (Welsh & Sandler, 1992; Corbett et al., 1993; Henningsson et al., 2002;

Immunohistochemical studies have shown that the expression of Platelet-activating factor (PAF) receptor (PAF-r) within the islet, is restricted to endothelial cells, providing potential target for therapeutic intervention (Biancone et al., 2006). PAF, is a phospholipid with diverse physiological effects that mediates a host of biochemical activities, including angiogenesis and inflammation. Islet endothelial cells have also been shown to rapidly produce PAF under stimulation with trombin, and PAF accelerated angiogenesis (Mattsson et al., 2006). These data suggest that intra-islet production of PAF, induced by inflammatory

Lastly, islet endothelial cells express genes encoding for a number of other factors involved in angiogenesis, including potent pro-angiogenic factors, such as VEGF, and angiostatic factors, such as endostatin and pigment epithelial-derived factor (Lammert et al., 2001).

**2.1 Endothelial signalling during development and interplay between endothelial and** 

Elegant experiments on early pancreatic development demonstrated that blood vessel endothelium in the dorsal aorta provides inductive signals for the differentiation of the primitive endoderm into islet cells (Lammert et al., 2001; Yoshitomi & Zaret, 2004). *In vivo* embryonic manipulation of frog embryos to block the formation of the dorsal aorta endothelium, leads to failure of pancreatic gene and insulin expression. To assay blood vessel-pancreas interactions later in development, VEGF-A was overexpressed in transgenic mice using the pancreatic promoter, *Pdx1*; this leads to hypervascularisation of the pancreas and hyperplasia of the pancreatic islets (threefold increase in islet area), at the expense of acinar cell types. Further, coculture experiments with endoderm and dorsal aortic endothelium from early mouse embryos, result in pancreatic gene *Pdx1* induction and insulin expression, indicating that endothelial signals are sufficient for the pancreatic organogenesis program. A successive study on pancreatic organogenesis, has shown that aortal endothelial cells induce in the dorsal pancreatic endoderm the crucial pancreatic transcription factor Ptf1a, that has been shown to be necessary for the development of

A two sep-model for islet development has been proposed (Konstantinova & Lammert, 2004): the first step involves signals from the endothelium to the pancreatic epithelium, the second involves signals in the opposite direction, with islets expressing VEGF-A at later stages of their development to attract capillaries. As for the molecular basis for such signals, recent studies indicate that cells, by using VEGF-A attract endothelial cells, which form capillaries with a vascular basement membrane next to the cells. In turn, laminins, amongst other vascular basement membrane proteins, regulate insulin gene expression and cell proliferation; these effects require 1 integrin on cells (Nikolova et

Studies on cell proliferation in humans are limited, but there is evidence that this process occurs at relatively high levels in the first 2 years of life, declining thereafter, with the possibility, at least in animals, of re-induction under conditions of insulin-resistance, such as pregnancy or obesity (Meier et al., 2008; Cnop et al., 2010). This suggests that cell may retain an intrinsic capacity to replicate, and an increase of the islet vasculature has been observed in association with conditions of expanded islet mass (Mizuno et al., 1999; Like

von Andrian & Mackay CR, 2000; Ostermann et al., 2002).

 **cells** 

al., 2006).

endocrine and duct cell lineages.

mediators, may contribute to the neovascularisation of transplanted islets.

mice not only displayed loss of endothelial fenestrations and thicker endothelial cell body but also defective blood glucose levels on glucose tolerance test, pointing to a possible defect in the release of insulin (Lammert et al., 2003). A more recent study indicated that mice with β cell reduced VEGF-A expression show impaired glucose-stimulated insulin secretion, related to vascular alterations of the islets (Brissova et al., 2006).

Fig. 2. Nephrin is expressed by islet endothelial cells.

Representative immunogold labelling micrograph of islet MECs stained with anti-nephrin Abs. By immunogold staining, nephrin appears distributed on the surface of islet MECs, without accumulation at cell-to-cell junctions (original magnification 1000X).

The microvasculature participates in sensing the environment of the islets and generates signals to affect adult islet endocrine function, being accepted that post-natal β cell mass is dynamic and can increase in function and mass to compensate for added demand (Bonner-Weir & Sharma, 2006). In an in vitro system, purified islet endothelial cells have been shown to stimulate cell proliferation, through secretion of hepatocyte growth factor (HGF) (Suschek et al., 1994). VEGF-A and insulin are the islet-derived factor that induce HGF secretion. In vivo experiments, using pancreas of pregnant rats in which a high physiological proliferation of β cell occurs, showed prominent expression of HGF, coinciding with the peak of cell proliferation.

Islet endothelium exhibits a unique phenotype also in the activities of the constitutive and cytokine-inducible endothelial nitric oxide (NO) synthases, forming the vasoactive mediator NO, since these enzymes are specifically regulated by the glucose level (Kolb & Kolb-Bachofen, 1992). This indicates an organ-specific control of NO production, whose role in islet cytotoxicity is well established (Kroncke et al., 1993; Steiner et al., 1997; Southern et al., 1990; Schmidt et al., 1992). The role of NO in the physiology of insulin release instead

mice not only displayed loss of endothelial fenestrations and thicker endothelial cell body but also defective blood glucose levels on glucose tolerance test, pointing to a possible defect in the release of insulin (Lammert et al., 2003). A more recent study indicated that mice with β cell reduced VEGF-A expression show impaired glucose-stimulated insulin secretion,

Representative immunogold labelling micrograph of islet MECs stained with anti-nephrin Abs. By immunogold staining, nephrin appears distributed on the surface of islet MECs,

The microvasculature participates in sensing the environment of the islets and generates signals to affect adult islet endocrine function, being accepted that post-natal β cell mass is dynamic and can increase in function and mass to compensate for added demand (Bonner-Weir & Sharma, 2006). In an in vitro system, purified islet endothelial cells have been shown to stimulate cell proliferation, through secretion of hepatocyte growth factor (HGF) (Suschek et al., 1994). VEGF-A and insulin are the islet-derived factor that induce HGF secretion. In vivo experiments, using pancreas of pregnant rats in which a high physiological proliferation of β cell occurs, showed prominent expression of HGF,

Islet endothelium exhibits a unique phenotype also in the activities of the constitutive and cytokine-inducible endothelial nitric oxide (NO) synthases, forming the vasoactive mediator NO, since these enzymes are specifically regulated by the glucose level (Kolb & Kolb-Bachofen, 1992). This indicates an organ-specific control of NO production, whose role in islet cytotoxicity is well established (Kroncke et al., 1993; Steiner et al., 1997; Southern et al., 1990; Schmidt et al., 1992). The role of NO in the physiology of insulin release instead

without accumulation at cell-to-cell junctions (original magnification 1000X).

related to vascular alterations of the islets (Brissova et al., 2006).

Fig. 2. Nephrin is expressed by islet endothelial cells.

coinciding with the peak of cell proliferation.

remains controversial (Welsh & Sandler, 1992; Corbett et al., 1993; Henningsson et al., 2002; von Andrian & Mackay CR, 2000; Ostermann et al., 2002).

Immunohistochemical studies have shown that the expression of Platelet-activating factor (PAF) receptor (PAF-r) within the islet, is restricted to endothelial cells, providing potential target for therapeutic intervention (Biancone et al., 2006). PAF, is a phospholipid with diverse physiological effects that mediates a host of biochemical activities, including angiogenesis and inflammation. Islet endothelial cells have also been shown to rapidly produce PAF under stimulation with trombin, and PAF accelerated angiogenesis (Mattsson et al., 2006). These data suggest that intra-islet production of PAF, induced by inflammatory mediators, may contribute to the neovascularisation of transplanted islets.

Lastly, islet endothelial cells express genes encoding for a number of other factors involved in angiogenesis, including potent pro-angiogenic factors, such as VEGF, and angiostatic factors, such as endostatin and pigment epithelial-derived factor (Lammert et al., 2001).

#### **2.1 Endothelial signalling during development and interplay between endothelial and cells**

Elegant experiments on early pancreatic development demonstrated that blood vessel endothelium in the dorsal aorta provides inductive signals for the differentiation of the primitive endoderm into islet cells (Lammert et al., 2001; Yoshitomi & Zaret, 2004). *In vivo* embryonic manipulation of frog embryos to block the formation of the dorsal aorta endothelium, leads to failure of pancreatic gene and insulin expression. To assay blood vessel-pancreas interactions later in development, VEGF-A was overexpressed in transgenic mice using the pancreatic promoter, *Pdx1*; this leads to hypervascularisation of the pancreas and hyperplasia of the pancreatic islets (threefold increase in islet area), at the expense of acinar cell types. Further, coculture experiments with endoderm and dorsal aortic endothelium from early mouse embryos, result in pancreatic gene *Pdx1* induction and insulin expression, indicating that endothelial signals are sufficient for the pancreatic organogenesis program. A successive study on pancreatic organogenesis, has shown that aortal endothelial cells induce in the dorsal pancreatic endoderm the crucial pancreatic transcription factor Ptf1a, that has been shown to be necessary for the development of endocrine and duct cell lineages.

A two sep-model for islet development has been proposed (Konstantinova & Lammert, 2004): the first step involves signals from the endothelium to the pancreatic epithelium, the second involves signals in the opposite direction, with islets expressing VEGF-A at later stages of their development to attract capillaries. As for the molecular basis for such signals, recent studies indicate that cells, by using VEGF-A attract endothelial cells, which form capillaries with a vascular basement membrane next to the cells. In turn, laminins, amongst other vascular basement membrane proteins, regulate insulin gene expression and cell proliferation; these effects require 1 integrin on cells (Nikolova et al., 2006).

Studies on cell proliferation in humans are limited, but there is evidence that this process occurs at relatively high levels in the first 2 years of life, declining thereafter, with the possibility, at least in animals, of re-induction under conditions of insulin-resistance, such as pregnancy or obesity (Meier et al., 2008; Cnop et al., 2010). This suggests that cell may retain an intrinsic capacity to replicate, and an increase of the islet vasculature has been observed in association with conditions of expanded islet mass (Mizuno et al., 1999; Like

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 61

interactions between generated human islet endothelial cells and autoreactive T cells, indicate that islet endothelial cells constitutively express the CD86 (B7-2) and ICOS-L, but not CD80 (B7-1) and CD40 costimulatory molecules. Such co-stimulatory molecules are capable of functionally co-stimulating CD4+ T cell activation, and to help activated memory (CD45R0+) CD4 T cells to migrate across the endothelial barrier (Lozanoska et al., 2008). These studies provide strong indication that islet endothelium actively participates in the recruitment of recently activated lymph node migrant autoreactive T cells. Blockade of the costimulation may represent a mode of *in vivo* action of intervention therapies that interfere with costimulation, such as CTLA-4 Ig (abatacept). Furthermore, analysis of the immunophenotype of endothelial cells, focusing on endothelial MHC class I molecule expression, in a range of different tissues and mouse strain, including the NOD mice, shows that MHC levels have a profound effect on activation, adhesion and transmigration of pathogenic, islet autoreactive CD8 T cells (Lozanoska-Ochser & Peakman, 2009). These finding have a direct relevance to the pathogenesis of autoimmune diabetes in the MOD mouse, and are in concert with those with Savinov *et al*. (Savinov et al., 2001) who demonstrated that homing of a diabetogenic insulin-specific CD8+ T cell clone was severely impaired when clone cells were infused in IFN- knock-out mice, despite normal adhesion to the microvasculature. More recently, the same authors showed that islet-specific homing of the same diabetogenic clone depends in part upon recognition of the cognate MHC/peptide complexes presented by pancreatic islet endothelial cells, which are

presumed to acquire insulin from adjacent cells (Savinov et al., 2003).

cells, processed and presented to autoreactive T cells.

2000).

Based on these observations, it is proposed the model in which, during islet inflammation due to as-yet non-defined environmental insult (possibly a viral infection), cytokines and other inflammatory mediators, such as IFN-, are released and elicit activation of vascular endothelium. Endothelial activation leads to increased adhesion and extravasation of leukocytes. Further, insulin, to high level of which endothelial cells are chronically exposed, and islet antigens released by damaged cells, may be taken up by activated endothelial

Furthermore, sustained and intermitted hyperglycemia has been shown to affect endothelial cellular survival and proliferation, including islet microendothelium (Favaro et al., 2008). Several metabolic mechanisms are involved, including oxidative stress, increased intracellular Ca++, mitochondrial dysfunction, changes in intracellular fatty-acid metabolism, impaired tyrosine phosphorylation and activation of PI3K/Akt and ERK1/2 pathways and reduced intracellular cAMP and its target, the cAMP-dependent PKA pathways (Datta et al., 1999; Favaro et al., 2010). These multifunctional pathways transmit signals that result in prevention of apoptosis or induction of cell cycle progression, depending on the cell type and can cross-regulate one another (Stork & Schmitt, 2002). Akt signaling cascade has also a role in insulin-mediated glucose transport and pancreatic β-cell mass and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006 ). Pro-survival Bcl-2 protein, which stabilizes the mitochondrial membrane and prevents the release of cytocrome c from the mitochondria and the activation of caspases (Choy et al., 2001), is also found to be down-regulated by high glucose in human islet microendothelial cells. In contrast, the pro-apoptotic member Bax, which antagonizes Bcl-2, is up-regulated (Favaro et al., 2010). It is noteworthy that over-expression of Bcl-2 in endothelial cells has been described to decrease T cell cytotoxicity, suggesting that this protein may also protect endothelial cells from apoptosis resulting from an immunological insult (Zheng et al.,

Also collagen IV and other basement membrane proteins, laminins, could potentiate insulin secretion, promote insulin gene expression and proliferation in cells, via interaction with integrin 11 on cell (Treutelaar et al., 2003; Kroncke et al., 1991).

These studies confirm the existence of an endothelial-endocrine axis within adult pancreatic islets.

#### **3. Islet endothelium and type 1 diabetes**

Islet endothelium forms the barrier across which autoreactive T cells transmigrate during the development of islet inflammation in autoimmune diabetes. Transendothelial migration and recruitment of autoreactive T cells into the pancreatic islets is a critical event during the development of chronic insulitis in type 1 diabetes. Transmigration is a complex, multistep process involving first selectins and their counter ligands that induce rolling of cells along the luminal surface of endothelial cells, followed by firm adhesion between cells and endothelium, and diapedesis (von Andrian & Mackay, 2000).

Several human and murine studies indicate that during autoimmune insulitis, the endothelial cells surrounding the inflamed islets adopt an activated phenotype, upregulate a variety of adhesion molecules, and are likely to be involved in regulating mononuclear cell accumulation (transmigration and homing) in the islets (Hanafusa et al., 1990; Hanninen et al., 1992; Hanninen et al., 1993; Itoh et al., 1993; Somoza et al.,1994). Activation of the islet endothelium may either initiate or enhance subsequent leukocyte infiltration of the islets. The islet endothelium is able to hyperexpress adhesion molecules, to secrete numerous cytochines and chemokines, and to hyperexpress class I and class II HLA molecules (Itoh et al., 1993; Somoza et al.,1994; Alejandro et al., 1982). Endothelial cells participate also in presentation of cognate antigens to T cells, which has potent effects on their migration in vitro and in vivo (Epperson & Pober, 1994; Marelli-Berg et al., 1999; Marelli-Berg et al., 2004; Pober et al., 2001) .

In particular, in humans, immunohistological studies of islets obtained near to the time of type 1 diabetes diagnosis, show abundant adhesion molecule expression on vessel and immune cells. In particular, bioptic studies showed that infiltrating mononuclear cells consisted of CD4+ T, predominant CD8+ T and B lymphocytes and macrophages, accompanied by increased expression class I and class II HLA antigens in endothelial cells (Itoh et al.,1993; Greening et al., 2003; Lozanoska-Ochser & Peakman, 2005). Pancreatic islet endothelial MHC class I hyperexpression has been observed also in NOD mice and the biobreeding rat model of autoimmune diabetes (Kay et al., 1991; Ono et al., 1988), and represents a mechanism through which tissue-specific migration of T cells is refined and promoted.

In support to this, human islet endothelial cells have been shown *in vitro* to be capable of internalizing, processing and presenting to autoreactive CD4 T cell clones, disease-relevant epitopes of the islet autoantigen GAD65 (Greening et al., 2003; Di Lorenzo et al., 2007). This resulted in markedly enhanced transmigration.

Islet endothelial cells have also been shown to possess the necessary repertoire of the costimulatory molecules for adequate T cell activation. In vitro studies on the molecular

1970; Predescu et al., 1998). Islet endothelium-derived hepatocyte growth factor (HGF) is one of the factors potentially involved in the stimulation of cell proliferation (Suschek et

Also collagen IV and other basement membrane proteins, laminins, could potentiate insulin secretion, promote insulin gene expression and proliferation in cells, via interaction with

These studies confirm the existence of an endothelial-endocrine axis within adult pancreatic

Islet endothelium forms the barrier across which autoreactive T cells transmigrate during the development of islet inflammation in autoimmune diabetes. Transendothelial migration and recruitment of autoreactive T cells into the pancreatic islets is a critical event during the development of chronic insulitis in type 1 diabetes. Transmigration is a complex, multistep process involving first selectins and their counter ligands that induce rolling of cells along the luminal surface of endothelial cells, followed by firm adhesion between cells and

Several human and murine studies indicate that during autoimmune insulitis, the endothelial cells surrounding the inflamed islets adopt an activated phenotype, upregulate a variety of adhesion molecules, and are likely to be involved in regulating mononuclear cell accumulation (transmigration and homing) in the islets (Hanafusa et al., 1990; Hanninen et al., 1992; Hanninen et al., 1993; Itoh et al., 1993; Somoza et al.,1994). Activation of the islet endothelium may either initiate or enhance subsequent leukocyte infiltration of the islets. The islet endothelium is able to hyperexpress adhesion molecules, to secrete numerous cytochines and chemokines, and to hyperexpress class I and class II HLA molecules (Itoh et al., 1993; Somoza et al.,1994; Alejandro et al., 1982). Endothelial cells participate also in presentation of cognate antigens to T cells, which has potent effects on their migration in vitro and in vivo (Epperson & Pober, 1994; Marelli-Berg et al., 1999; Marelli-Berg et al., 2004;

In particular, in humans, immunohistological studies of islets obtained near to the time of type 1 diabetes diagnosis, show abundant adhesion molecule expression on vessel and immune cells. In particular, bioptic studies showed that infiltrating mononuclear cells consisted of CD4+ T, predominant CD8+ T and B lymphocytes and macrophages, accompanied by increased expression class I and class II HLA antigens in endothelial cells (Itoh et al.,1993; Greening et al., 2003; Lozanoska-Ochser & Peakman, 2005). Pancreatic islet endothelial MHC class I hyperexpression has been observed also in NOD mice and the biobreeding rat model of autoimmune diabetes (Kay et al., 1991; Ono et al., 1988), and represents a mechanism through which tissue-specific migration of T cells is refined and

In support to this, human islet endothelial cells have been shown *in vitro* to be capable of internalizing, processing and presenting to autoreactive CD4 T cell clones, disease-relevant epitopes of the islet autoantigen GAD65 (Greening et al., 2003; Di Lorenzo et al., 2007). This

Islet endothelial cells have also been shown to possess the necessary repertoire of the costimulatory molecules for adequate T cell activation. In vitro studies on the molecular

integrin 11 on cell (Treutelaar et al., 2003; Kroncke et al., 1991).

endothelium, and diapedesis (von Andrian & Mackay, 2000).

**3. Islet endothelium and type 1 diabetes** 

resulted in markedly enhanced transmigration.

al., 1994).

islets.

Pober et al., 2001) .

promoted.

interactions between generated human islet endothelial cells and autoreactive T cells, indicate that islet endothelial cells constitutively express the CD86 (B7-2) and ICOS-L, but not CD80 (B7-1) and CD40 costimulatory molecules. Such co-stimulatory molecules are capable of functionally co-stimulating CD4+ T cell activation, and to help activated memory (CD45R0+) CD4 T cells to migrate across the endothelial barrier (Lozanoska et al., 2008). These studies provide strong indication that islet endothelium actively participates in the recruitment of recently activated lymph node migrant autoreactive T cells. Blockade of the costimulation may represent a mode of *in vivo* action of intervention therapies that interfere with costimulation, such as CTLA-4 Ig (abatacept). Furthermore, analysis of the immunophenotype of endothelial cells, focusing on endothelial MHC class I molecule expression, in a range of different tissues and mouse strain, including the NOD mice, shows that MHC levels have a profound effect on activation, adhesion and transmigration of pathogenic, islet autoreactive CD8 T cells (Lozanoska-Ochser & Peakman, 2009). These finding have a direct relevance to the pathogenesis of autoimmune diabetes in the MOD mouse, and are in concert with those with Savinov *et al*. (Savinov et al., 2001) who demonstrated that homing of a diabetogenic insulin-specific CD8+ T cell clone was severely impaired when clone cells were infused in IFN- knock-out mice, despite normal adhesion to the microvasculature. More recently, the same authors showed that islet-specific homing of the same diabetogenic clone depends in part upon recognition of the cognate MHC/peptide complexes presented by pancreatic islet endothelial cells, which are presumed to acquire insulin from adjacent cells (Savinov et al., 2003).

Based on these observations, it is proposed the model in which, during islet inflammation due to as-yet non-defined environmental insult (possibly a viral infection), cytokines and other inflammatory mediators, such as IFN-, are released and elicit activation of vascular endothelium. Endothelial activation leads to increased adhesion and extravasation of leukocytes. Further, insulin, to high level of which endothelial cells are chronically exposed, and islet antigens released by damaged cells, may be taken up by activated endothelial cells, processed and presented to autoreactive T cells.

Furthermore, sustained and intermitted hyperglycemia has been shown to affect endothelial cellular survival and proliferation, including islet microendothelium (Favaro et al., 2008). Several metabolic mechanisms are involved, including oxidative stress, increased intracellular Ca++, mitochondrial dysfunction, changes in intracellular fatty-acid metabolism, impaired tyrosine phosphorylation and activation of PI3K/Akt and ERK1/2 pathways and reduced intracellular cAMP and its target, the cAMP-dependent PKA pathways (Datta et al., 1999; Favaro et al., 2010). These multifunctional pathways transmit signals that result in prevention of apoptosis or induction of cell cycle progression, depending on the cell type and can cross-regulate one another (Stork & Schmitt, 2002). Akt signaling cascade has also a role in insulin-mediated glucose transport and pancreatic β-cell mass and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006 ). Pro-survival Bcl-2 protein, which stabilizes the mitochondrial membrane and prevents the release of cytocrome c from the mitochondria and the activation of caspases (Choy et al., 2001), is also found to be down-regulated by high glucose in human islet microendothelial cells. In contrast, the pro-apoptotic member Bax, which antagonizes Bcl-2, is up-regulated (Favaro et al., 2010). It is noteworthy that over-expression of Bcl-2 in endothelial cells has been described to decrease T cell cytotoxicity, suggesting that this protein may also protect endothelial cells from apoptosis resulting from an immunological insult (Zheng et al., 2000).

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 63

Several mechanisms, including molecular mimicry, bystander activation of autoreactive T cells, superantigenic activity of viral proteins, viral infection and persistence, not mutually exclusive, have been proposed to explain the relationship between EV infections and induction of autoimmune diseases (extensively reviewed in Varela-Calvino & Peakman,

As for a role in type 1 diabetes, results have been somewhat conflicting and not conclusive (von Herrath, 2009; Tauriainen et al., 2010). Autoantibodies to islet autoantigens are detected years prior to diagnosis of type 1 diabetes and prospective studies evaluating whether EV could predict islet autoimmunity have yielded conflicting results, with positive associations in the Finnish studies (Lönnrot et al., 2000; Salminen et al., 2003; Sadeharju et al., 2003), and no association in other reports (Graves et al., 2003; Füchtenbusch et al., 2001). Discrepancies could be related to the fact that in most studies the determination of EV infection was carried out indirectly through the determination of IgM and IgG anti-EV antibodies, while studies using multiple approaches to identify EV infection (serology, RT-PCR, faeces analysis) appear more likely to report an association with type 1 diabetes or islet autoimmunity. A higher frequency of EV RNA has been consistently shown in the serum of patients with diabetes compared to healthy control subjects, demonstrating a recent or a persistent infection (Lönnrot, M., Salminen, K., et al., 2000; Lönnrot, M., Korpela, K., et al., 2000), and in some of the cases the detection of EV RNA preceded the synthesis of islet cell autoantibodies. In most studies viruses of the CVB group, usually CVB3 and CVB4 were identified (Clements et al., 1995;

Andréoletti et al., 1997; Chehadeh et al., 2000), in agreement with serological studies.

diagnosis appears thus to be active, indicating recent or prolonged exposure.

have etiological implications for the development of type 1 diabetes.

As for T cell responses to EV, studies are inconclusive. However, it has been shown that CD4 T cells from newly diagnosed patients up-regulate CD69 early activation marker after exposure to CVB4-infected lysates (Varela-Calvino et al., 2001) and produce more IFN- a proinflammatory cytokine generated by effector memory CD4 T cells, but show less T cell proliferation (Varela-Calvino & Peakman, 2003). Proliferation is dependent upon IL-2 secretion associated with central memory T cells. This implies that anti-CVB4 effector cells are mobilized from the central memory pool, which may be depleted. Response to CVB4 antigens at diabetes

Results from animal models indicate that viral infections *per se* usually cannot initiate the autoimmune disease process leading to diabetes, but may accelerate an already ongoing disease process. Studies in various NOD mice strains show that EV infections may accelerate the progression to diabetes only if they occur after autoreactive T cells have been accumulated in the islets (Hiltunen et al.,1997; Lönnrot et al., 1998; Lönnrot et al., 2000; Otonkoski et al., 2000). CVB infection appears to accelerate the development of the disease via bystander activation of autoreactive T cells, due to inflammation of the pancreas, tissue damage, release of sequestered autoantigens in concert with production of proinflammatory cytokines, all leading to activation of autoreactive T cells, but apparently only when a certain threshold of these autoreactive T cells have already accumulated in the pancreas. Timing of a CVB infection, rather than its simple presence or absence, may thus

A recent report evaluating whether such a general model of disease progression rather than initiation by EV applies to human type 1 diabetes (Stene et al., 2010), suggests that progression from islet autoimmunity to type 1 diabetes in high-risk individuals may increase after an EV infection characterised by the presence of viral RNA in blood. Indeed, most EV are avid triggers of production of pro-inflammatory cytokines by human

2003; Ercolini & Miller, 2009).

Due to the established interdependent physical and functional relationship between islet endothelium and β cells, from pancreatic organogenesis to adult life (Zanone et al., 2008), and the notion that post-natal β -cell mass is dynamic and can increase in function and mass for added demand by replication or neogenesis, possibly through endothelial inductive signals (Nikolova et al., 2006; Johansson et al., 2006; Bonner-Weir & Sharma, 2002; Dor et al., 2004), these high glucose-induced changes in islet endothelium carry relevant consequences on β cells. In fact, production of the vasoactive mediator NO (Meier, 2008; Favaro et al., 2008) to upregulate CD40L expression in human islet microendetelial cells *in vitro* (Favaro et al., 2010). Functional CD40L is expressed on vascular endothelium (Mach et al., 1997) and contributes to B cell activation, isotype switching, costimulation in T cell mediated immunity, activation of extravasating monocytes (Yang & Wilson, 1996; Wagner et al., 2004), with an impact in atherosclerosis and in chronic inflammatory and autoimmune diseases. Blockers of the CD40L have been strikingly effective in animal models of autoimmune diseases, such as systemic lupus erythematosus and type 1 diabetes (Homann et al., 2002). Therefore, high glucoseinduced overexpression of CD40L on islet endothelial cells might accelerate the targeting and loss of the remaining β-cell capacity during ongoing autoimmune insulitis.

In fact, production of the vasoactive mediator NO by islet endothelium (Meier, 2008; Favaro et al., 2008) is increased in hyperglycaemic conditions and has an established direct cytotoxicity on islets and potentially impairs insulin release (Corbett JA et al., 1993). Islet microendothelial cells also are source of the proinflammatory cytokine IL-1β under hyperglycaemic conditions, independently of any viral or immune-mediated process. IL-1β impairs insulin release in human islet, induces Fas expression enabling Fas-mediated apoptosis and it is implicated as a mediator of glucotoxicity (Maedler K, et al. 2002). The high glucose condition is also reported to upregulate CD40L expression in human islet microendothelial cells in vitro (Favaro E et al., 2010).

#### **4. Enteroviruses and type 1 diabetes**

Viral infection has been long implicated in the development of type 1 diabetes and evidences of a link between viral infections and initiation or acceleration of pancreatic islet autoimmunity have been under investigation for more than 30 years. Rubella virus (Karvonen et al., 1993), mumps virus (Hyoty et al., 1988), cytomegalovirus (Ward et a., 1979), rotavirus (Honeyman et al., 2000) and enteroviruses (EV) (Lonnrot et al., 2000; Stene et al., 2010) have all been suggested as environmental factors contributing to type 1 diabetes. EV, especially those of the Coxsackievirus B (CVB) group (Hyöty et al., 1988; Varela-Calvino & Peakman , 2003; Green et al., 2004), are historically the prime suspects as important aetiological determinants and seroepidemiological, histopathological, animal studies, and *in vitro* experiments have provided the strongest overall evidence for these viruses. The EV genus of the Picornaviridae family is a large group of human pathogens traditionally divided into polioviruses, coxsackieviruses, echoviruses and the new EV, and each group contains a range of serotypes (King et al., 2000; Roivainen, 2006). Human EV are the most common cause of viral infection in humans, are associated with a wide variety of clinical syndromes and have long been considered possible culprits of inflammatory conditions and immune-mediated pathological processes, such as chronic myocarditis, dilated cardiomyopathy and chronic myositis (Tam, 2006; Luppi et al., 2000). In the cardiac context, injury is caused by a direct cytopathic effect of the virus, an immune response to viral infection or autoimmunity triggered by the viral infection (Huber, 2006).

Due to the established interdependent physical and functional relationship between islet endothelium and β cells, from pancreatic organogenesis to adult life (Zanone et al., 2008), and the notion that post-natal β -cell mass is dynamic and can increase in function and mass for added demand by replication or neogenesis, possibly through endothelial inductive signals (Nikolova et al., 2006; Johansson et al., 2006; Bonner-Weir & Sharma, 2002; Dor et al., 2004), these high glucose-induced changes in islet endothelium carry relevant consequences on β cells. In fact, production of the vasoactive mediator NO (Meier, 2008; Favaro et al., 2008) to upregulate CD40L expression in human islet microendetelial cells *in vitro* (Favaro et al., 2010). Functional CD40L is expressed on vascular endothelium (Mach et al., 1997) and contributes to B cell activation, isotype switching, costimulation in T cell mediated immunity, activation of extravasating monocytes (Yang & Wilson, 1996; Wagner et al., 2004), with an impact in atherosclerosis and in chronic inflammatory and autoimmune diseases. Blockers of the CD40L have been strikingly effective in animal models of autoimmune diseases, such as systemic lupus erythematosus and type 1 diabetes (Homann et al., 2002). Therefore, high glucoseinduced overexpression of CD40L on islet endothelial cells might accelerate the targeting and

In fact, production of the vasoactive mediator NO by islet endothelium (Meier, 2008; Favaro et al., 2008) is increased in hyperglycaemic conditions and has an established direct cytotoxicity on islets and potentially impairs insulin release (Corbett JA et al., 1993). Islet microendothelial cells also are source of the proinflammatory cytokine IL-1β under hyperglycaemic conditions, independently of any viral or immune-mediated process. IL-1β impairs insulin release in human islet, induces Fas expression enabling Fas-mediated apoptosis and it is implicated as a mediator of glucotoxicity (Maedler K, et al. 2002). The high glucose condition is also reported to upregulate CD40L expression in human islet

Viral infection has been long implicated in the development of type 1 diabetes and evidences of a link between viral infections and initiation or acceleration of pancreatic islet autoimmunity have been under investigation for more than 30 years. Rubella virus (Karvonen et al., 1993), mumps virus (Hyoty et al., 1988), cytomegalovirus (Ward et a., 1979), rotavirus (Honeyman et al., 2000) and enteroviruses (EV) (Lonnrot et al., 2000; Stene et al., 2010) have all been suggested as environmental factors contributing to type 1 diabetes. EV, especially those of the Coxsackievirus B (CVB) group (Hyöty et al., 1988; Varela-Calvino & Peakman , 2003; Green et al., 2004), are historically the prime suspects as important aetiological determinants and seroepidemiological, histopathological, animal studies, and *in vitro* experiments have provided the strongest overall evidence for these viruses. The EV genus of the Picornaviridae family is a large group of human pathogens traditionally divided into polioviruses, coxsackieviruses, echoviruses and the new EV, and each group contains a range of serotypes (King et al., 2000; Roivainen, 2006). Human EV are the most common cause of viral infection in humans, are associated with a wide variety of clinical syndromes and have long been considered possible culprits of inflammatory conditions and immune-mediated pathological processes, such as chronic myocarditis, dilated cardiomyopathy and chronic myositis (Tam, 2006; Luppi et al., 2000). In the cardiac context, injury is caused by a direct cytopathic effect of the virus, an immune response to viral

loss of the remaining β-cell capacity during ongoing autoimmune insulitis.

infection or autoimmunity triggered by the viral infection (Huber, 2006).

microendothelial cells in vitro (Favaro E et al., 2010).

**4. Enteroviruses and type 1 diabetes** 

Several mechanisms, including molecular mimicry, bystander activation of autoreactive T cells, superantigenic activity of viral proteins, viral infection and persistence, not mutually exclusive, have been proposed to explain the relationship between EV infections and induction of autoimmune diseases (extensively reviewed in Varela-Calvino & Peakman, 2003; Ercolini & Miller, 2009).

As for a role in type 1 diabetes, results have been somewhat conflicting and not conclusive (von Herrath, 2009; Tauriainen et al., 2010). Autoantibodies to islet autoantigens are detected years prior to diagnosis of type 1 diabetes and prospective studies evaluating whether EV could predict islet autoimmunity have yielded conflicting results, with positive associations in the Finnish studies (Lönnrot et al., 2000; Salminen et al., 2003; Sadeharju et al., 2003), and no association in other reports (Graves et al., 2003; Füchtenbusch et al., 2001). Discrepancies could be related to the fact that in most studies the determination of EV infection was carried out indirectly through the determination of IgM and IgG anti-EV antibodies, while studies using multiple approaches to identify EV infection (serology, RT-PCR, faeces analysis) appear more likely to report an association with type 1 diabetes or islet autoimmunity. A higher frequency of EV RNA has been consistently shown in the serum of patients with diabetes compared to healthy control subjects, demonstrating a recent or a persistent infection (Lönnrot, M., Salminen, K., et al., 2000; Lönnrot, M., Korpela, K., et al., 2000), and in some of the cases the detection of EV RNA preceded the synthesis of islet cell autoantibodies. In most studies viruses of the CVB group, usually CVB3 and CVB4 were identified (Clements et al., 1995; Andréoletti et al., 1997; Chehadeh et al., 2000), in agreement with serological studies.

As for T cell responses to EV, studies are inconclusive. However, it has been shown that CD4 T cells from newly diagnosed patients up-regulate CD69 early activation marker after exposure to CVB4-infected lysates (Varela-Calvino et al., 2001) and produce more IFN- a proinflammatory cytokine generated by effector memory CD4 T cells, but show less T cell proliferation (Varela-Calvino & Peakman, 2003). Proliferation is dependent upon IL-2 secretion associated with central memory T cells. This implies that anti-CVB4 effector cells are mobilized from the central memory pool, which may be depleted. Response to CVB4 antigens at diabetes diagnosis appears thus to be active, indicating recent or prolonged exposure.

Results from animal models indicate that viral infections *per se* usually cannot initiate the autoimmune disease process leading to diabetes, but may accelerate an already ongoing disease process. Studies in various NOD mice strains show that EV infections may accelerate the progression to diabetes only if they occur after autoreactive T cells have been accumulated in the islets (Hiltunen et al.,1997; Lönnrot et al., 1998; Lönnrot et al., 2000; Otonkoski et al., 2000). CVB infection appears to accelerate the development of the disease via bystander activation of autoreactive T cells, due to inflammation of the pancreas, tissue damage, release of sequestered autoantigens in concert with production of proinflammatory cytokines, all leading to activation of autoreactive T cells, but apparently only when a certain threshold of these autoreactive T cells have already accumulated in the pancreas. Timing of a CVB infection, rather than its simple presence or absence, may thus have etiological implications for the development of type 1 diabetes.

A recent report evaluating whether such a general model of disease progression rather than initiation by EV applies to human type 1 diabetes (Stene et al., 2010), suggests that progression from islet autoimmunity to type 1 diabetes in high-risk individuals may increase after an EV infection characterised by the presence of viral RNA in blood. Indeed, most EV are avid triggers of production of pro-inflammatory cytokines by human

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 65

amino acids or nucleotides potentially most critical for the pathogenesis of type 1 diabetes

Another work (Chehadeh et al., 2000) indicates that the CVB group is capable to replicate at a low level in human islet cells *in vitro,* persisting without cytolytic effect. This replication is associated with chronic synthesis of IFN- by the islet cells. Neutralization of the IFN-

Type I interferons induce an anti-viral state in infected cells, providing an early defense against viral infections (Stark et al., 1998), and it appears that cells may depend on interferons to lower their permissiveness to CVB4 infection, thus regulating the susceptibility to virus-induced diabetes (Flodstrom et a., 2002; Flodstrom et al., 2003). In this model, NOD mice that expressed the suppressor of cytokine signalling 1 (SOCS-1) in cells developed diabetes, due to the replication of the virus in the cells. A critical link between

Furthermore, due to its immunoregulatory properties, IFN- represents a link between the innate and the adaptive immunity: a pathological event may commence with activation of the innate immune system in order to avoid cytolytic destruction, followed by T cell activation and expansion, including autoreactive T cells. Viral expansion of non–specific T cell responses has been shown to be mimicked by injection of IFN- (Tough & Sprent, 1996), or IFN- expression by pancreatic cells (Stewart et al., 1993; Chakrabarti et al., 1996). In humans, IFN- has been detected in β cells (Foulis et al., 1987; Huang et al., 1995) and in

A very recent report, indicates that rare genetic variations occurring in the gene IFIH1 and affecting the expression and structure of its protein product IFIH, lower the risk of developing type 1 diabetes (Nejentsev et al., 2009). IFIH1 triggers the secretion of interferons. Another study showed that IFIH1 expression in peripheral blood cells is associated with type 1 diabetes (Liu et al., 2009). These data allows to speculate that, on viral infection, interferon-response genes are activated in insulin-producing cells, leading to increased levels of interferons. Interferons inhibit viral replication, but also enhance the expression of surface MHC-I molecules. Cytotoxic CD8 T cells recognize infected cells, through the MHC-I molecules, damaging and eventually killing them. Thus, viral infection

As for i*n vivo* studies in humans, the isolation of an EV has been documented only few times. Historically, CVB4 was successfully cultured from the pancreas of a diabetic child at disease onset and it induced diabetes in susceptible animals, more than 30 years ago (Yoon et al., 1979). The diabetogenic E2 strain was likewise obtained by plaque purification of the human isolate Edwards of CVB4, that was isolated from a child with widespread CVB4

In contrast with the apparent success in the detection of EV mRNA from blood, no EV genome could be detected when pancreas from cases of type 1 diabetes were analysed post-mortem during the first year after diagnosis (Foulis et al., 1997). However, in the same study EV genome could be detected in the pancreas of children who died of acute myocarditis in which the heart was EV positive. These discrepancies may be explained with the argument that there is a critical window for virus detection in the pancreas, and this is only potentially achieved when there is an acute presentation of viral illness, as in the case of myocarditis. By *in situ* hybridisation studies on post-mortem pancreatic tissues of several type 1 diabetic patients, EV RNA positive

infection, presenting with acute myocarditis and pancreatitis (Chatterjee et al., 1988).

cells were for the first time detected, and exclusively in islets (Ylipaasto et al., 2004).

the target cell antiviral responses and susceptibility to disease is thus established.

have to be identified amongst the microvariants of relevant virus strains.

leads to a rise in viral replication and rapid islet destruction.

blood of type 1 diabetic patients (Chehadeh et al., 2000).

can contribute to the development of type 1 diabetes.

leukocytes (Vreugdenhil et al., 2000), notably type I interferons, and it is noteworthy that increased levels of IFN-γ have been detected in the blood of newly diagnosed patients (Chehadeh et al., 2000), and EV RNA was detected in half of the IFN- positive patients. These data are consistent with a recent EV infection.

#### **4.1 Enteroviruses and pancreatic islets**

Major determinants of the different clinicopathological manifestations of EV infections, ranging from silent infections to autoimmune diseases, are represented by the viral variants, the nature of the infection, acute, chronic or re-infection, and the distinct tissue tropism of the viral strain, modulated by the local expression of appropriate cellular receptors and coreceptors. The first step in viral infection is the attachment of the virus to its receptor, a cell surface molecule which viruses have adapted to use for their entry into the cells. These include the Coxsackie-Adenovirus receptor (CAR), integrin VLA-2, v3, v5, ICAM-1 and decayaccelerating factor (DAF) (Bergelson et al., 1997; Noutsias et al., 2001; Shafren, 1998). In cultured cells, CVB have been found to interact with at least three receptors. CAR is a 46kD adhesion molecule and all tested clinical and laboratory isolates bind to this receptor (Bergelson, 2002; Kallewaard et al., 2009). A large subset of CVB isolates also binds to DAF, a complement regulatory protein (Coyne & Bergelson, 2006) which appears to act as a receptor for cell attachment (Shafren et al., 1995), and some CVB3 isolates have been shown to use a third receptor, heparan sulfate, to infect CAR-deficient cells *in vitro* (Zautner et al., 2003).

These receptor molecules do not simply bind viruses, but may activate a series of events influencing the organ-specific outcome of disease (Ito et al., 2000; Selinka et al., 2004 ). CAR expression, for instance, is positively related to the extent of inflammation in the cardiac myosin-induced myocarditis model (Ito et al., 2000), and knockout of MyD88, an adaptor involved in toll-like receptor signaling, causes reduced cardiac expression of CAR and proinflammatory cytokines (Fuse et al., 2005) or TGF- reduced CAR levels inhibit CVB3 infection of cardiac myocytes (Shi et al., 2010). CAR appears as the major receptor mediating CVB infection also in the pancreas *in vitro* and *in vivo*, since tissue-specific CAR gene deletion generated a 1000-fold reduction in virus titres within the pancreas during infection, and a significant reduction in virus-induced pancreatic tissue damage and inflammation (Kallewaard et al., 2009).

While acute infection in the pancreas has been clearly detected among the cells of the exocrine tissue, cell infection by EV has been extensively studied and the issue of whether microvariants of EV can directly infect, replicate and persist in, and cause damage of cells remains controversial (Flodstrom et al., 2002). More than three decade ago Yoon et al. showed that CVB4 is capable of replicating in cultured human islets (Yoon et al., 1978). Other works suggested that the CVB group has variants that can replicate in islet cells (Harting et al., 1983; Chatterjee et al.,1988), and by growing viruses on islets of Langerhans it is possible to isolate strains that can induce insulitis experimentally in animals and replicate in islet cells *in vivo*. Prototype CVB3, CVB4 and CVB5 as wells as CVA9 can replicate *in vitro* in purified insulin-producing cells, and infection may result in functional impairment or cytolytic death of the cells, but it may also have no apparent adverse effect (Roivainen et al., 2000; Roivainen et al., 2002). It appears that the consequences of the virus replication on cell survival and function are not entirely dependent on the serotype but on a as-yet unidentified characteristics of the virus strain. For instance, between the diabetogenic strain E2 of CVB4 and the prototype CVB4, a 111 amino acid difference has been identified, and

leukocytes (Vreugdenhil et al., 2000), notably type I interferons, and it is noteworthy that increased levels of IFN-γ have been detected in the blood of newly diagnosed patients (Chehadeh et al., 2000), and EV RNA was detected in half of the IFN- positive patients.

Major determinants of the different clinicopathological manifestations of EV infections, ranging from silent infections to autoimmune diseases, are represented by the viral variants, the nature of the infection, acute, chronic or re-infection, and the distinct tissue tropism of the viral strain, modulated by the local expression of appropriate cellular receptors and coreceptors. The first step in viral infection is the attachment of the virus to its receptor, a cell surface molecule which viruses have adapted to use for their entry into the cells. These include the Coxsackie-Adenovirus receptor (CAR), integrin VLA-2, v3, v5, ICAM-1 and decayaccelerating factor (DAF) (Bergelson et al., 1997; Noutsias et al., 2001; Shafren, 1998). In cultured cells, CVB have been found to interact with at least three receptors. CAR is a 46kD adhesion molecule and all tested clinical and laboratory isolates bind to this receptor (Bergelson, 2002; Kallewaard et al., 2009). A large subset of CVB isolates also binds to DAF, a complement regulatory protein (Coyne & Bergelson, 2006) which appears to act as a receptor for cell attachment (Shafren et al., 1995), and some CVB3 isolates have been shown to use a third receptor, heparan sulfate, to infect CAR-deficient cells *in vitro* (Zautner et al., 2003). These receptor molecules do not simply bind viruses, but may activate a series of events influencing the organ-specific outcome of disease (Ito et al., 2000; Selinka et al., 2004 ). CAR expression, for instance, is positively related to the extent of inflammation in the cardiac myosin-induced myocarditis model (Ito et al., 2000), and knockout of MyD88, an adaptor involved in toll-like receptor signaling, causes reduced cardiac expression of CAR and proinflammatory cytokines (Fuse et al., 2005) or TGF- reduced CAR levels inhibit CVB3 infection of cardiac myocytes (Shi et al., 2010). CAR appears as the major receptor mediating CVB infection also in the pancreas *in vitro* and *in vivo*, since tissue-specific CAR gene deletion generated a 1000-fold reduction in virus titres within the pancreas during infection, and a significant reduction in virus-induced pancreatic tissue damage and inflammation

While acute infection in the pancreas has been clearly detected among the cells of the exocrine tissue, cell infection by EV has been extensively studied and the issue of whether microvariants of EV can directly infect, replicate and persist in, and cause damage of cells remains controversial (Flodstrom et al., 2002). More than three decade ago Yoon et al. showed that CVB4 is capable of replicating in cultured human islets (Yoon et al., 1978). Other works suggested that the CVB group has variants that can replicate in islet cells (Harting et al., 1983; Chatterjee et al.,1988), and by growing viruses on islets of Langerhans it is possible to isolate strains that can induce insulitis experimentally in animals and replicate in islet cells *in vivo*. Prototype CVB3, CVB4 and CVB5 as wells as CVA9 can replicate *in vitro* in purified insulin-producing cells, and infection may result in functional impairment or cytolytic death of the cells, but it may also have no apparent adverse effect (Roivainen et al., 2000; Roivainen et al., 2002). It appears that the consequences of the virus replication on cell survival and function are not entirely dependent on the serotype but on a as-yet unidentified characteristics of the virus strain. For instance, between the diabetogenic strain E2 of CVB4 and the prototype CVB4, a 111 amino acid difference has been identified, and

These data are consistent with a recent EV infection.

**4.1 Enteroviruses and pancreatic islets** 

(Kallewaard et al., 2009).

amino acids or nucleotides potentially most critical for the pathogenesis of type 1 diabetes have to be identified amongst the microvariants of relevant virus strains.

Another work (Chehadeh et al., 2000) indicates that the CVB group is capable to replicate at a low level in human islet cells *in vitro,* persisting without cytolytic effect. This replication is associated with chronic synthesis of IFN- by the islet cells. Neutralization of the IFN leads to a rise in viral replication and rapid islet destruction.

Type I interferons induce an anti-viral state in infected cells, providing an early defense against viral infections (Stark et al., 1998), and it appears that cells may depend on interferons to lower their permissiveness to CVB4 infection, thus regulating the susceptibility to virus-induced diabetes (Flodstrom et a., 2002; Flodstrom et al., 2003). In this model, NOD mice that expressed the suppressor of cytokine signalling 1 (SOCS-1) in cells developed diabetes, due to the replication of the virus in the cells. A critical link between the target cell antiviral responses and susceptibility to disease is thus established.

Furthermore, due to its immunoregulatory properties, IFN- represents a link between the innate and the adaptive immunity: a pathological event may commence with activation of the innate immune system in order to avoid cytolytic destruction, followed by T cell activation and expansion, including autoreactive T cells. Viral expansion of non–specific T cell responses has been shown to be mimicked by injection of IFN- (Tough & Sprent, 1996), or IFN- expression by pancreatic cells (Stewart et al., 1993; Chakrabarti et al., 1996). In humans, IFN- has been detected in β cells (Foulis et al., 1987; Huang et al., 1995) and in blood of type 1 diabetic patients (Chehadeh et al., 2000).

A very recent report, indicates that rare genetic variations occurring in the gene IFIH1 and affecting the expression and structure of its protein product IFIH, lower the risk of developing type 1 diabetes (Nejentsev et al., 2009). IFIH1 triggers the secretion of interferons. Another study showed that IFIH1 expression in peripheral blood cells is associated with type 1 diabetes (Liu et al., 2009). These data allows to speculate that, on viral infection, interferon-response genes are activated in insulin-producing cells, leading to increased levels of interferons. Interferons inhibit viral replication, but also enhance the expression of surface MHC-I molecules. Cytotoxic CD8 T cells recognize infected cells, through the MHC-I molecules, damaging and eventually killing them. Thus, viral infection can contribute to the development of type 1 diabetes.

As for i*n vivo* studies in humans, the isolation of an EV has been documented only few times. Historically, CVB4 was successfully cultured from the pancreas of a diabetic child at disease onset and it induced diabetes in susceptible animals, more than 30 years ago (Yoon et al., 1979). The diabetogenic E2 strain was likewise obtained by plaque purification of the human isolate Edwards of CVB4, that was isolated from a child with widespread CVB4 infection, presenting with acute myocarditis and pancreatitis (Chatterjee et al., 1988).

In contrast with the apparent success in the detection of EV mRNA from blood, no EV genome could be detected when pancreas from cases of type 1 diabetes were analysed post-mortem during the first year after diagnosis (Foulis et al., 1997). However, in the same study EV genome could be detected in the pancreas of children who died of acute myocarditis in which the heart was EV positive. These discrepancies may be explained with the argument that there is a critical window for virus detection in the pancreas, and this is only potentially achieved when there is an acute presentation of viral illness, as in the case of myocarditis. By *in situ* hybridisation studies on post-mortem pancreatic tissues of several type 1 diabetic patients, EV RNA positive cells were for the first time detected, and exclusively in islets (Ylipaasto et al., 2004).

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 67

The host factors modulating viral infections include not only the host immune response, but also types and characteristics of cells that become infected in different tissues. Parenchymal cells of an organ are rarely in direct contact with the circulatory system, and viruses in the circulation must either circumvent or infect vascular endothelial cells to reach secondary organs. Vascular endothelial cells act in fact as important interface between the vascular space and the organ parenchyma, and, as previously stated, endothelial cells in different organs exhibit diverse structural and functional characteristics that can influence biological and pathological functions. Amongst these, vascular endothelial cells have an established role as mediators of tissue tropism and access for virus, influencing organ and tissue specific susceptibility to viral infection. Therefore, they can modulate the pathological expression of virus-induced diseases (Friedman et al., 1981; Huber et al., 1990; Conaldi et al., 1997; Zanone et al., 2003). For instance, in murine studies on CVB infectivity of different organs, CVB3 isolates from the heart showed greater infectivity and replication in heart endothelial cells than endothelial cells derived from liver or lung (Huber et al., 1990), thus confirming the

In line with this scenario, it is essential that, to gain access to secondary organs, viruses pass through the vascular endothelium by transcytosis or infection, or via infected circulating cells migrating into the target tissues. Endothelial cells derived from different organs show distinct susceptibility to CVB infections, and the behaviour against a viral challenge of endothelial cells in large vessels and microvessels may differ (Friedman et al., 1981; Huber et al., 1990; Conaldi et al., 1997; Zanone et al., 2003; Saijets et al., 2003). Endothelial cells expressing appropriate receptors would fail to act as effective barrier to infections, allowing

Human umbilical vein-derived endothelial cells have been shown to be persistently infected by different CVB strains (Flodstrom et al., 2000; Huber et al., 1990; Conaldi et al., 1997). However, physiological and pathological events take place mainly at the level of the microvasculature. Using a dermal microvascular endothelial cell line, we have provided evidence that small vessel endothelial cells can harbour a persistent CVB viral infection (Zanone et al., 2003). All 3 CVB tested productively infected microvascular endothelial cells for up to 3 months without obvious cytolysis. A small proportion of the cells, approximately 10%, appeared to be involved in viral replication during chronic infection, suggesting that persistence is probably established through a mechanism of carrier-state culture, as proposed to explain CVB persistence in other cell types (Flodstrom et al., 2002; Greening et al., 2003). In addition, the infection increased production of proinflammatory cytokines IL-6 and IL-8, indicating endothelial cell activation by virus, and induced quantitative modification of adhesion molecule expression (ICAM-1, VCAM-1). These upregulation may influence the pattern of migration and extravasation of leucocytes in inflammation and immunity. These data add weight to the view that common CV infections are able to trigger complex pathophysiological processes, rather than simple cell lysis, as is becoming increasingly evident in clinical and experimental settings. These viruses can in fact persist for a considerable time in infected patients and cause chronic pathology or trigger immunopathological damage to infected and uninfected tissues (Muir et al., 1989; Stone, 1994). Furthermore, chronic infection of endothelial cells *in vivo* could provide better viral access to tissues underlying the endothelial layer and subsequent parenchymal cell

**4.2 Coxsackievirus infection and endothelial cells** 

essential role of host factors in developing specific diseases.

infection.

viral particles to pass through, and replicate in, the vascular endothelium.

In recent years, other studies have eventually indicated the presence of EV in pancreatic tissue in a sizable proportion of patients dying soon after diabetes onset (Tauriainen et al., 2009; Dotta et al., 2007; Richardson et al., 2009; Ylipaasto et al., 2004). A cell infectious CVB4 was isolated in the pancreas of three patients at disease onset, by immunohistochemical, electron microscopy, genome nucleotide sequencing, cell culture and immunological studies (Dotta et al., 2007). Infection was specific to cells, which showed islet inflammation mediated mainly by natural killer cells, reduced insulin secretion. The virus was also able to infect cells from human islets of non diabetic donors.

A strain of echovirus 3 was isolated from an individual currently with appearance of islet cell and IA-2 autoantibodies (Williams at al., 2006). Richardson et al (Richardson et al., 2009) identified EV VP1 capsid protein in islets of 44 out of 72 recent-onset type 1 diabetic patients, and the staining was restricted to cells. A recent report suggests that the virus is present in the intestinal mucosa of diabetic patients (Oikarinen et al., 2008).

These detection reports strengthen the case for a viral role in the pathogenesis of type 1 diabetes.

Fig. 3. Representative confocal immunofluorescence micrographs of islet MECs, stained with polyclonal anti-HCAR Ab, showing a diffuse expression in a fine punctate pattern in islet MECs (original magnification 630X, nuclei stained in blue with DAPI).

#### **4.2 Coxsackievirus infection and endothelial cells**

66 Type 1 Diabetes Complications

In recent years, other studies have eventually indicated the presence of EV in pancreatic tissue in a sizable proportion of patients dying soon after diabetes onset (Tauriainen et al., 2009; Dotta et al., 2007; Richardson et al., 2009; Ylipaasto et al., 2004). A cell infectious CVB4 was isolated in the pancreas of three patients at disease onset, by immunohistochemical, electron microscopy, genome nucleotide sequencing, cell culture and immunological studies (Dotta et al., 2007). Infection was specific to cells, which showed islet inflammation mediated mainly by natural killer cells, reduced insulin secretion. The

A strain of echovirus 3 was isolated from an individual currently with appearance of islet cell and IA-2 autoantibodies (Williams at al., 2006). Richardson et al (Richardson et al., 2009) identified EV VP1 capsid protein in islets of 44 out of 72 recent-onset type 1 diabetic patients, and the staining was restricted to cells. A recent report suggests that the virus is present in

These detection reports strengthen the case for a viral role in the pathogenesis of type 1

Fig. 3. Representative confocal immunofluorescence micrographs of islet MECs, stained with polyclonal anti-HCAR Ab, showing a diffuse expression in a fine punctate pattern in

islet MECs (original magnification 630X, nuclei stained in blue with DAPI).

virus was also able to infect cells from human islets of non diabetic donors.

the intestinal mucosa of diabetic patients (Oikarinen et al., 2008).

diabetes.

The host factors modulating viral infections include not only the host immune response, but also types and characteristics of cells that become infected in different tissues. Parenchymal cells of an organ are rarely in direct contact with the circulatory system, and viruses in the circulation must either circumvent or infect vascular endothelial cells to reach secondary organs. Vascular endothelial cells act in fact as important interface between the vascular space and the organ parenchyma, and, as previously stated, endothelial cells in different organs exhibit diverse structural and functional characteristics that can influence biological and pathological functions. Amongst these, vascular endothelial cells have an established role as mediators of tissue tropism and access for virus, influencing organ and tissue specific susceptibility to viral infection. Therefore, they can modulate the pathological expression of virus-induced diseases (Friedman et al., 1981; Huber et al., 1990; Conaldi et al., 1997; Zanone et al., 2003). For instance, in murine studies on CVB infectivity of different organs, CVB3 isolates from the heart showed greater infectivity and replication in heart endothelial cells than endothelial cells derived from liver or lung (Huber et al., 1990), thus confirming the essential role of host factors in developing specific diseases.

In line with this scenario, it is essential that, to gain access to secondary organs, viruses pass through the vascular endothelium by transcytosis or infection, or via infected circulating cells migrating into the target tissues. Endothelial cells derived from different organs show distinct susceptibility to CVB infections, and the behaviour against a viral challenge of endothelial cells in large vessels and microvessels may differ (Friedman et al., 1981; Huber et al., 1990; Conaldi et al., 1997; Zanone et al., 2003; Saijets et al., 2003). Endothelial cells expressing appropriate receptors would fail to act as effective barrier to infections, allowing viral particles to pass through, and replicate in, the vascular endothelium.

Human umbilical vein-derived endothelial cells have been shown to be persistently infected by different CVB strains (Flodstrom et al., 2000; Huber et al., 1990; Conaldi et al., 1997).

However, physiological and pathological events take place mainly at the level of the microvasculature. Using a dermal microvascular endothelial cell line, we have provided evidence that small vessel endothelial cells can harbour a persistent CVB viral infection (Zanone et al., 2003). All 3 CVB tested productively infected microvascular endothelial cells for up to 3 months without obvious cytolysis. A small proportion of the cells, approximately 10%, appeared to be involved in viral replication during chronic infection, suggesting that persistence is probably established through a mechanism of carrier-state culture, as proposed to explain CVB persistence in other cell types (Flodstrom et al., 2002; Greening et al., 2003). In addition, the infection increased production of proinflammatory cytokines IL-6 and IL-8, indicating endothelial cell activation by virus, and induced quantitative modification of adhesion molecule expression (ICAM-1, VCAM-1). These upregulation may influence the pattern of migration and extravasation of leucocytes in inflammation and immunity. These data add weight to the view that common CV infections are able to trigger complex pathophysiological processes, rather than simple cell lysis, as is becoming increasingly evident in clinical and experimental settings. These viruses can in fact persist for a considerable time in infected patients and cause chronic pathology or trigger immunopathological damage to infected and uninfected tissues (Muir et al., 1989; Stone, 1994). Furthermore, chronic infection of endothelial cells *in vivo* could provide better viral access to tissues underlying the endothelial layer and subsequent parenchymal cell infection.

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 69

The CVB infection upregulates in islet endothelial cells the expression of adhesion molecules and increases the production of proinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8 as well as IFN-, once again pointing to endothelial cell activation (Zanone et al., 2007), in line with studies that suggest that a low-grade inflammation may cause profound

In time course analyses, infected cells transiently upregulated expression of two major adhesion molecules, which may have *in vivo* functional consequences, enhancing cellular

Fig. 4. Schematic representation of the relationship between Coxsackievirus infection, islet

The highly fenestrated endothelial cells exhibit expression of classical endothelial markers, adhesion and co-stimulatory molecules, with the potential of being involved in autoreactive T cell adhesion, activation and transmigration in type 1 diabetes. They express specific markers, such as nephrin, whose functions at this site remain to be unravelled. Human islet endothelial cells express receptors and coreceptors for Coxsackievirus (such as HCAR, DAF, integrin and ICAM-1). These cells are potential target of an acute or persistent CVB infection, that activates the endothelium, upregulates expression of adhesion molecules, increases the production of proinflammatory cytokines and chemokines, and provides better viral access to tissues underlying the endothelial layer. Moreover, the increased production of IFN-α may enhance the expression of surface class I and II HLA molecules involved in viral and self antigen presentation, with selective recruitment and expansion of cytotoxic CD8+ T cells, which recognize infected endothelial and β cells, eventually damaging and killing them.

impairments of endothelial function (Hingonari et al., 2000; Charakida et al., 2005).

recruitment and leading to persistent tissue inflammation.

endothelial cells and β cells.

The mechanisms of CVB persistence is not clear. It is possible that the infected cells undergo cytolysis and release virions to infect more cells, thus maintaining a chronic infection of the culture without massive cell destruction. Alternatively, it could be hypothesized that the cells can cure themselves of viruses, e.g. by limiting production of cell host products required for viral replication, or by production of anti-viral mediators. Stability of the cell membrane could also be another important factor in the ability of infected cells to survive infection, without lysis. In previous studies, the distinct susceptibility of different cell types to long-term infection has been related to the production of interferons (Conaldi et al., 1997**;** Heim et al., 1992).

It has also been suggested that the persistent infection of cultured HUVEC may be due to down-regulation of viral receptors in infected cells. However, a study indicates that the expression of the specific CVB receptor, CAR, in these cells was not quantitatively altered by infection with CVB but rather by culture confluence (Huber et al., 1990).

Human islet endothelial cells have been more recently shown to express the specific human Coxsackievirus and Adenovirus receptor (HCAR) (Figure 3) and CVB co-receptors, such as DAF, integrins and ICAM-1, that have differentiated functions on virus attachment and entry into target cells (Zanone et al., 2007). Islet endothelial cells can harbour a persistent, low level infection by CVB, assessed as detection of VP1 capsid protein and release of infectious particles. The infection has no obvious effects on cell morphology or viability and can provide better viral access to the underlying islet tissue. Under experimental conditions to avoid massive cytolysis and possibly to mimic silent *in vivo* infection, as EV infections can cause little or no clinical symptoms, only a proportion of cells appeared to be involved in viral replication, suggesting a mechanism of carrier-state culture (Conaldi et al., 1997; Zanone et al., 2003).

Notably, the infection of islet enodothelial cells upregulates the expression of DAF, HCAR and integrin v3, in contrast to the behaviour of other macro- and microvascular endothelial cells lines, i.e. HUVEC, HMEC-1 and human aortic ECs. In fact, it has been shown that CVB infection downregulates DAF on HUVEC and HMEC-1 (Zanone et al., 2003), leaves HCAR expression unchanged on HUVEC (Carson et al., 1999), and downregulates HCAR expression and upregulates DAF expression on human aortic endothelial cells (Zanone et al., 2007).

This differential behaviour underlines the widely accepted heterogeneity of phenotype and function amongst endothelial cells derived from different vascular beds (Swerlick et al., 1992; Lidington et al., 1999), and may be relevant for the pathological sequelae of the infection. Despite detailed knowledge of the molecular structure and virus interaction of HCAR, its biological and possible pathogenic relevance are uncertain. HCAR belongs to the immunoglobulin superfamily and appears to have signalling functions (Bergelson et al., 1997; Noutsias et al., 2001; Fechner et al., 2003). Remarkably, CAR has been shown to be upregulated on affected cardiomyocytes in a rat model of experimental autoimmune myocarditis (Ito et al., 2000) and in human idiopatic dilated cardiomyopathy (Noutsias et al., 2001), for which EVs are the most frequently implicated pathogens (Feldman, 2000). CAR expression could therefore represent a key determinant of cardiac susceptibility to viral infections and have a pathogenic relevance in chronic cardiomyopathies. It has also been suggested that cell-to-cell contact modulates CAR-to-CAR interaction-based signals (Carson et al., 1999**;** Fechner et al., 2003).

The mechanisms of CVB persistence is not clear. It is possible that the infected cells undergo cytolysis and release virions to infect more cells, thus maintaining a chronic infection of the culture without massive cell destruction. Alternatively, it could be hypothesized that the cells can cure themselves of viruses, e.g. by limiting production of cell host products required for viral replication, or by production of anti-viral mediators. Stability of the cell membrane could also be another important factor in the ability of infected cells to survive infection, without lysis. In previous studies, the distinct susceptibility of different cell types to long-term infection has been related to the production of interferons (Conaldi et al., 1997**;**

It has also been suggested that the persistent infection of cultured HUVEC may be due to down-regulation of viral receptors in infected cells. However, a study indicates that the expression of the specific CVB receptor, CAR, in these cells was not quantitatively altered by

Human islet endothelial cells have been more recently shown to express the specific human Coxsackievirus and Adenovirus receptor (HCAR) (Figure 3) and CVB co-receptors, such as DAF, integrins and ICAM-1, that have differentiated functions on virus attachment and entry into target cells (Zanone et al., 2007). Islet endothelial cells can harbour a persistent, low level infection by CVB, assessed as detection of VP1 capsid protein and release of infectious particles. The infection has no obvious effects on cell morphology or viability and can provide better viral access to the underlying islet tissue. Under experimental conditions to avoid massive cytolysis and possibly to mimic silent *in vivo* infection, as EV infections can cause little or no clinical symptoms, only a proportion of cells appeared to be involved in viral replication, suggesting a mechanism of carrier-state culture (Conaldi et al., 1997;

Notably, the infection of islet enodothelial cells upregulates the expression of DAF, HCAR and integrin v3, in contrast to the behaviour of other macro- and microvascular endothelial cells lines, i.e. HUVEC, HMEC-1 and human aortic ECs. In fact, it has been shown that CVB infection downregulates DAF on HUVEC and HMEC-1 (Zanone et al., 2003), leaves HCAR expression unchanged on HUVEC (Carson et al., 1999), and downregulates HCAR expression and upregulates DAF expression on human aortic

This differential behaviour underlines the widely accepted heterogeneity of phenotype and function amongst endothelial cells derived from different vascular beds (Swerlick et al., 1992; Lidington et al., 1999), and may be relevant for the pathological sequelae of the infection. Despite detailed knowledge of the molecular structure and virus interaction of HCAR, its biological and possible pathogenic relevance are uncertain. HCAR belongs to the immunoglobulin superfamily and appears to have signalling functions (Bergelson et al., 1997; Noutsias et al., 2001; Fechner et al., 2003). Remarkably, CAR has been shown to be upregulated on affected cardiomyocytes in a rat model of experimental autoimmune myocarditis (Ito et al., 2000) and in human idiopatic dilated cardiomyopathy (Noutsias et al., 2001), for which EVs are the most frequently implicated pathogens (Feldman, 2000). CAR expression could therefore represent a key determinant of cardiac susceptibility to viral infections and have a pathogenic relevance in chronic cardiomyopathies. It has also been suggested that cell-to-cell contact modulates CAR-to-CAR interaction-based signals (Carson

infection with CVB but rather by culture confluence (Huber et al., 1990).

Heim et al., 1992).

Zanone et al., 2003).

endothelial cells (Zanone et al., 2007).

et al., 1999**;** Fechner et al., 2003).

The CVB infection upregulates in islet endothelial cells the expression of adhesion molecules and increases the production of proinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8 as well as IFN-, once again pointing to endothelial cell activation (Zanone et al., 2007), in line with studies that suggest that a low-grade inflammation may cause profound impairments of endothelial function (Hingonari et al., 2000; Charakida et al., 2005).

In time course analyses, infected cells transiently upregulated expression of two major adhesion molecules, which may have *in vivo* functional consequences, enhancing cellular recruitment and leading to persistent tissue inflammation.

Fig. 4. Schematic representation of the relationship between Coxsackievirus infection, islet endothelial cells and β cells.

The highly fenestrated endothelial cells exhibit expression of classical endothelial markers, adhesion and co-stimulatory molecules, with the potential of being involved in autoreactive T cell adhesion, activation and transmigration in type 1 diabetes. They express specific markers, such as nephrin, whose functions at this site remain to be unravelled. Human islet endothelial cells express receptors and coreceptors for Coxsackievirus (such as HCAR, DAF, integrin and ICAM-1). These cells are potential target of an acute or persistent CVB infection, that activates the endothelium, upregulates expression of adhesion molecules, increases the production of proinflammatory cytokines and chemokines, and provides better viral access to tissues underlying the endothelial layer. Moreover, the increased production of IFN-α may enhance the expression of surface class I and II HLA molecules involved in viral and self antigen presentation, with selective recruitment and expansion of cytotoxic CD8+ T cells, which recognize infected endothelial and β cells, eventually damaging and killing them.

Islet Endothelium: Role in Type 1 Diabetes and in Coxsackievirus Infections 71

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**6. References** 

Infection also increased the production of pro-inflammatory cytokines, IL-1, IL-6 and IL-8, further contributing to viral pathogenetic sequelae and to an indirect amplification of virus specific and non-specific responses. In this scenario, an exacerbated local inflammatory response secondary to viral infection represents an attempt to restrict virus replication, but it could promote chemoattraction and homing of circulating viral or, in susceptible individuals, islet antigen-specific T cells, in a bystander activation model (Horwitz et al., 1998). Cytokines may also be directly toxic to cells, leading to release of sequestered antigens, presentation by professional dendritic cells, and activation of autoantigen-specific T cells. Endothelial cells themselves may serve as antigen-presenting cells (Greening et al., 2003; Savinov et al., 2003).

The infection was also accompanied by increased production of IFN-, that has a role in initiation and maintenance of chronic CVB infection, as shown for other infected cell lines including islet cells, and in line with the extensive studies documenting abnormal localization of IFN- in the pancreas of type 1 diabetic patients (Chehadeh et al., 2000, Huber et al., 1990, Conaldi et al., 1997; Heim et al., 1992). As stated above, IFN- may be responsible for a viral expansion of non–specific T cell responses, including autoreactive T cells.

Again, in dilated cardiomyopathy inflammatory endothelial activation is present, and endothelial CAM expression correlates with the intramyocardial counterreceptor-bearing lymphocyte infiltrates (Noutsias et al., 1999; Seko et al., 1993). In this model, it is likely that endothelial cells are infected before cardiotropic viruses invade the myocardium (Klingel et al., 1992).

An increased production of lymphotactin RNA by the infected cells is also reported. Lymphotactin is a chemokine with the ability to chemoattract highly specifically CD4+ and CD8+ T cells and NK cells (Kennedy et al., 1995; Hedrick et al., 1997), with possible anti-viral and anti-tumor effects. An inappropriate T cell infiltration, drawn by lymphotactin, is present in other inflammatory conditions (Middel et al., 2001; Blaschke et al., 2003), and lymphotactin exposed on infected islet endothelial cells could, therefore, play a role in islet infiltration by T cells.

The endothelium infection may thus contribute to selective recruitment and expansion of subsets of leukocytes during inflammatory immune responses in type 1 diabetes. These findings add to a body of work that highlights the possible role of human EVs as environmental triggers that are capable of influencing the incidence of type 1 diabetes, the susceptibility of which to environmental influences is well established.

#### **5. Conclusion**

There is a body of work that highlights the possible role of human EVs as environmental triggers that are capable of influencing the incidence of type 1 diabetes, the susceptibility of which to environmental influences is well established (Hyöty & Taylor, 2002; Varela-Calvino & Peakman, 2003). Vascular endothelial cells have a major role in viral tropism and disease pathogenesis. Islet endothelium appears to be endowed of distinctive structural and functional features, and is acquiring a role in type 1 and type 2 diabetes (Figure 4). An interaction between islet endothelium and an EV, CVB in particular, infection might trigger a series of pro-inflammatory events that could be important in islet inflammation and possibly influence the development of autoimmune diabetes, through the initiation or acceleration of islet autoimmunity in susceptible individuals.

#### **6. References**

70 Type 1 Diabetes Complications

Infection also increased the production of pro-inflammatory cytokines, IL-1, IL-6 and IL-8, further contributing to viral pathogenetic sequelae and to an indirect amplification of virus specific and non-specific responses. In this scenario, an exacerbated local inflammatory response secondary to viral infection represents an attempt to restrict virus replication, but it could promote chemoattraction and homing of circulating viral or, in susceptible individuals, islet antigen-specific T cells, in a bystander activation model (Horwitz et al., 1998). Cytokines may also be directly toxic to cells, leading to release of sequestered antigens, presentation by professional dendritic cells, and activation of autoantigen-specific T cells. Endothelial cells themselves may serve as antigen-presenting cells (Greening et al.,

The infection was also accompanied by increased production of IFN-, that has a role in initiation and maintenance of chronic CVB infection, as shown for other infected cell lines including islet cells, and in line with the extensive studies documenting abnormal localization of IFN- in the pancreas of type 1 diabetic patients (Chehadeh et al., 2000, Huber et al., 1990, Conaldi et al., 1997; Heim et al., 1992). As stated above, IFN- may be responsible

Again, in dilated cardiomyopathy inflammatory endothelial activation is present, and endothelial CAM expression correlates with the intramyocardial counterreceptor-bearing lymphocyte infiltrates (Noutsias et al., 1999; Seko et al., 1993). In this model, it is likely that endothelial cells are infected before cardiotropic viruses invade the myocardium (Klingel et

An increased production of lymphotactin RNA by the infected cells is also reported. Lymphotactin is a chemokine with the ability to chemoattract highly specifically CD4+ and CD8+ T cells and NK cells (Kennedy et al., 1995; Hedrick et al., 1997), with possible anti-viral and anti-tumor effects. An inappropriate T cell infiltration, drawn by lymphotactin, is present in other inflammatory conditions (Middel et al., 2001; Blaschke et al., 2003), and lymphotactin exposed on infected islet endothelial cells could, therefore, play a role in islet

The endothelium infection may thus contribute to selective recruitment and expansion of subsets of leukocytes during inflammatory immune responses in type 1 diabetes. These findings add to a body of work that highlights the possible role of human EVs as environmental triggers that are capable of influencing the incidence of type 1 diabetes, the

There is a body of work that highlights the possible role of human EVs as environmental triggers that are capable of influencing the incidence of type 1 diabetes, the susceptibility of which to environmental influences is well established (Hyöty & Taylor, 2002; Varela-Calvino & Peakman, 2003). Vascular endothelial cells have a major role in viral tropism and disease pathogenesis. Islet endothelium appears to be endowed of distinctive structural and functional features, and is acquiring a role in type 1 and type 2 diabetes (Figure 4). An interaction between islet endothelium and an EV, CVB in particular, infection might trigger a series of pro-inflammatory events that could be important in islet inflammation and possibly influence the development of autoimmune diabetes, through the initiation or

susceptibility of which to environmental influences is well established.

acceleration of islet autoimmunity in susceptible individuals.

for a viral expansion of non–specific T cell responses, including autoreactive T cells.

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**4** 

*Italy* 

**Type 1 Diabetes Mellitus and Co-Morbidities** 

Co-morbid conditions are relatively frequent in Type 1 Diabetes Mellitus (T1DM). They can

Furthermore, these conditions could present very interesting ethiopatogenetic mechanisms.

Patients with type 1 diabetes (T1D) have an increased risk of other autoimmune conditions, such as autoimmune thyroid disease (AIT), celiac disease (CD), Addison's disease (AD) and vitiligo. These diseases are associated with organ-specific autoantibodies: AIT with thyroid peroxidase (TPO) and thyroglobulin autoantibodies (TG), CD with endomysial (EMA) and transglutaminase (TTG) autoantibodies, and AD with adrenal autoantibodies. Using these autoantibodies, organ-specific autoimmunity may be often detected before the development of clinical disease, in order to prevent significant morbidity related to unrecognized disease (Barker, 2006). The probable mechanism of these associations involves a shared genetic

The majority of autoimmune endocrinopathies, including T1D, are inherited as complex genetic traits. Multiple genetic and environmental factors interact with each other to confer susceptibility to these disorders. Genetic risk factors associated with T1D, ATD, CD and AD

The major histocompatibility complex (MHC) has been extensively studied in these diseases. HLA molecules are highly polymorphic and multiple different peptides can be presented to T cells by these molecules. In general it appears that the alleles associated with autoimmunity are not abnormal, but functional variants, that aid in determining specific targets of autoimmunity. The leading hypothesis is that these molecules contribute to determine risk through the peptides they bind and present to T-lymphocytes, either by influencing thymic selection, or peripheral antigen presentation. (Ide & Eisenbarth, 2003). HLA DR4 and DR3 are strongly associated with T1D and approximately 30-50% of patients are DR3/DR4 heterozygotes. The DR3/DR4 genotype confers the highest diabetes risk with a synergistic mode of action, followed by DR4 and DR3 homozygosity, respectively. The

severely affect clinical management of the disease, especially in pediatric age.

**1. Introduction** 

**2.1 Genetic associations** 

**2. Associated autoimmune conditions** 

background (Myśliwiec et al., 2008; Smyth et al., 2008).

include HLA genes and non-HLA genes.

**2.1.1 HLA genes** 

Adriana Franzese, Enza Mozzillo, Rosa Nugnes, Mariateresa Falco and Valentina Fattorusso *Department of Pediatrics, University Federico II of Naples* 


## **Type 1 Diabetes Mellitus and Co-Morbidities**

Adriana Franzese, Enza Mozzillo, Rosa Nugnes, Mariateresa Falco and Valentina Fattorusso *Department of Pediatrics, University Federico II of Naples Italy* 

#### **1. Introduction**

84 Type 1 Diabetes Complications

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#### **2. Associated autoimmune conditions**

#### **2.1 Genetic associations**

Patients with type 1 diabetes (T1D) have an increased risk of other autoimmune conditions, such as autoimmune thyroid disease (AIT), celiac disease (CD), Addison's disease (AD) and vitiligo. These diseases are associated with organ-specific autoantibodies: AIT with thyroid peroxidase (TPO) and thyroglobulin autoantibodies (TG), CD with endomysial (EMA) and transglutaminase (TTG) autoantibodies, and AD with adrenal autoantibodies. Using these autoantibodies, organ-specific autoimmunity may be often detected before the development of clinical disease, in order to prevent significant morbidity related to unrecognized disease (Barker, 2006). The probable mechanism of these associations involves a shared genetic background (Myśliwiec et al., 2008; Smyth et al., 2008).

The majority of autoimmune endocrinopathies, including T1D, are inherited as complex genetic traits. Multiple genetic and environmental factors interact with each other to confer susceptibility to these disorders. Genetic risk factors associated with T1D, ATD, CD and AD include HLA genes and non-HLA genes.

#### **2.1.1 HLA genes**

The major histocompatibility complex (MHC) has been extensively studied in these diseases. HLA molecules are highly polymorphic and multiple different peptides can be presented to T cells by these molecules. In general it appears that the alleles associated with autoimmunity are not abnormal, but functional variants, that aid in determining specific targets of autoimmunity. The leading hypothesis is that these molecules contribute to determine risk through the peptides they bind and present to T-lymphocytes, either by influencing thymic selection, or peripheral antigen presentation. (Ide & Eisenbarth, 2003).

HLA DR4 and DR3 are strongly associated with T1D and approximately 30-50% of patients are DR3/DR4 heterozygotes. The DR3/DR4 genotype confers the highest diabetes risk with a synergistic mode of action, followed by DR4 and DR3 homozygosity, respectively. The

Type 1 Diabetes Mellitus and Co-Morbidities 87

specific polymorphism, changing an arginine to tryptophan at position 620, has been associated with T1D (Bottini et al., 2004; Smyth et al., 2004) and also other autoimmune disorders, such as rheumatoid arthritis, systemic lupus erythematosus, Graves' disease and weakly with AD. The association with many autoimmune diseases suggests that this gene

Another non-HLA gene associated with T1D which has a generic role in susceptibility to autoimmunity is CTLA-4 (Cytotoxic T lymphocyte-associated antigen-4) (Vaidya & Pearce, 2004). CTLA-4 gene is an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders, including T1D (Ueda et al., 2003). The CTLA-4 gene, which is located on chromosome 2, encodes a costimulatory molecule that is expressed on the surface of activated T cells. It plays a critical role in the T-cell response to antigen presentation, binding costimulatory molecules and inhibiting T-cell activation. (Vaidya & Pearce, 2004). The inhibitory effect of CTLA-4 on T-cell activation has led the investigations into its role in different human autoimmune disorders. Polymorphisms within the CTLA-4 gene have been linked to AIT (Vaidya et al., 1999). CTLA-4 has also been linked to AD and more strongly to subjects affected by AD in association with T1D and AIT compared with AD alone (Vaidya et al., 2000). CTLA-4 has been associated with a wide range of other autoimmune disorders, including primary biliary cirrhosis, multiple sclerosis, CD and rheumatoid arthritis. These observations have suggested that CTLA-4 is a general autoimmune locus, and that the susceptibility polymorphisms within the gene may lead to general defects in the immune regulation, while other tissue-specific (e.g. insulin gene polymorphisms) or antigen-specific (e.g. MHC) genetic factors and environmental factors determine the involvement of particular

may be playing a role in susceptibility to autoimmunity in general.

target organs (Vaidya & Pearce, 2004).

**2.2 Type 1 diabetes and celiac disease 2.2.1 Prevalence and age at starting** 

Gene Associated diseases MIC-A T1D, CD, AD PTPN22 AIT, AD CTLA-4 T1D, AIT

Table 1. Non-HLA genes associated with T1D and other autoimmune diseases

Traditional studies, both in children and adults, have shown that CD occurs in patients with T1D with a prevalence that varies from 1,5 to 10 % compared with 0.5 % of the general population (Cronin & Shanahan, 2007; Vaarala, 2000). The mean age at diagnosis of classical CD is commonly around 2-3 years, while the mean age at diagnosis of DM1 is 7-8 years. The age at onset of T1D is younger in patients with the double disease than in those with only T1D (Kaspers et al., 2004). The risk of CD is negatively and independently associated with age at onset of diabetes, with an higher risk being seen in children age < 4 years than in those age > 9 years (Cerutti et al., 2004). In patients with T1D, diabetes is usually diagnosed first, CD precedes diabetes onset only in 10-25% (Cerutti et al., 2004; Valerio et al., 2002), while generally CD diagnosis in T1D patients occurs, trough the screening performed at diabetes onset, in 70-80% of patients with a median age >8 years. Some authors hypothized that in genetically susceptible patients one disease could predispose to another. Particularly, it has been suggested that untreated (latent or silent) CD could be an immunological trigger and induce diabetes and/or thyroid disorders due to gluten as a driving antigen (Pocecco &

HLA-DQ (particularly DQ 2 and DQ8) locus has been found to be the most important determinant of diabetes susceptibility. Approximately 90% of individuals with T1D have either DQ2 or DQ8, compared to 40% of the general population (Ide & Eisenbarth, 2003). So, the highest-risk human leukocyte antigen (HLA) genotype for T1D is DR3-DQ2, DR4-DQ8. DR3-DQ2 shows a strong association with CD; homozygosity for DR3-DQ2 in a population with T1D carries a 33% risk for the presence of TTG autoantibodies (Bao et al., 1999). Moreover, in families with multiple members affected with T1D and AIT, DR3-DQ2 has been linked with AIT and T1D (Levin et al, 2004). AD has been associated with the presence of a rare subtype of DR3-DQ2, DR4-DQ8 in which the DR4 subtype is DRB1\*0404. This subtype is found in less than 1% of the general population compared with 30% of the population with AD (Barker et al., 2005; Myhre et al., 2002; Yu et al., 1999). A schematic representation of the HLA region and its association with T1D is shown in the Figure 1.

(from Pugliese A. and Eisenbarth G.S., Chapter 7, Type 1 Diabetes: Molecular, Cellular, and Clinical Immunology, www.barbaradaviscenter.org)

Fig. 1. The HLA Region and T1D susceptibility. Schematic representation of the HLA region showing microsatellite markers, loci, and alleles associated with T1D susceptibility. Distances between loci are grossly approximated.

#### **2.1.2 Non-HLA genes**

Non-HLA genes are also involved in the predisposition to T1D and other autoimmune diseases, such as MIC-A, PTPN22, CTLA-4 (Barker, 2006).

Polymorphisms of MIC-A (MHC I-related gene A) have been associated with T1D, CD and AD. This gene encodes for a protein that is expressed in the thymus and interacts with the receptor NKG2D, which is important for thymic maturation of T cells (Hue et al., 2003). It is hypothesized that the loss of this interaction is a way in which immunological tolerance may be lost. NKG2D also regulates the priming of human naïve CD8+ T cells, providing an alternative explanation for associations with autoimmune diseases (Maasho et al., 2005).

The PTPN22 gene is expressed in T cells and encodes lymphoid tyrosine phosphatase (LYP). LYP appears to be important in the signal cascade downstream from the T-cell receptor. A

HLA-DQ (particularly DQ 2 and DQ8) locus has been found to be the most important determinant of diabetes susceptibility. Approximately 90% of individuals with T1D have either DQ2 or DQ8, compared to 40% of the general population (Ide & Eisenbarth, 2003). So, the highest-risk human leukocyte antigen (HLA) genotype for T1D is DR3-DQ2, DR4-DQ8. DR3-DQ2 shows a strong association with CD; homozygosity for DR3-DQ2 in a population with T1D carries a 33% risk for the presence of TTG autoantibodies (Bao et al., 1999). Moreover, in families with multiple members affected with T1D and AIT, DR3-DQ2 has been linked with AIT and T1D (Levin et al, 2004). AD has been associated with the presence of a rare subtype of DR3-DQ2, DR4-DQ8 in which the DR4 subtype is DRB1\*0404. This subtype is found in less than 1% of the general population compared with 30% of the population with AD (Barker et al., 2005; Myhre et al., 2002; Yu et al., 1999). A schematic representation of the HLA region and its association with T1D is shown in the Figure 1.

(from Pugliese A. and Eisenbarth G.S., Chapter 7, Type 1 Diabetes: Molecular, Cellular, and Clinical

showing microsatellite markers, loci, and alleles associated with T1D susceptibility.

Fig. 1. The HLA Region and T1D susceptibility. Schematic representation of the HLA region

Non-HLA genes are also involved in the predisposition to T1D and other autoimmune

Polymorphisms of MIC-A (MHC I-related gene A) have been associated with T1D, CD and AD. This gene encodes for a protein that is expressed in the thymus and interacts with the receptor NKG2D, which is important for thymic maturation of T cells (Hue et al., 2003). It is hypothesized that the loss of this interaction is a way in which immunological tolerance may be lost. NKG2D also regulates the priming of human naïve CD8+ T cells, providing an alternative explanation for associations with autoimmune diseases (Maasho et al., 2005). The PTPN22 gene is expressed in T cells and encodes lymphoid tyrosine phosphatase (LYP). LYP appears to be important in the signal cascade downstream from the T-cell receptor. A

Immunology, www.barbaradaviscenter.org)

**2.1.2 Non-HLA genes** 

Distances between loci are grossly approximated.

diseases, such as MIC-A, PTPN22, CTLA-4 (Barker, 2006).

specific polymorphism, changing an arginine to tryptophan at position 620, has been associated with T1D (Bottini et al., 2004; Smyth et al., 2004) and also other autoimmune disorders, such as rheumatoid arthritis, systemic lupus erythematosus, Graves' disease and weakly with AD. The association with many autoimmune diseases suggests that this gene may be playing a role in susceptibility to autoimmunity in general.

Another non-HLA gene associated with T1D which has a generic role in susceptibility to autoimmunity is CTLA-4 (Cytotoxic T lymphocyte-associated antigen-4) (Vaidya & Pearce, 2004). CTLA-4 gene is an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders, including T1D (Ueda et al., 2003). The CTLA-4 gene, which is located on chromosome 2, encodes a costimulatory molecule that is expressed on the surface of activated T cells. It plays a critical role in the T-cell response to antigen presentation, binding costimulatory molecules and inhibiting T-cell activation. (Vaidya & Pearce, 2004). The inhibitory effect of CTLA-4 on T-cell activation has led the investigations into its role in different human autoimmune disorders. Polymorphisms within the CTLA-4 gene have been linked to AIT (Vaidya et al., 1999). CTLA-4 has also been linked to AD and more strongly to subjects affected by AD in association with T1D and AIT compared with AD alone (Vaidya et al., 2000). CTLA-4 has been associated with a wide range of other autoimmune disorders, including primary biliary cirrhosis, multiple sclerosis, CD and rheumatoid arthritis. These observations have suggested that CTLA-4 is a general autoimmune locus, and that the susceptibility polymorphisms within the gene may lead to general defects in the immune regulation, while other tissue-specific (e.g. insulin gene polymorphisms) or antigen-specific (e.g. MHC) genetic factors and environmental factors determine the involvement of particular target organs (Vaidya & Pearce, 2004).


Table 1. Non-HLA genes associated with T1D and other autoimmune diseases

#### **2.2 Type 1 diabetes and celiac disease 2.2.1 Prevalence and age at starting**

Traditional studies, both in children and adults, have shown that CD occurs in patients with T1D with a prevalence that varies from 1,5 to 10 % compared with 0.5 % of the general population (Cronin & Shanahan, 2007; Vaarala, 2000). The mean age at diagnosis of classical CD is commonly around 2-3 years, while the mean age at diagnosis of DM1 is 7-8 years. The age at onset of T1D is younger in patients with the double disease than in those with only T1D (Kaspers et al., 2004). The risk of CD is negatively and independently associated with age at onset of diabetes, with an higher risk being seen in children age < 4 years than in those age > 9 years (Cerutti et al., 2004). In patients with T1D, diabetes is usually diagnosed first, CD precedes diabetes onset only in 10-25% (Cerutti et al., 2004; Valerio et al., 2002), while generally CD diagnosis in T1D patients occurs, trough the screening performed at diabetes onset, in 70-80% of patients with a median age >8 years. Some authors hypothized that in genetically susceptible patients one disease could predispose to another. Particularly, it has been suggested that untreated (latent or silent) CD could be an immunological trigger and induce diabetes and/or thyroid disorders due to gluten as a driving antigen (Pocecco &

Type 1 Diabetes Mellitus and Co-Morbidities 89

before the development of villous atrophy (Troncone et al., 1996). No definite consensus exists among experts about to treat pot-CD patients with GFD. No data are available on the natural history of these patients in the long term, nor on the risks they are exposed if left on normal gluten-containing diet, while a recent paper provided evidence that pot-CD children

Other studies have shown intestinal inflammation also in T1D patients without CD-related antibodies and structurally normal intestinal mucosa (Westerholm-Ormio et al., 2003). According to this, our group has observed a gluten-related inflammation either in rectal either in small bowel mucosa of children with T1D (Maglio et al,. 2009; Troncone et al., 2003). It can be speculated that gluten could be an optimal candidate to stimulate an abnormal innate immune reaction in intestinal mucosa due to its pro-inflammatory characteristics. It remains a crucial issue to estabilish to what the extented intestinal inflammation in T1D is gluten-dependent and whether it precedes the occurrence of the

Antithyroid antibodies have been shown to occur during the first years of diabetes in 11- 16.9% of individuals with T1D (Kordonouri et al., 2002). Long-term follow up suggests that as much as 30 % of patients with T1D develop AIT (Umpierrez et al., 2003). The range of prevalence of AIT in patients with T1D is unusually wide (3.4-50%) (Burek et al., 1990; Radetti et al., 1995). Thyroid antibodies are observed more frequently in girls than in boys,

Hyperthyroidism is less common than hypothyroidism in association with T1D (Umpierrez et al., 2003), but still more common than in the general population. It may be due to Grave's disease or the hyperthyroid phase of Hashimoto's thyroiditis. The presence of abnormal thyroid function related to AIT in the population with T1D has the potential to affect growth, weight gain, diabetes control, menstrual regularity, and overall well-being. In particular clinical features of hypothyroidism may include the presence of a painless goitre, increased weight gain, retarded growth, tiredness, lethargy, cold intolerance and bradycardia while diabetic control may not be significantly affected. Clinical features of hyperthyroidism may include unexplained difficulty in maintaining glycaemic control, weight loss without loss of appetite, agitation, tachycardia, tremor, heat intolerance, thyroid enlargement or characteristic eye signs. The treatment of hypothyroidism is based on replacement with oral L-thyroxine (T4) sufficient to normalise TSH levels and usually this allows regression of the goitre if present. The treatment of hyperthyroidism is based on the

There are studies showing worse diabetes control in patients with a second autoimmunity, including AIT and CD (Franzese et al., 2000; Iafusco et al., 1998). The factors responsible for the worsened control have not been completely elucidated. Thyroid dysfunction could be responsible of variations in absorption of carbohydrates and increased insulin resistance. There are studies showing similar diabetes control in patients with and without a second autoimmunity, in these studies thyroid autoimmunity does not lead to worsening of diabetic metabolic control in children with T1D (Kordonouri et al., 2002; Rami et al., 2005; Sumnik et al., 2006). The thyroid status is not different between diabetic patients with and

often emerging along during pubertal maturation (Kordonouri et al., 2005).

use of carbimazole and beta-adrenergic blocking drugs, if necessary.

may benefit from GFD treatment (Kurppa et al., 2010).

**2.3 Type 1 diabetes and autoimmune thyroid disease** 

**2.3.1 Prevalence and age at starting** 

**2.3.2 Clinical features and follow-up** 

disease.

Ventura, 1995). In accordance with this, the prevalence of autoimmune disorders in CD is closely related to age at diagnosis or, in other words, to the duration of exposure to gluten (Ventura et al., 1999) and thyroid-related antibodies tend to disappear during twelve months of gluten-free diet, like CD-related antibodies (Ventura et al., 2000). However, at present, it is unknown whether treatment of CD reduces the likelihood of developing autoimmune disorders, or changes their natural history and actually others found no correlation between duration of gluten exposure in adult CD and risk of autoimmune disorders (Viljamaa et al., 2005).

#### **2.2.2 Clinical features and follow up**

The classic presentation of CD describes symptoms related to gastrointestinal malabsorption and includes malnutrition, failure to thrive, diarrhea, anorexia, constipation, vomiting, abdominal distension, and pain. This predominance of gastrointestinal symptoms is more common in children younger than three years of age. Non-gastrointestinal or atypical symptoms of CD include short stature, pubertal delay, fatigue, vitamin deficiencies, and iron deficiency anemia and are more commonly observed in older children. The classical presentation of CD can occur in T1D patients, but many patients with CD and T1D are either asymptomatic (silent CD) or present with only mild symptoms (Holmes, 2001a; Ventura et al., 2000). Diagnosis of CD is regularly performed because screening protocols are universally recommended and performed. In patients with overt CD, identifying and treating CD with gluten free diet (GFD) surely confer benefit in reducing complications such as malabsorption, infertility, osteoporosis, poor nutrition, impaired growth and reducing long-term malignancy risks and mortality rates (Collin et al., 2002; Freemark & Levitsky, 2003; Rubio-Tapia et al., 2009), while no evidence exists on long-term morbidity in silent CD. Similarly, children with T1D with evidence of symptomatic CD benefit from GFD (Hansen et al., 2006; Saadah et al., 2004); in symptom-free cases the demonstrated benefit is limited to weight gain and bone mineral density (BMD) changes.( Artz et al., 2008; Rami et al., 2005; Simmons et al., 2007). Recently a 2-year prospective follow up study has provided additional evidence that only in some of the children with T1D and few classical symptoms of CD, identified by screening as being TG+ present, the demonstrated benefit of GFD is limited to weight gain and BMD changes (Simmons et al., 2011); moreover, other authors have reported an improved glycemic control in GFD-compliant celiac patients (Sanchez-Albisua et al., 2005). On the contrary, silent untreated CD has no obvious effect on metabolic control in T1D patients, but could negatively influence weight gain (Rami et al., 2005). In any case, the adherence to GFD by children with T1D has been reported generally below 50% (Acerini et al., 1998; Crone et al., 2003; Hansen et al., 2006; Saadah et al., 2004, Westman et al., 1999). The different viewpoints highlight the need of a long follow up of patients affected by T1D and asymptomatic CD to clarify the role of a GFD. Actually some authors argument against the need to stress GFD in nonsymptomatic T1D patients (Franzese et al., 2007; Van Koppen et al., 2009). However, the wide spectrum of CD include also subjects with positive celiac-related antibodies without diagnostic small-bowel mucosal villous atrophy. This condition is defined as potential celiac disease (pot-CD) (Holmes, 2001b; Paparo et al., 2005; Troncone et al., 1996). Some authors described that the prevalence of pot-CD among patients with T1D recruited from the majority of childhood diabetes care centers in Italy is 12.2 %, with an higher prevalence of females. The prevalence of pot-CD in the CD control population is 8.4 % (Franzese et al., 2011). Case reports and small follow-up studies indicated that only few pot-CD patients may suffer from CD-related symptoms

Ventura, 1995). In accordance with this, the prevalence of autoimmune disorders in CD is closely related to age at diagnosis or, in other words, to the duration of exposure to gluten (Ventura et al., 1999) and thyroid-related antibodies tend to disappear during twelve months of gluten-free diet, like CD-related antibodies (Ventura et al., 2000). However, at present, it is unknown whether treatment of CD reduces the likelihood of developing autoimmune disorders, or changes their natural history and actually others found no correlation between duration of gluten exposure in adult CD and risk of autoimmune

The classic presentation of CD describes symptoms related to gastrointestinal malabsorption and includes malnutrition, failure to thrive, diarrhea, anorexia, constipation, vomiting, abdominal distension, and pain. This predominance of gastrointestinal symptoms is more common in children younger than three years of age. Non-gastrointestinal or atypical symptoms of CD include short stature, pubertal delay, fatigue, vitamin deficiencies, and iron deficiency anemia and are more commonly observed in older children. The classical presentation of CD can occur in T1D patients, but many patients with CD and T1D are either asymptomatic (silent CD) or present with only mild symptoms (Holmes, 2001a; Ventura et al., 2000). Diagnosis of CD is regularly performed because screening protocols are universally recommended and performed. In patients with overt CD, identifying and treating CD with gluten free diet (GFD) surely confer benefit in reducing complications such as malabsorption, infertility, osteoporosis, poor nutrition, impaired growth and reducing long-term malignancy risks and mortality rates (Collin et al., 2002; Freemark & Levitsky, 2003; Rubio-Tapia et al., 2009), while no evidence exists on long-term morbidity in silent CD. Similarly, children with T1D with evidence of symptomatic CD benefit from GFD (Hansen et al., 2006; Saadah et al., 2004); in symptom-free cases the demonstrated benefit is limited to weight gain and bone mineral density (BMD) changes.( Artz et al., 2008; Rami et al., 2005; Simmons et al., 2007). Recently a 2-year prospective follow up study has provided additional evidence that only in some of the children with T1D and few classical symptoms of CD, identified by screening as being TG+ present, the demonstrated benefit of GFD is limited to weight gain and BMD changes (Simmons et al., 2011); moreover, other authors have reported an improved glycemic control in GFD-compliant celiac patients (Sanchez-Albisua et al., 2005). On the contrary, silent untreated CD has no obvious effect on metabolic control in T1D patients, but could negatively influence weight gain (Rami et al., 2005). In any case, the adherence to GFD by children with T1D has been reported generally below 50% (Acerini et al., 1998; Crone et al., 2003; Hansen et al., 2006; Saadah et al., 2004, Westman et al., 1999). The different viewpoints highlight the need of a long follow up of patients affected by T1D and asymptomatic CD to clarify the role of a GFD. Actually some authors argument against the need to stress GFD in nonsymptomatic T1D patients (Franzese et al., 2007; Van Koppen et al., 2009). However, the wide spectrum of CD include also subjects with positive celiac-related antibodies without diagnostic small-bowel mucosal villous atrophy. This condition is defined as potential celiac disease (pot-CD) (Holmes, 2001b; Paparo et al., 2005; Troncone et al., 1996). Some authors described that the prevalence of pot-CD among patients with T1D recruited from the majority of childhood diabetes care centers in Italy is 12.2 %, with an higher prevalence of females. The prevalence of pot-CD in the CD control population is 8.4 % (Franzese et al., 2011). Case reports and small follow-up studies indicated that only few pot-CD patients may suffer from CD-related symptoms

disorders (Viljamaa et al., 2005).

**2.2.2 Clinical features and follow up** 

before the development of villous atrophy (Troncone et al., 1996). No definite consensus exists among experts about to treat pot-CD patients with GFD. No data are available on the natural history of these patients in the long term, nor on the risks they are exposed if left on normal gluten-containing diet, while a recent paper provided evidence that pot-CD children may benefit from GFD treatment (Kurppa et al., 2010).

Other studies have shown intestinal inflammation also in T1D patients without CD-related antibodies and structurally normal intestinal mucosa (Westerholm-Ormio et al., 2003). According to this, our group has observed a gluten-related inflammation either in rectal either in small bowel mucosa of children with T1D (Maglio et al,. 2009; Troncone et al., 2003). It can be speculated that gluten could be an optimal candidate to stimulate an abnormal innate immune reaction in intestinal mucosa due to its pro-inflammatory characteristics. It remains a crucial issue to estabilish to what the extented intestinal inflammation in T1D is gluten-dependent and whether it precedes the occurrence of the disease.

#### **2.3 Type 1 diabetes and autoimmune thyroid disease 2.3.1 Prevalence and age at starting**

Antithyroid antibodies have been shown to occur during the first years of diabetes in 11- 16.9% of individuals with T1D (Kordonouri et al., 2002). Long-term follow up suggests that as much as 30 % of patients with T1D develop AIT (Umpierrez et al., 2003). The range of prevalence of AIT in patients with T1D is unusually wide (3.4-50%) (Burek et al., 1990; Radetti et al., 1995). Thyroid antibodies are observed more frequently in girls than in boys, often emerging along during pubertal maturation (Kordonouri et al., 2005).

#### **2.3.2 Clinical features and follow-up**

Hyperthyroidism is less common than hypothyroidism in association with T1D (Umpierrez et al., 2003), but still more common than in the general population. It may be due to Grave's disease or the hyperthyroid phase of Hashimoto's thyroiditis. The presence of abnormal thyroid function related to AIT in the population with T1D has the potential to affect growth, weight gain, diabetes control, menstrual regularity, and overall well-being. In particular clinical features of hypothyroidism may include the presence of a painless goitre, increased weight gain, retarded growth, tiredness, lethargy, cold intolerance and bradycardia while diabetic control may not be significantly affected. Clinical features of hyperthyroidism may include unexplained difficulty in maintaining glycaemic control, weight loss without loss of appetite, agitation, tachycardia, tremor, heat intolerance, thyroid enlargement or characteristic eye signs. The treatment of hypothyroidism is based on replacement with oral L-thyroxine (T4) sufficient to normalise TSH levels and usually this allows regression of the goitre if present. The treatment of hyperthyroidism is based on the use of carbimazole and beta-adrenergic blocking drugs, if necessary.

There are studies showing worse diabetes control in patients with a second autoimmunity, including AIT and CD (Franzese et al., 2000; Iafusco et al., 1998). The factors responsible for the worsened control have not been completely elucidated. Thyroid dysfunction could be responsible of variations in absorption of carbohydrates and increased insulin resistance. There are studies showing similar diabetes control in patients with and without a second autoimmunity, in these studies thyroid autoimmunity does not lead to worsening of diabetic metabolic control in children with T1D (Kordonouri et al., 2002; Rami et al., 2005; Sumnik et al., 2006). The thyroid status is not different between diabetic patients with and

Type 1 Diabetes Mellitus and Co-Morbidities 91

not usually cosmetically acceptable. Treatment is difficult and multiple therapies have been

The tendency of autoimmune diseases to aggregate is well known as clusters of autoimmune diseases within families and individuals. Analysis of susceptible genetic loci for the distinct autoimmune disease shows considerable overlap that suggests the possibility of shared pathways in their pathogenesis. Reports on the clustering of T1D, AIT, CD and rheumatoid arthritis (RA) in the same patient are very scarce. The major genetic predisposition to RA is contributed by variants of the class II HLA gene, HLA DRB1. In exploring the overlap between T1D, CD and RA, there is strong evidence that variation within the TAGAP gene is associated with all three autoimmune diseases. Relatively little is known about the TAGAP gene, which encodes a protein transiently expressed in activated T cells, suggesting that it may have a role in immune regulation. So the TAGAP gene, previously associated with both T1D and CD, is also associated with RA susceptibility. Interestingly a number of loci appear to be specific to one of the three diseases currently studied suggesting that they may play a role in determining the particular autoimmune phenotype at presentation (Eyre et al., 2010). The majority of the published case reports are girls. The predominance of females among the affected individuals may reflect that certain genes play role in the pathogenesis as gender-specific factors or the penetrance of multiple risk genes are enhanced in females. In most reported patients, diabetes is diagnosed first, thyroid autoimmunity and juvenile rheumatoid arthritis develop after a period of several

months to years. (Nagy et al., 2010; Pignata et al., 2000; Valerio et al., 2000).

CD28) to the development of T1D, SLE or other autoimmune diseases.

The association of T1D with Systemic Lupus Erythematosus (SLE) and Sclerodermia is rare but reported in literature (Inuo et al., 2009, Zeglaoui et al., 2010). Some authors found a significant association between DQ2 allele and the presence of anti-SSA antibodies, while others described an association between CD and the presence of A1B8DR3 haplotype, which seems to be frequent in SLE and in Sclerodermia (Black et al., 1983; Mark, 2000; Sollid & Thorsby, 1993). In human, the CTLA-4 and PD-1 genes significantly contributed to the development of various autoimmune diseases in different genetic backgrounds (Inuo et al., 2009). ). It has been suggest the involvement of CTLA-4 and PD-1 (inhibitor receptors of

Juvenile sclerodermia is present in 3% of sclerodermia cases, SLE in children is present in 9% of cases of SLE; one case of a 15 years girl with CD and SLE and Sclerodermia has been

Since Type 1 Diabetes is associated with the presence of additional autoimmune disease, such as AIT, CD and AD, which are associated with the production of organ-specific antibodies, it is possible to screen patients with T1D by means of these ones. However, only a subset of the subjects with organ-specific antibodies develops clinical disease. The frequency of screening and follow up of patients with positive antibodies remain controversial. The current American Diabetes Association (ADA) recommendations are to

**2.6.2 Sclerodermia, systemic lupus erythematosus** 

**2.7 Screening for associated autoimmune disorders** 

reported (Zeglaoui et al., 2010).

tried with little success . (Ho et al., 2011)

**2.6.1 Rheumatoid arthritis** 

**2.6 Type 1 diabetes and collagenopathies** 

without CD: children with both T1D and CD do not have an increased risk of AIT development compared to diabetic patients without CD (Sumnik et al., 2006).

#### **2.4 Type 1 diabetes, Addison disease and polyglandular syndromes 2.4.1 Prevalence and age at starting**

Addison's disease (AD) affects approximately 1 in 10,000 of the general population. The autoimmune process resulting in AD can be identified by the detection of autoantibodies against the adrenal cortex (Anderson et al., 1957; Lovas & Husebye, 2002). Up 2 % of patients with T1D have antiadrenal autoantibodies (De Block et al.; 2001, Falorni et al., 1997; Peterson et al., 1997).

AD is occasionally associated with T1D in the Autoimmune Polyglandular Syndromes (APS I and II). APS I, also known as autoimmune polyendocrinopathy candidiasis ectodermal dysplasia (APECED), is a rare polyendocrine autoimmune disease caused by mutations of the autoimmune regulator gene (AIRE) on chromosome 21q22.3 (Aaltonen et al., 1994; Ahonen et al., 1990), which is characterized by the association of mucocutaneous candidiasis, adrenal insufficiency, and/or hypoparathyroidism. Follow-up of subjects with this disorder has revealed that many organ systems may be involved in the autoimmune process including the pancreatic β cell. Approximately 20% of subjects with APS-I develop T1D (Barker, 2006). APS II is more common in adults, but is also observed in children in association with autoimmune thyroiditis (Dittmar & Kahaly, 2003). Other less common disorders observed in APSII include Addison's disease, hypogonadism, vitiligo, alopecia, pernicious anemia and myasthenia gravis. Another rare disorder associated with T1D in early childhood is the Immunodysregulation Polyendocrinopathy X-linked Syndrome (IPEX), which is characterized also by severe enteropathy and autoimmune symptoms due to a clear genetic defect (FOX-P3) (Chatila et al., 2000). FOX-P3 is expressed in CD4+CD25+ regulatory T cells; mutations result in the inability to generate these regulatory T cells resulting in multiorgan autoimmunity (Barker, 2006).

#### **2.4.2 Clinical features and follow-up**

The condition of AD is suspected by the clinical picture of frequent hypoglycaemia, unexplained decrease in insulin requirements, increased skin pigmentation, lassitude, weight loss, hyponatraemia and hyperkalaemia. The diagnosis is based on the demonstration of a low cortisol, especially in response to ACTH test. Treatment with a glucocorticoid is urgent and life-threatening. In some cases the therapy has to be supplemented with a mineralocorticoid. In asymptomatic children with positive adrenal antibodies, detected on routine screening, a rising ACTH level suggests a failing adrenal cortex and the development of primary adrenal insufficiency (Kordonouri et al., 2009). There are no current recommendations for screening of adrenal autoimmunity.

#### **2.5 Type 1 diabetes and vitiligo**

Vitiligo is an acquired pigmentary disorder characterized by a loss of melanocytes resulting in white spots or leukoderma. The association of vitiligo with other autoimmune disorders, including thyroid disease, adrenal insufficiency, gonadal dysfunction, polyendocrine failure, diabetes mellitus, pernicious anemia, myasthenia gravis and alopecia areata, has been well documented (Bystryn, 1997; Handa & Dogra, 2003). This condition is present in about 6% of diabetic children (Hanas et al., 2009). Spontaneous re-pigmentation is rare and

without CD: children with both T1D and CD do not have an increased risk of AIT

Addison's disease (AD) affects approximately 1 in 10,000 of the general population. The autoimmune process resulting in AD can be identified by the detection of autoantibodies against the adrenal cortex (Anderson et al., 1957; Lovas & Husebye, 2002). Up 2 % of patients with T1D have antiadrenal autoantibodies (De Block et al.; 2001, Falorni et al., 1997;

AD is occasionally associated with T1D in the Autoimmune Polyglandular Syndromes (APS I and II). APS I, also known as autoimmune polyendocrinopathy candidiasis ectodermal dysplasia (APECED), is a rare polyendocrine autoimmune disease caused by mutations of the autoimmune regulator gene (AIRE) on chromosome 21q22.3 (Aaltonen et al., 1994; Ahonen et al., 1990), which is characterized by the association of mucocutaneous candidiasis, adrenal insufficiency, and/or hypoparathyroidism. Follow-up of subjects with this disorder has revealed that many organ systems may be involved in the autoimmune process including the pancreatic β cell. Approximately 20% of subjects with APS-I develop T1D (Barker, 2006). APS II is more common in adults, but is also observed in children in association with autoimmune thyroiditis (Dittmar & Kahaly, 2003). Other less common disorders observed in APSII include Addison's disease, hypogonadism, vitiligo, alopecia, pernicious anemia and myasthenia gravis. Another rare disorder associated with T1D in early childhood is the Immunodysregulation Polyendocrinopathy X-linked Syndrome (IPEX), which is characterized also by severe enteropathy and autoimmune symptoms due to a clear genetic defect (FOX-P3) (Chatila et al., 2000). FOX-P3 is expressed in CD4+CD25+ regulatory T cells; mutations result in the inability to generate these regulatory T cells

The condition of AD is suspected by the clinical picture of frequent hypoglycaemia, unexplained decrease in insulin requirements, increased skin pigmentation, lassitude, weight loss, hyponatraemia and hyperkalaemia. The diagnosis is based on the demonstration of a low cortisol, especially in response to ACTH test. Treatment with a glucocorticoid is urgent and life-threatening. In some cases the therapy has to be supplemented with a mineralocorticoid. In asymptomatic children with positive adrenal antibodies, detected on routine screening, a rising ACTH level suggests a failing adrenal cortex and the development of primary adrenal insufficiency (Kordonouri et al., 2009). There

Vitiligo is an acquired pigmentary disorder characterized by a loss of melanocytes resulting in white spots or leukoderma. The association of vitiligo with other autoimmune disorders, including thyroid disease, adrenal insufficiency, gonadal dysfunction, polyendocrine failure, diabetes mellitus, pernicious anemia, myasthenia gravis and alopecia areata, has been well documented (Bystryn, 1997; Handa & Dogra, 2003). This condition is present in about 6% of diabetic children (Hanas et al., 2009). Spontaneous re-pigmentation is rare and

are no current recommendations for screening of adrenal autoimmunity.

development compared to diabetic patients without CD (Sumnik et al., 2006).

**2.4 Type 1 diabetes, Addison disease and polyglandular syndromes** 

**2.4.1 Prevalence and age at starting** 

resulting in multiorgan autoimmunity (Barker, 2006).

**2.4.2 Clinical features and follow-up** 

**2.5 Type 1 diabetes and vitiligo** 

Peterson et al., 1997).

not usually cosmetically acceptable. Treatment is difficult and multiple therapies have been tried with little success . (Ho et al., 2011)

#### **2.6 Type 1 diabetes and collagenopathies 2.6.1 Rheumatoid arthritis**

The tendency of autoimmune diseases to aggregate is well known as clusters of autoimmune diseases within families and individuals. Analysis of susceptible genetic loci for the distinct autoimmune disease shows considerable overlap that suggests the possibility of shared pathways in their pathogenesis. Reports on the clustering of T1D, AIT, CD and rheumatoid arthritis (RA) in the same patient are very scarce. The major genetic predisposition to RA is contributed by variants of the class II HLA gene, HLA DRB1. In exploring the overlap between T1D, CD and RA, there is strong evidence that variation within the TAGAP gene is associated with all three autoimmune diseases. Relatively little is known about the TAGAP gene, which encodes a protein transiently expressed in activated T cells, suggesting that it may have a role in immune regulation. So the TAGAP gene, previously associated with both T1D and CD, is also associated with RA susceptibility. Interestingly a number of loci appear to be specific to one of the three diseases currently studied suggesting that they may play a role in determining the particular autoimmune phenotype at presentation (Eyre et al., 2010). The majority of the published case reports are girls. The predominance of females among the affected individuals may reflect that certain genes play role in the pathogenesis as gender-specific factors or the penetrance of multiple risk genes are enhanced in females. In most reported patients, diabetes is diagnosed first, thyroid autoimmunity and juvenile rheumatoid arthritis develop after a period of several months to years. (Nagy et al., 2010; Pignata et al., 2000; Valerio et al., 2000).

#### **2.6.2 Sclerodermia, systemic lupus erythematosus**

The association of T1D with Systemic Lupus Erythematosus (SLE) and Sclerodermia is rare but reported in literature (Inuo et al., 2009, Zeglaoui et al., 2010). Some authors found a significant association between DQ2 allele and the presence of anti-SSA antibodies, while others described an association between CD and the presence of A1B8DR3 haplotype, which seems to be frequent in SLE and in Sclerodermia (Black et al., 1983; Mark, 2000; Sollid & Thorsby, 1993). In human, the CTLA-4 and PD-1 genes significantly contributed to the development of various autoimmune diseases in different genetic backgrounds (Inuo et al., 2009). ). It has been suggest the involvement of CTLA-4 and PD-1 (inhibitor receptors of CD28) to the development of T1D, SLE or other autoimmune diseases.

Juvenile sclerodermia is present in 3% of sclerodermia cases, SLE in children is present in 9% of cases of SLE; one case of a 15 years girl with CD and SLE and Sclerodermia has been reported (Zeglaoui et al., 2010).

#### **2.7 Screening for associated autoimmune disorders**

Since Type 1 Diabetes is associated with the presence of additional autoimmune disease, such as AIT, CD and AD, which are associated with the production of organ-specific antibodies, it is possible to screen patients with T1D by means of these ones. However, only a subset of the subjects with organ-specific antibodies develops clinical disease. The frequency of screening and follow up of patients with positive antibodies remain controversial. The current American Diabetes Association (ADA) recommendations are to

Type 1 Diabetes Mellitus and Co-Morbidities 93

needed to support normal serum concentrations of IGFs and IGFBPs and indirectly to promote growth. Poor gain of height and weight, hepatomegaly, non alcoholic steatosis hepatis (NASH) and late pubertal development might be seen in children with persistently poorly controlled diabetes. Similar to healthy adolescents, the pubertal growth spurt represents the most critical phase for linear growth and final height in children with T1D. The pubertal phase is characteristically associated with reduction in insulin sensitivity, which is known to be more severe in patients with T1D, and might negatively influence growth and height gain (Chiarelli et al., 2004). Although the chronological age at onset of puberty and the duration of the pubertal growth spurt is not significantly different between subjects with T1D and healthy adolescents, several studies have shown a blunted pubertal growth spurt which seems to be associated with a reduced peak of height velocity SDS (Vanelli et al., 1992). Although loss of height from the onset of diabetes has been widely reported, an impaired final height has not been reported in children with T1D. In fact, while some studies, especially those performed in the pre-intensive insulin therapy era, showed an impaired final height in children with diabetes (Penfold et al., 1995), more recent studies

The Diabetes Control and Complications Trial (DCCT) and other studies have reported increased weight gain as a side effect of intensive insulin therapy with improved metabolic control (DCCT Research Group, 1993). As obesity is a modifiable cardiovascular risk factor, careful monitoring and management of weight gain should be emphasised in diabetes care.

Monitoring of growth and development and the use of percentile charts is a crucial element in the care of children and adolescents with diabetes. Improvements in diabetes care and management and especially newer insulin schedules based on multiple daily injections or insulin pumps have led to a reduction in diabetic complications and seem to ameliorate growth in children with T1D. Start an intensive insulin regimen since the onset of diabetes might prevent the induction of abnormalities of the GH–IGF-I–IGFBP-3 axis potentially achieving near-normal portal insulin concentrations and thereby leading to normal IGF-I

Eating disorders (EDs) are a significant health problem for many children and adolescents with T1D similar to that observed in other high risk groups, such as competitive athletes, models and ballet dancers. EDs and subclinical disordered eating behaviors (DEBs) have been described in adolescents with T1D with a higher prevalence than in a non-diabetic population. The start of insulin treatment and the need to comply with dietary recommendations both lead to weight gain, which in turn leads to body dissatisfaction and a drive for thinness. Since the dietary restraint usually requires ignoring internal cues of hunger and satiety, it has been suggested that it may be a triggering factor in the development of cycles of binge eating and purging. The concurrence of T1D and EDs can greatly increase morbidity and mortality. In diabetic subjects, EDs are associated with insulin omission for weight loss and impaired metabolic control. On the contrary, in a five year longitudinal study, the expected relationship between ED and poor metabolic control was not evident, although there was a trend for higher haemoglobin A1c in individuals with an EDs (Colton et al., 2007). This offers hope that early interventions might prevent the worsening metabolic control that is often associated with EDs. In addition subclinical DEBs

and IGFBP-3 levels and physiological growth in children and adolescents with T1D.

**3.2 Type 1 diabetes and eating disorders** 

show a normal or only slightly reduced final height (Salerno et al., 1997).

Girls seem to be more at risk of overweight and as well of eating disorders.

screen for CD-associated antibodies at diagnosis of T1D and in presence of symptoms. The International Society of Pediatric Adolescent Diabetes (ISPAD) recommends to screen for CD at the time of diagnosis, annually for the first five years and every second year thereafter. More frequent assessment is indicated if the clinical situation suggests the possibility of CD or the child has a first-degree relative with CD. Respect to the screening for thyroid disease, current recommendations from the ADA are for screening TSH after stabilization at onset of diabetes, with symptoms of hypo- or hyperthyroidism, and every 1– 2 yr thereafter. ISPAD recommends to screen by circulating TSH and antibodies at the diagnosis of T1D and, thereafter, every second year in asymptomatic individuals without goitre or in the absence of thyroid autoantibodies. More frequent assessment is indicated otherwise, subjects with positive TPO autoantibodies and normal thyroid function are screened on a more frequent basis (every 6 months to 1 yr). There are no current recommendations for screening of adrenal autoimmunity (Barker, 2006). Authors observed that the prevalence of adrenal antibodies in diabetic patients with thyroid antibodies compared with those without thyroid antibodies is increased (5,1 vs 0,6%) (Riley et al., 1981). It is possible conclude that routine screening for AD in children with T1D is not warranted unless there is a strong clinical suspicion or family history of AD (Marks et al., 2003)


Table 2. Autoimmune diseases associated with T1D, recommended systems and frequency of the screening

#### **3. Associated non-autoimmune conditions**

#### **3.1 Type 1 diabetes and growth**

Type 1 diabetes and other chronic diseases are well known to adversely affect linear growth and pubertal development, this can include a wide spectrum of different conditions, from poor gain of weight to Mauriac Syndrome (MS); MS classically involves hepatomegaly, growth impairment, and Cushingoid features in poorly controlled diabetic patients. Although MS, the most important expression of growth alteration due to severe insulin deficiency in diabetic patients, is now rare, impaired growth in children with T1D is still reported. This is particularly true in patients with poor metabolic control (Chiarelli et al., 2004; Franzese et al., 2001). Some studies report that poorly controlled patients show a decrease in height standard deviation score over the next few years, while better controlled patients maintain their height advantage (Gunczler & Lanes, 1999; Holl et al., 1998).

Longitudinal bone growth is a complex phenomenon involving a multitude of regulatory mechanisms strongly influenced by growth hormone (GH) (Chiarelli et al., 2004) and by the interaction between insulin-like growth factors (IGF-I and IGF-II), that circulate bounded to specific insulin-like growth factor binding proteins (IGFBPs). IGFBP-3, the major circulating binding protein during post-natal life, is GH-dependent. Insulin is an important regulator of this complex. In fact, adequate insulin secretion and normal portal insulin concentrations are

screen for CD-associated antibodies at diagnosis of T1D and in presence of symptoms. The International Society of Pediatric Adolescent Diabetes (ISPAD) recommends to screen for CD at the time of diagnosis, annually for the first five years and every second year thereafter. More frequent assessment is indicated if the clinical situation suggests the possibility of CD or the child has a first-degree relative with CD. Respect to the screening for thyroid disease, current recommendations from the ADA are for screening TSH after stabilization at onset of diabetes, with symptoms of hypo- or hyperthyroidism, and every 1– 2 yr thereafter. ISPAD recommends to screen by circulating TSH and antibodies at the diagnosis of T1D and, thereafter, every second year in asymptomatic individuals without goitre or in the absence of thyroid autoantibodies. More frequent assessment is indicated otherwise, subjects with positive TPO autoantibodies and normal thyroid function are screened on a more frequent basis (every 6 months to 1 yr). There are no current recommendations for screening of adrenal autoimmunity (Barker, 2006). Authors observed that the prevalence of adrenal antibodies in diabetic patients with thyroid antibodies compared with those without thyroid antibodies is increased (5,1 vs 0,6%) (Riley et al., 1981). It is possible conclude that routine screening for AD in children with T1D is not warranted unless there is a strong clinical suspicion or family history of AD (Marks et al.,

Celiac disease Transglutaminase antibodies Yearly Thyroiditis TSH, FT4, thyroid antibodies Yearly

Collagenopathies Specific auto-antibodies No screening

Table 2. Autoimmune diseases associated with T1D, recommended systems and frequency

Type 1 diabetes and other chronic diseases are well known to adversely affect linear growth and pubertal development, this can include a wide spectrum of different conditions, from poor gain of weight to Mauriac Syndrome (MS); MS classically involves hepatomegaly, growth impairment, and Cushingoid features in poorly controlled diabetic patients. Although MS, the most important expression of growth alteration due to severe insulin deficiency in diabetic patients, is now rare, impaired growth in children with T1D is still reported. This is particularly true in patients with poor metabolic control (Chiarelli et al., 2004; Franzese et al., 2001). Some studies report that poorly controlled patients show a decrease in height standard deviation score over the next few years, while better controlled

patients maintain their height advantage (Gunczler & Lanes, 1999; Holl et al., 1998).

Longitudinal bone growth is a complex phenomenon involving a multitude of regulatory mechanisms strongly influenced by growth hormone (GH) (Chiarelli et al., 2004) and by the interaction between insulin-like growth factors (IGF-I and IGF-II), that circulate bounded to specific insulin-like growth factor binding proteins (IGFBPs). IGFBP-3, the major circulating binding protein during post-natal life, is GH-dependent. Insulin is an important regulator of this complex. In fact, adequate insulin secretion and normal portal insulin concentrations are

antibodies Screening if AD in family

Addison disease Cortisolemia, adrenal

**3. Associated non-autoimmune conditions** 

**3.1 Type 1 diabetes and growth** 

2003)

of the screening

needed to support normal serum concentrations of IGFs and IGFBPs and indirectly to promote growth. Poor gain of height and weight, hepatomegaly, non alcoholic steatosis hepatis (NASH) and late pubertal development might be seen in children with persistently poorly controlled diabetes. Similar to healthy adolescents, the pubertal growth spurt represents the most critical phase for linear growth and final height in children with T1D. The pubertal phase is characteristically associated with reduction in insulin sensitivity, which is known to be more severe in patients with T1D, and might negatively influence growth and height gain (Chiarelli et al., 2004). Although the chronological age at onset of puberty and the duration of the pubertal growth spurt is not significantly different between subjects with T1D and healthy adolescents, several studies have shown a blunted pubertal growth spurt which seems to be associated with a reduced peak of height velocity SDS (Vanelli et al., 1992). Although loss of height from the onset of diabetes has been widely reported, an impaired final height has not been reported in children with T1D. In fact, while some studies, especially those performed in the pre-intensive insulin therapy era, showed an impaired final height in children with diabetes (Penfold et al., 1995), more recent studies show a normal or only slightly reduced final height (Salerno et al., 1997).

The Diabetes Control and Complications Trial (DCCT) and other studies have reported increased weight gain as a side effect of intensive insulin therapy with improved metabolic control (DCCT Research Group, 1993). As obesity is a modifiable cardiovascular risk factor, careful monitoring and management of weight gain should be emphasised in diabetes care. Girls seem to be more at risk of overweight and as well of eating disorders.

Monitoring of growth and development and the use of percentile charts is a crucial element in the care of children and adolescents with diabetes. Improvements in diabetes care and management and especially newer insulin schedules based on multiple daily injections or insulin pumps have led to a reduction in diabetic complications and seem to ameliorate growth in children with T1D. Start an intensive insulin regimen since the onset of diabetes might prevent the induction of abnormalities of the GH–IGF-I–IGFBP-3 axis potentially achieving near-normal portal insulin concentrations and thereby leading to normal IGF-I and IGFBP-3 levels and physiological growth in children and adolescents with T1D.

#### **3.2 Type 1 diabetes and eating disorders**

Eating disorders (EDs) are a significant health problem for many children and adolescents with T1D similar to that observed in other high risk groups, such as competitive athletes, models and ballet dancers. EDs and subclinical disordered eating behaviors (DEBs) have been described in adolescents with T1D with a higher prevalence than in a non-diabetic population. The start of insulin treatment and the need to comply with dietary recommendations both lead to weight gain, which in turn leads to body dissatisfaction and a drive for thinness. Since the dietary restraint usually requires ignoring internal cues of hunger and satiety, it has been suggested that it may be a triggering factor in the development of cycles of binge eating and purging. The concurrence of T1D and EDs can greatly increase morbidity and mortality. In diabetic subjects, EDs are associated with insulin omission for weight loss and impaired metabolic control. On the contrary, in a five year longitudinal study, the expected relationship between ED and poor metabolic control was not evident, although there was a trend for higher haemoglobin A1c in individuals with an EDs (Colton et al., 2007). This offers hope that early interventions might prevent the worsening metabolic control that is often associated with EDs. In addition subclinical DEBs

Type 1 Diabetes Mellitus and Co-Morbidities 95

steroids, aspirin, cyclosporin, mycophenolate, becaplermin, excision and grafting, laser surgery, hyperbaric oxygen, topical granulocytemacrophage colony-stimulating factor and photochemotherapy with topical PUVA (Hanas et al., 2009). A recent study suggests the use of TNF inhibitors in selected patients for treatment of NBL (ulcerative forms) unresponsive to prior conventional therapies (Suárez-Amor et al., 2010). NBL in children can be hard to manage and may be associated with a long-term risk of malignant transformation to squamous cell carcinoma. Systemic therapies, such as corticosteroids and azathioprine are immunosuppressive and immunomodulatory and could facilitate malignant transformation (Beattie et al., 2006). Therefore, although NBL is not clearly related to poor metabolic control, we believe that the diabetic control may also be useful. Effective primary prevention strategies and new treatment options are needed to adequately control the disease and its

Children and adolescents with T1D can show several impairment of bone metabolism and structure, resulting in a higher risk of decreased bone mass and its related complications later in life. Consequently an assessment of quality of the bone through non-invasive methods (phalangeal ultrasonography) seems to be opportune in the care of diabetic patients, specially the ones with clusters of autoimmune diseases to define a possible

Bone impairment in multiple autoimmune diseases might be considered not only a complication due to endocrine or nutritional mechanisms, but also a consequence of an

Alterations of bone mineral density (BMD) are especially observed when diabetes is associated with CD and/or AIT. Bone loss, described in patients with T1D, AIT or CD is usually viewed as a complication of these diseases and is related to duration of diabetes and quality of metabolic control. The exact mechanisms accounting for bone loss in these diseases have been variably explained by metabolic derangements due to the impaired hormonal function in T1D or AIT (McCabe, 2007), or calcium malabsorption and secondary hyperparathyroidism in untreated CD patients (Selby et al., 1999). Alterations of homeostatic mechanisms might explain an imbalance of osteoclast activity leading to

Bone remodeling involves complex interactions between osteoclasts and other cells in their microenvironment (marrow stromal cells, osteoblasts, macrophages, T-lymphocytes and marrow cells) (Kollet et al., 2007; Teitelbaum, 2007). Besides their role in calcium mobilization from bone and initiation of bone remodeling, osteoclasts are now considered as the innate immune cells in the bone, since they are able to produce and respond to cytokines and chemokines. Some authors found altered levels of plasma Osteoprotegerin (OPG) in children with T1D. Osteoprotegerin is a circulating secretory glycoprotein and is a member of the tumor necrosis factor receptor (TNFR) family. It works as a decoy receptor for the cytokine receptor activator of NFkB ligand (RANKL). RANKL and OPG are a key agonist/antagonist cytokine system: RANKL increases the pool of active osteoclasts thus

progression.

**3.4 Osteopenia** 

involvement of the bone (Lombardi et al.,2010).

osteopenia (Lombardi et al.,2010; Wu et al., 2008).

immunoregulatory imbalance.

**3.4.1 Metabolic causes** 

**3.4.2 Immune causes** 

among youth with T1D have been associated with increased risk of poor metabolic control and increased prevalence of microvascular complications such as retinopathy and nephropathy (Rydall et al., 1997). Some studies have examined the prevalence of EDs and DEBs in youth with T1D. Prevalence rates vary considerably from study to study possibly due to differences in sample, screening tools, and data collection methods. In a multi-site, cross sectional case-control study, the prevalence of ED meeting DSM-IV diagnostic criteria was about 10% and that of their sub-threshold variants about 14%: both were about twice as common in adolescent females with T1D than in their non-diabetic peers. (Jones et al., 2000). However there are also rare cases in childhood (Franzese et al., 2002a).

#### **3.2.1 Management**

Nutritional treatment is one of the main difficulties in managing diabetes in the young. Diabetes clinicians should be aware of the potential warning signs in an adolescent with diabetes as well as assessment and treatment options for eating disorders with concomitant T1D. Clinical approaches should focuse on normalizing eating behaviour and enhancing self-esteem based on personal attributes unrelated to weight and eating, with a low threshold for referral for specialized EDs services (Colton et al., 2007). A multidisciplinary team, composed by clinicians, psychologist/psychiatric, dietitian/nutrition therapist, especially one with a background in EDs, is opportune to identify and treat unhealthy EDs and DEBs in T1D. Treatment for adolescents with T1D should include both diabetes management treatment and mental health treatment. The diabetes team and the mental health team have separate responsibilities but work collaboratively to address disordered eating in patients with T1D. Treatment begins with emphasis on nutritional rehabilitation, weight restoration, and adequate diabetes control. Psychotherapy should begin immediately for the patient and family (S.D. Kelly et al., 2005).

#### **3.3 Necrobiosis lipoidica diabeticorum**

Necrobiosis lipoidica diabeticorum (NBL) is an infrequent skin affection in pediatric age. The etiology is not clearly understood. The reported prevalence in children varies from 0.06% to 10% (De Silva et al., 1999). The female/male ratio is 3:1(Hammami et al., 2008). The average age of onset is 30–40 years. In the past, it has been described as a complication of diabetes and associated with microvascular complications (W.F. Kelly et al., 1993), but NBL has been observed also at the beginning of diabetes. NBL typically appears on the anterior lower legs. The lesions are usually bilateral and are characterized by well circumscribed yellow brown inflammatory plaques with raised borders and an atrophic center. Ulceration occurs in up to 35% of cases and is notoriously difficult to treat (Elmholdt et al., 2008). This complication negatively affects quality of life and implies a greater risk for secondary infection. Although NBL is usually observed in diabetic patients, there is some controversy regarding the degree of this association and it has been hypothesized that the strength of this association may have been overestimated in the past. Some authors have studied the effect of glucose control on NBL and found no correlation with glycosylated hemoglobin A1c levels (Dandona et al., 1981), while others found an association with a poor glucose control (Cohen et al., 1996).

#### **3.3.1 Management**

There is currently no standardized effective treatment of NBL. A wide variety of treatments have been used over the years in adults. These include: topical, systemic or intra-lesional steroids, aspirin, cyclosporin, mycophenolate, becaplermin, excision and grafting, laser surgery, hyperbaric oxygen, topical granulocytemacrophage colony-stimulating factor and photochemotherapy with topical PUVA (Hanas et al., 2009). A recent study suggests the use of TNF inhibitors in selected patients for treatment of NBL (ulcerative forms) unresponsive to prior conventional therapies (Suárez-Amor et al., 2010). NBL in children can be hard to manage and may be associated with a long-term risk of malignant transformation to squamous cell carcinoma. Systemic therapies, such as corticosteroids and azathioprine are immunosuppressive and immunomodulatory and could facilitate malignant transformation (Beattie et al., 2006). Therefore, although NBL is not clearly related to poor metabolic control, we believe that the diabetic control may also be useful. Effective primary prevention strategies and new treatment options are needed to adequately control the disease and its progression.

#### **3.4 Osteopenia**

94 Type 1 Diabetes Complications

among youth with T1D have been associated with increased risk of poor metabolic control and increased prevalence of microvascular complications such as retinopathy and nephropathy (Rydall et al., 1997). Some studies have examined the prevalence of EDs and DEBs in youth with T1D. Prevalence rates vary considerably from study to study possibly due to differences in sample, screening tools, and data collection methods. In a multi-site, cross sectional case-control study, the prevalence of ED meeting DSM-IV diagnostic criteria was about 10% and that of their sub-threshold variants about 14%: both were about twice as common in adolescent females with T1D than in their non-diabetic peers. (Jones et al.,

Nutritional treatment is one of the main difficulties in managing diabetes in the young. Diabetes clinicians should be aware of the potential warning signs in an adolescent with diabetes as well as assessment and treatment options for eating disorders with concomitant T1D. Clinical approaches should focuse on normalizing eating behaviour and enhancing self-esteem based on personal attributes unrelated to weight and eating, with a low threshold for referral for specialized EDs services (Colton et al., 2007). A multidisciplinary team, composed by clinicians, psychologist/psychiatric, dietitian/nutrition therapist, especially one with a background in EDs, is opportune to identify and treat unhealthy EDs and DEBs in T1D. Treatment for adolescents with T1D should include both diabetes management treatment and mental health treatment. The diabetes team and the mental health team have separate responsibilities but work collaboratively to address disordered eating in patients with T1D. Treatment begins with emphasis on nutritional rehabilitation, weight restoration, and adequate diabetes control. Psychotherapy should begin immediately

Necrobiosis lipoidica diabeticorum (NBL) is an infrequent skin affection in pediatric age. The etiology is not clearly understood. The reported prevalence in children varies from 0.06% to 10% (De Silva et al., 1999). The female/male ratio is 3:1(Hammami et al., 2008). The average age of onset is 30–40 years. In the past, it has been described as a complication of diabetes and associated with microvascular complications (W.F. Kelly et al., 1993), but NBL has been observed also at the beginning of diabetes. NBL typically appears on the anterior lower legs. The lesions are usually bilateral and are characterized by well circumscribed yellow brown inflammatory plaques with raised borders and an atrophic center. Ulceration occurs in up to 35% of cases and is notoriously difficult to treat (Elmholdt et al., 2008). This complication negatively affects quality of life and implies a greater risk for secondary infection. Although NBL is usually observed in diabetic patients, there is some controversy regarding the degree of this association and it has been hypothesized that the strength of this association may have been overestimated in the past. Some authors have studied the effect of glucose control on NBL and found no correlation with glycosylated hemoglobin A1c levels (Dandona et al., 1981), while others found an association with a poor glucose control (Cohen et al., 1996).

There is currently no standardized effective treatment of NBL. A wide variety of treatments have been used over the years in adults. These include: topical, systemic or intra-lesional

2000). However there are also rare cases in childhood (Franzese et al., 2002a).

**3.2.1 Management** 

**3.3.1 Management** 

for the patient and family (S.D. Kelly et al., 2005).

**3.3 Necrobiosis lipoidica diabeticorum** 

Children and adolescents with T1D can show several impairment of bone metabolism and structure, resulting in a higher risk of decreased bone mass and its related complications later in life. Consequently an assessment of quality of the bone through non-invasive methods (phalangeal ultrasonography) seems to be opportune in the care of diabetic patients, specially the ones with clusters of autoimmune diseases to define a possible involvement of the bone (Lombardi et al.,2010).

Bone impairment in multiple autoimmune diseases might be considered not only a complication due to endocrine or nutritional mechanisms, but also a consequence of an immunoregulatory imbalance.

#### **3.4.1 Metabolic causes**

Alterations of bone mineral density (BMD) are especially observed when diabetes is associated with CD and/or AIT. Bone loss, described in patients with T1D, AIT or CD is usually viewed as a complication of these diseases and is related to duration of diabetes and quality of metabolic control. The exact mechanisms accounting for bone loss in these diseases have been variably explained by metabolic derangements due to the impaired hormonal function in T1D or AIT (McCabe, 2007), or calcium malabsorption and secondary hyperparathyroidism in untreated CD patients (Selby et al., 1999). Alterations of homeostatic mechanisms might explain an imbalance of osteoclast activity leading to osteopenia (Lombardi et al.,2010; Wu et al., 2008).

#### **3.4.2 Immune causes**

Bone remodeling involves complex interactions between osteoclasts and other cells in their microenvironment (marrow stromal cells, osteoblasts, macrophages, T-lymphocytes and marrow cells) (Kollet et al., 2007; Teitelbaum, 2007). Besides their role in calcium mobilization from bone and initiation of bone remodeling, osteoclasts are now considered as the innate immune cells in the bone, since they are able to produce and respond to cytokines and chemokines. Some authors found altered levels of plasma Osteoprotegerin (OPG) in children with T1D. Osteoprotegerin is a circulating secretory glycoprotein and is a member of the tumor necrosis factor receptor (TNFR) family. It works as a decoy receptor for the cytokine receptor activator of NFkB ligand (RANKL). RANKL and OPG are a key agonist/antagonist cytokine system: RANKL increases the pool of active osteoclasts thus

Type 1 Diabetes Mellitus and Co-Morbidities 97

contracture of the finger joints and large joints, associated with tight waxy skin. Changes begin in the metacarpophalangeal and proximal interphalangeal joints of the fifth finger and extend radially with involvement of the distal interphalangeal joints as well. Involvement of larger joints includes particularly the wrist and elbow, but also ankles and cervical and thoracolumbar spine (Komatsu et al., 2004). The limitation is only mildly disabling even when severe. With rare exception, LJM appears after the age of 10 years. The prevalence of LJM in T1D, evaluated in several studies ranges from 9 to 58% in paediatric and adult

The biochemical basis of LJM may be a consequence of changes in the connective tissue, probably due to alterations in the structural macromolecules of the extracellular matrix. The hyperglycaemia can alterate the glycation of protein with the formation of advanced glycation end products (AGEs), which resist to protein degradation and consequently increase thickness of basal membranes in the periarticular tissues (Shimbargger, 1987). Development of LJM is related to both age and diabetes duration (Cagliero et al., 2002), while others showed that it can be compromised also in a precocious age and with a short duration of diabetes (Komatsu et al., 2004). Of note, fluorescence of skin collagen, which reflects the accumulation of stable AGEs, increases linearly with age, but with abnormal rapidity in T1D and in correlation with the presence of retinopathy, nephropathy and

Some authors have showed that there is a clear link between upper limb musculoskeletal abnormalities and poor metabolic control (Ramchurn et al., 2009). It has been observed a reduction in frequency of LJM between the mid-70s and mid-90s in children, most likely due to the improved glucose control during this era (Infante et al., 2001; Lindsay et al., 2005).

Insulin oedema is a well-recognized and extremely rare complication of insulin therapy. It was found to occur equally in both sexes in adults, but a clear female predominance was noted in younger ages. The condition is self-limiting, but a progression to overt cardiac failure and development of pleural effusion has been reported. (Chelliah &

The pathophysiology remains vague. Intensive fluid resuscitation in an insulin-deficient catabolic state may lead to extravasation of fluid to the subcutaneous tissue, resulting in peripheral oedema. This may be exacerbated by the increased capillary permeability associated with chronic hyperglycemia. Renal tubular sodium reabsorption is enhanced by insulin therapy via stimulating the Na+/K+-ATPase as well as the expression of Na+/H+ exchanger 3 in the proximal tubule. Transient inappropriate hyperaldosteronism has also been suggested to contribute to the fluid retention (Bas et al., 2010). Loss of albumin from the circulation due to increased transcapillary leakage probably contributed to the formation of oedema and the decreased serum albumin, but was not severe enough to account for the magnitude of oedema (Wheatly & Edwards 1985). Cases with normal serum albumin have

Clinically, insulin oedema may present with a spectrum of severity until to frank anasarca. Pleural effusions have uncommonly been reported, although some of these patients were elderly and may have had pre-existing cardiac disease. Rarely, the oedema extended from peripheral tissues to serosal cavities with ascites and cardiac failure (Bas et al., 2010). Fluid and salt restriction should be implemented and this may be all that is necessary. Diuretic

patients (Lindsay et al., 2005).

neuropathy (Monnier et al., 1986).

**3.7 Type 1 diabetes and oedema** 

Burge, 2004).

also been reported.

increasing bone resorption, whereas OPG, which neutralizes RANKL, has the opposite effect. Alterations or abnormalities of the RANKL/OPG system have been implicated in different metabolic bone diseases characterized by increased osteoclast differentiation and activation, and by enhanced bone resorption (Galluzzi et al., 2005). Therefore, bone could be an additional target of immune dysregulation.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA4), a well-known susceptibility gene for autoimmune disorders, might also represent a possible link between immune system and bone. In animal studies CTLA4 expressed on T regulatory (Treg) cells impairs osteoclast formation (Zaiss et al., 2007). Therefore the failure of Treg cell function in clustering of multiple autoimmune diseases could represent a mechanism to explain both the occurrence of poly-reactive autoimmune processes and the increase of bone resorption in the same individuals.

In patients affected by both T1D and CD, the risk of developing osteopenia is probably influenced by the compliance to gluten-free diet. Osteopenia occurs more frequently in patients with diabetes and CD with poor compliance to GFD. Interestingly, recent observations indicate also an imbalance of cytokines relevant to bone metabolism in untreated celiac patients' sera and the direct effect of these sera on in vitro bone cell activity. In particular the RANKL/osteoprotegerin (OPG) ratio was increased in patients not on gluten-free diet. Actually, the only presence of a second disease, either AIT or CD, do not seems to increase the frequency of osteopenia, provided a good compliance to GFD in CD patients, while the association of three autoimmune diseases significantly increases the occurrence of osteopenia (37.5%). In addition, poor compliance to GFD of CD patients could increase the occurrence of osteopenia more in patients with three autoimmune diseases (80%) than in those with two autoimmune diseases (18.8%) (Valerio et al., 2008).

#### **3.5 Gastropathy**

Gastrointestinal motility disorders are found in a consistent proportion of children with T1D and are associated with significant morbidity: they are usually associated with dyspeptic symptoms, such as nausea, vomiting, fullness and epigastric discomfort, and could be an important cause of morbidity in diabetic patients. Gastroparesis has been shown to be significantly correlated with a poor metabolic control in a population of T1D children with gastric electrical abnormalities. (Cucchiara et al., 1998). Furthermore it is conceivable that delayed gastric emptying may cause a mismatch between the onset of insulin action and the delivery of nutrients into the small intestine (Rayner et al., 2001). Diabetic children with unexplained poor glycemic control should be investigated for abnormalities in gastric motility (Shen & Soffer 2000). On the other hand, hyperglycaemia itself can affect the neuromuscular mechanisms regulating gastrointestinal motility and delay the gastric emptying process (Jebbink et al., 1994). Therefore, it is of great importance to try to reverse abnormalities of gastric motility and improve gastric emptying in patients with T1D and gastroparesis by the use of domperidone in children with T1D. (Franzese et al., 2002b).

#### **3.6 Type 1 diabetes and limited joint mobility**

Type 1 diabetes can be associated with other less common disabling conditions of locomotor system: Dupuytren's contracture, stiff hand, carpal tunnel syndrome, and limited joint mobility (LJM). Limited joint mobility is one of the earliest clinically apparent long-term complications of T1D in childhood and adolescence, characterized by a bilateral painless

increasing bone resorption, whereas OPG, which neutralizes RANKL, has the opposite effect. Alterations or abnormalities of the RANKL/OPG system have been implicated in different metabolic bone diseases characterized by increased osteoclast differentiation and activation, and by enhanced bone resorption (Galluzzi et al., 2005). Therefore, bone could be

Cytotoxic T lymphocyte-associated antigen-4 (CTLA4), a well-known susceptibility gene for autoimmune disorders, might also represent a possible link between immune system and bone. In animal studies CTLA4 expressed on T regulatory (Treg) cells impairs osteoclast formation (Zaiss et al., 2007). Therefore the failure of Treg cell function in clustering of multiple autoimmune diseases could represent a mechanism to explain both the occurrence of poly-reactive autoimmune processes and the increase of bone resorption in the same

In patients affected by both T1D and CD, the risk of developing osteopenia is probably influenced by the compliance to gluten-free diet. Osteopenia occurs more frequently in patients with diabetes and CD with poor compliance to GFD. Interestingly, recent observations indicate also an imbalance of cytokines relevant to bone metabolism in untreated celiac patients' sera and the direct effect of these sera on in vitro bone cell activity. In particular the RANKL/osteoprotegerin (OPG) ratio was increased in patients not on gluten-free diet. Actually, the only presence of a second disease, either AIT or CD, do not seems to increase the frequency of osteopenia, provided a good compliance to GFD in CD patients, while the association of three autoimmune diseases significantly increases the occurrence of osteopenia (37.5%). In addition, poor compliance to GFD of CD patients could increase the occurrence of osteopenia more in patients with three autoimmune diseases

Gastrointestinal motility disorders are found in a consistent proportion of children with T1D and are associated with significant morbidity: they are usually associated with dyspeptic symptoms, such as nausea, vomiting, fullness and epigastric discomfort, and could be an important cause of morbidity in diabetic patients. Gastroparesis has been shown to be significantly correlated with a poor metabolic control in a population of T1D children with gastric electrical abnormalities. (Cucchiara et al., 1998). Furthermore it is conceivable that delayed gastric emptying may cause a mismatch between the onset of insulin action and the delivery of nutrients into the small intestine (Rayner et al., 2001). Diabetic children with unexplained poor glycemic control should be investigated for abnormalities in gastric motility (Shen & Soffer 2000). On the other hand, hyperglycaemia itself can affect the neuromuscular mechanisms regulating gastrointestinal motility and delay the gastric emptying process (Jebbink et al., 1994). Therefore, it is of great importance to try to reverse abnormalities of gastric motility and improve gastric emptying in patients with T1D and gastroparesis by the use of domperidone in children with T1D. (Franzese et al., 2002b).

Type 1 diabetes can be associated with other less common disabling conditions of locomotor system: Dupuytren's contracture, stiff hand, carpal tunnel syndrome, and limited joint mobility (LJM). Limited joint mobility is one of the earliest clinically apparent long-term complications of T1D in childhood and adolescence, characterized by a bilateral painless

(80%) than in those with two autoimmune diseases (18.8%) (Valerio et al., 2008).

an additional target of immune dysregulation.

**3.6 Type 1 diabetes and limited joint mobility** 

individuals.

**3.5 Gastropathy** 

contracture of the finger joints and large joints, associated with tight waxy skin. Changes begin in the metacarpophalangeal and proximal interphalangeal joints of the fifth finger and extend radially with involvement of the distal interphalangeal joints as well. Involvement of larger joints includes particularly the wrist and elbow, but also ankles and cervical and thoracolumbar spine (Komatsu et al., 2004). The limitation is only mildly disabling even when severe. With rare exception, LJM appears after the age of 10 years. The prevalence of LJM in T1D, evaluated in several studies ranges from 9 to 58% in paediatric and adult patients (Lindsay et al., 2005).

The biochemical basis of LJM may be a consequence of changes in the connective tissue, probably due to alterations in the structural macromolecules of the extracellular matrix. The hyperglycaemia can alterate the glycation of protein with the formation of advanced glycation end products (AGEs), which resist to protein degradation and consequently increase thickness of basal membranes in the periarticular tissues (Shimbargger, 1987). Development of LJM is related to both age and diabetes duration (Cagliero et al., 2002), while others showed that it can be compromised also in a precocious age and with a short duration of diabetes (Komatsu et al., 2004). Of note, fluorescence of skin collagen, which reflects the accumulation of stable AGEs, increases linearly with age, but with abnormal rapidity in T1D and in correlation with the presence of retinopathy, nephropathy and neuropathy (Monnier et al., 1986).

Some authors have showed that there is a clear link between upper limb musculoskeletal abnormalities and poor metabolic control (Ramchurn et al., 2009). It has been observed a reduction in frequency of LJM between the mid-70s and mid-90s in children, most likely due to the improved glucose control during this era (Infante et al., 2001; Lindsay et al., 2005).

#### **3.7 Type 1 diabetes and oedema**

Insulin oedema is a well-recognized and extremely rare complication of insulin therapy. It was found to occur equally in both sexes in adults, but a clear female predominance was noted in younger ages. The condition is self-limiting, but a progression to overt cardiac failure and development of pleural effusion has been reported. (Chelliah & Burge, 2004).

The pathophysiology remains vague. Intensive fluid resuscitation in an insulin-deficient catabolic state may lead to extravasation of fluid to the subcutaneous tissue, resulting in peripheral oedema. This may be exacerbated by the increased capillary permeability associated with chronic hyperglycemia. Renal tubular sodium reabsorption is enhanced by insulin therapy via stimulating the Na+/K+-ATPase as well as the expression of Na+/H+ exchanger 3 in the proximal tubule. Transient inappropriate hyperaldosteronism has also been suggested to contribute to the fluid retention (Bas et al., 2010). Loss of albumin from the circulation due to increased transcapillary leakage probably contributed to the formation of oedema and the decreased serum albumin, but was not severe enough to account for the magnitude of oedema (Wheatly & Edwards 1985). Cases with normal serum albumin have also been reported.

Clinically, insulin oedema may present with a spectrum of severity until to frank anasarca. Pleural effusions have uncommonly been reported, although some of these patients were elderly and may have had pre-existing cardiac disease. Rarely, the oedema extended from peripheral tissues to serosal cavities with ascites and cardiac failure (Bas et al., 2010). Fluid and salt restriction should be implemented and this may be all that is necessary. Diuretic

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Table 3. Non autoimmune associated conditions to Type 1 diabetes, causes and detection

#### **4. References**


therapy may be indicated in more severe decompensated cases. Administration of an aldosterone antagonist such as spironolactone may be considered from a pathophysiological point of view in the presence of inappropriate hyperaldosteronism (Kalambokis et al., 2004).

Eating disorders Dietary restriction Ameliorating of nutritional

Gastropathy Poor metabolic control Investigating of dyspeptic

Oedema Unknown Clinical examination Table 3. Non autoimmune associated conditions to Type 1 diabetes, causes and detection

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Monitoring of growth and physical development using

examination of the skin

Eventually controlled by Bone ultrasonography/

examination of the joint

growth charts

assistance

DEXA

symptoms

mobility

Routine clinical

In most instances no specific therapy is needed and spontaneous recovery is noted.

diabeticorum Parallel dermopathy Routine clinical

Probably even present, but worsened by poor metabolic

control/comorbidity

Impaired growth Poor metabolic control

mobility Parallel condition

Necrobiosis lipoidica

Osteopenia

Limited joint

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**5** 

*Serbia* 

**Hypoglycemia as a Pathological** 

G. Bjelakovic1,I. Stojanovic1, T. Jevtovic-Stoimenov1, Lj. Saranac2,

*2Department of Pediatrics, Clinical Center Nis, Faculty of Medicine, University of Niš* 

Maintenance of blood glucose homeostasis is fundamentally important for health. The maintain of stable levels of glucose in the blood is one of the most finely regulated of all homeostatic mechanisms and one in which the liver, the extrahepatic tissues, and several hormones play a part. Even mild disruptions of glucose homeostasis can have adverse

The physiological post absorptive serum glucose concentration in healthy humans range is 4, 4-5,8 mmol/L (80 to 105 mg/dL). The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose

Generally, hypoglycemia is defined as a serum glucose level below 3. 8 mmol/L (70 mg/dL).As a relatively rare disorder, hypoglycemia most often affects those humans at the

As a medical problem, hypoglycemia is diagnosed by the presence of three key features

1. Inborn error of metabolism (more common in the pediatric patient than in adults). Disturbance in carbohydrates metabolism: Malabsorption of glucose/galactose, alactasia, asucrasia, galactosemia, fructose intolerance. Glycogen storage disease, Type I ,or von Gierke Disease, glycogen storage disease, Type III, a deficiency of glycogen disbranching enzyme activity ( limit dextrinosis), Type VI glycogen storage disease, a

2. **Hormonal disturbance** .The hormone insulin plays a central role in the regulation of the blood glucose concentration. It is produced by the cells of islets of Langerhance in the pancreas. Insulin exerts hypoglycemic effect. Glucagon is the hormone produced by the

extremes of age, such as infants and the elderly, but may happen at any age.

3. relief of symptoms after the plasma glucose level is raised.

(known as Whipple's triad). Whipple's triad is: 1. symptoms consistent with hypoglycemia, 2. a low plasma glucose concentration, and

The etiology of hypoglycemia is numerous:

deficiency of liver phosphorylase )

**1. Introduction** 

consequences.

utilization.

B. Bjelakovic2, D. Pavlovic1, G. Kocic1 and B.G. Bjelakovic3 *1Institute of Biochemistry, Faculty of Medicine, University of Niš* 

*3Clinic of Internal Medicine, Department of Hepato-Gastroenterology,* 

**Result in Medical Praxis** 

*Faculty of Medicine, University of Niš* 


### **Hypoglycemia as a Pathological Result in Medical Praxis**

G. Bjelakovic1,I. Stojanovic1, T. Jevtovic-Stoimenov1, Lj. Saranac2, B. Bjelakovic2, D. Pavlovic1, G. Kocic1 and B.G. Bjelakovic3 *1Institute of Biochemistry, Faculty of Medicine, University of Niš 2Department of Pediatrics, Clinical Center Nis, Faculty of Medicine, University of Niš 3Clinic of Internal Medicine, Department of Hepato-Gastroenterology, Faculty of Medicine, University of Niš Serbia* 

#### **1. Introduction**

108 Type 1 Diabetes Complications

Ventura, A., Magazzù, G. & Greco, L. (1999). Duration of exposure to gluten and risk for

Ventura, A., Neri, E., Ughi, C., Leopaldi, A., Citta, A. & Not, T. (2000). Gluten-dependent

Westerholm-Ormio, M., Vaarala, O., Pihkala, P., Ilonen, J. & Savilahti, E. (2003).

Wheatly, T. & Edwards, O.M. (1985). Insulin oedema and its clinical significance: metabolic

Wu, Y., Humphrey, M.B. & Nakamura, M.C. (2008). Osteoclasts—the innate cells of the bone. *Autoimmunity*, Vol. 41, No. 3, (Apr 2008), pp. (183–194), 0891-6934 Yu, L., Brewer, K.W., Gates, S., Wu, A., Wang, T., Babu, S., Gottlieb, P., Freed, B.M., Noble,

Zaiss, M.M., Axmann, R., Zwerina, J., Polzer, K., Gückel, E., Skapenko, A., Schulze-Koops,

Zeglaoui, H., Landolsi, H., Mankai, A., Ghedira, I. & Bouajina, E. (2010). Type 1 diabetes

*Rheumatism*, Vol. 56, No. 12, (Dec 2007), pp. (4104–4112), 0004-3591

*Journal of Pediatrics*, Vol. 137, No. 2, (Aug 2000), pp. (263-265), 0022-3476 Viljamaa, M., Kaukinen, K., Huhtala, H., Kyronpalo, S., Rasmussen, M. & Collin, P. (2005).

*Gastroenterology*, Vol. 40, No. 4, (Apr 2005), pp. (437-443), 0036-5521

1999), pp. (297-303), 0016-5085

(May-Jun 1999), pp. (433-442), 0334-018X

0742-3071

pp. (328-335), 0021-972X

795), 0172-8172

autoimmune disorders in patients with celiac disease. SIGEP Study Group for Autoimmune Disorders in Celiac Disease. *Gastroenterology*, Vol. 117, No. 2, (Aug

diabetes-related and thyroid-related autoantibodies in patients with celiac disease.

Coeliac disease, autoimmune diseases and gluten exposure. *Scandinavian Journal of* 

Immunologic Activity in the Small Intestinal Mucosa of Pediatric Patients With Type 1 Diabetes. *Diabetes*, Vol. 52, No. 9, (Sep 2003), pp. (2287-2295), 0012-1797 Westman, E., Ambler, G.R., Royle, M., Peat, J. & Chan, A. (1999). Children with celiac

disease and insulin dependent diabetes mellitus–growth, diabetes control and dietary intake. *Journal of Pediatric Endocrinology and Metabolism*, Vol. 12, No. 3,

studies in three cases. *Diabetic Medicine*, Vol. 2, No. 5, (Sep 1985), pp. (400-404),

J., Erlich, H., Rewers, M. & Eisenbarth, G. (1999). DRB1\*04 and DQ alleles: expression of 21 hydroxylase autoantibodies and risk of progression to Addison's disease. *Journal of Clinical Endocrinology and Metabolism*, Vol. 84, No. 1, (Jan 1999),

H., Horwood, N., Cope, A. & Schett, G. (2007). Treg cells suppress osteoclast formation. A new link between the immune system and bone. *Arthritis and* 

mellitus, celiac disease, systemic lupus erythematosus and systemic scleroderma in a 15-year-old girl. *Rheumatology International*, Vol. 30, No. 6, (Apr 2010), pp. (793Maintenance of blood glucose homeostasis is fundamentally important for health. The maintain of stable levels of glucose in the blood is one of the most finely regulated of all homeostatic mechanisms and one in which the liver, the extrahepatic tissues, and several hormones play a part. Even mild disruptions of glucose homeostasis can have adverse consequences.

The physiological post absorptive serum glucose concentration in healthy humans range is 4, 4-5,8 mmol/L (80 to 105 mg/dL). The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose utilization.

Generally, hypoglycemia is defined as a serum glucose level below 3. 8 mmol/L (70 mg/dL).As a relatively rare disorder, hypoglycemia most often affects those humans at the extremes of age, such as infants and the elderly, but may happen at any age.

As a medical problem, hypoglycemia is diagnosed by the presence of three key features (known as Whipple's triad). Whipple's triad is:


The etiology of hypoglycemia is numerous:


Hypoglycemia as a Pathological Result in Medical Praxis 111

childhood. In adults, low blood glucose in the fasting state is almost always due to a serious underlying condition (Caraway & Watts,1986; King, 2011; Mayer, 1975; Service,1992)**.** 

Carbohydrates are important components of the diet. The carbohydrates that we ingest range from simple monosaccharides (glucose, fructose and galactose) to disaccharides (lactose, sucrose) and complex polysaccharides, starch and glycogen. Most carbohydrates are digested by salivary and pancreatic amylases, and are further broken down into monosaccharides by enzymes in the brush border membrane (BBM) of enterocytes. Maltase, lactase and sucrase-isomaltase are disaccharidases involved in the hydrolysis of nutritionally important disaccharides, maltose, lactose, saccharose. Once monosaccharides are presented to the BBM, mature enterocytes, expressing nutrient transporters, transport

The resultant glucose and other simple carbohydrates, galactose (from lactose) and fructose (from succrose) are transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells. Both fructose and galactose are readily converted to glucose by hepatocytes Absorption of glucose and galactose occurs by an active carrier-mediated transfer process. Fructose is absorbed by facilitated diffusion (Harper,1975; King, 2011;

Fructose and galactose are phosphorylated by specific enzymes, fructokinase and galactokinase, presented only in the liver, and converted to glucose. Glucose is transported from the liver via the bloodstream to be used by all the body cells as the most important source of energy. Glucose, as unique sugar in systemic blood circulation, leaves the blood, enters cells through specific transport proteins and has one principal fate: it is phosphorylated by ATP to form glucose-6-phosphate by hexokinase in all human body cells or by the action of glucokinase in hepatocytes. This step is notable because glucose-6 phosphate cannot diffuse through the membrane out of the cells (Haris, 1997; Harper,1979;

Maintenance of blood glucose homeostasis is fundamentally important for health. The plasma glucose level is tightly controlled throughout life in the normal individual, in spite of intermittent food ingestion and periods of fasting, as the net balance between the rates of glucose production and utilization. The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose utilization. The amount of plasma glucose level in healthy humans is usually maintained within a range of 4.4 to 5.8 mmol/L , 80 to 110 mg/dL), (Carraway & Watts,1986; King, 2011;

Glycogen is the storage form of glucose and serves as a tissue reserve for the body's glucose needs. Glycogen synthesis occurs in virtually all animal tissues, but it is especially prominent in the liver and skeletal muscles. In the liver, glycogen serves as a reservoir of

**2. Digestion and absorption of carbohydrates** 

the sugars into the enterocytes, (Drozdowski & Thomson, 2006).

www.deo.ucsf.edu/type1/understanding-diabetes).

King,2011; Tietz,1986; Voet & Voet, 2004a).

**3. Glycemia - Physiological regulation** 

Mayes,1975; Service, 1992; Voet & Voet, 2004a).

**4.1 Glycogen synthesis** 

**4. Intermediary metabolism of carbohydrates** 

cells of Langerhance islets in the pancreas. Its secretion is stimulated by hypoglycemia. The hormone glucagon, epinephrine, norepinephrine, growth hormone, and cortisol exert the opposite effects to insulin and they belong in the counter-regulatory hormones


The central nervous system requires glucose as its primary fuel. The brain uses more than 30% of blood glucose. The brain does not produce the glucose required for its functioning and it is completely dependent on the rest of the body for its supply. So, fluctuations in blood sugar levels can prove to be harmful for the brain; a continual supply of glucose is necessary as a source of energy for the nervous system and some other organs like erythrocytes, testes and kidney medulla .Gluconeogenesis, the biosynthesis of new glucose, (i.e. not glucose from glycogen) from other metabolites (lactic acid, amino acids and glycerol) is necessary for use as a fuel, since glucose is the sole energy source for these organs.

The symptoms caused by low blood sugar come from two sources and may resemble other medical conditions. The first symptoms are caused by the release of epinephrine from the nervous system. These include sweating, pale skin color, shakiness, trembling, rapid heart rate, a feeling of anxiety, nervousness, weakness, hunger, nausea and vomiting. Lowering of the brain's glucose causes: headache, difficulty in thinking, changes in vision, lethargy, restlessness, inability to concentrate or pay attention, mental confusion, sleepiness, stupor, and personality changes.

To treat low blood sugar immediately the patients should eat or drink something that has sugar in it, such as orange juice, milk, or a hard candy. It is need to find out the causes of hypoglycemia.

Laboratory diagnosis of hypoglycemia is very important in medical praxis especially in pediatric, internal medicine (hepatology, renal failure, and cardiology) neuropsychiatry disorders and so on.

Glucose is the name of the simple sugar found in plant and animal tissues. It is made within plants as a product of photosynthesis. Although glucose can be produced within the human body, most of it is supplied to people by dietary carbohydrate intake principally as starch. Once consumed and digested, glucose will either be used immediately or stored as glycogen for future use, (Caraway & Watts,1986; Mayer,1975).

Glucose is the major energy source for human body and is derived primarily from dietary carbohydrates (grains, starchy vegetables, and legumes), from body stores of carbohydrates (glycogen) and from the synthesis of glucose from protein and glycerol moiety of triglycerides (gluconeogenesis) (King, 2011).

The glucose level in blood is kept within narrow range through a variety of influences. While there is some variation in blood glucose as circumstance changes (feeding, prolonged fasting), levels above or below the normal range usually indicate disease.

High blood glucose due to diabetes mellitus is the most commonly encountered disorder of carbohydrate metabolism. Low blood glucose is an uncommon cause of serious diseases. There are numerous rare conditions that cause hypoglycemia in neonatal period and early

the opposite effects to insulin and they belong in the counter-regulatory hormones 3. The disorders of some organs (Liver and Kidney Disorders especially). Any abnormality in the functioning of the liver can disturb the process of blood-sugar regulation, resulting in hypoglycemia. On the other hand, kidney disorder can be one of

The central nervous system requires glucose as its primary fuel. The brain uses more than 30% of blood glucose. The brain does not produce the glucose required for its functioning and it is completely dependent on the rest of the body for its supply. So, fluctuations in blood sugar levels can prove to be harmful for the brain; a continual supply of glucose is necessary as a source of energy for the nervous system and some other organs like erythrocytes, testes and kidney medulla .Gluconeogenesis, the biosynthesis of new glucose, (i.e. not glucose from glycogen) from other metabolites (lactic acid, amino acids and glycerol) is necessary for use as

The symptoms caused by low blood sugar come from two sources and may resemble other medical conditions. The first symptoms are caused by the release of epinephrine from the nervous system. These include sweating, pale skin color, shakiness, trembling, rapid heart rate, a feeling of anxiety, nervousness, weakness, hunger, nausea and vomiting. Lowering of the brain's glucose causes: headache, difficulty in thinking, changes in vision, lethargy, restlessness, inability to concentrate or pay attention, mental confusion, sleepiness, stupor,

To treat low blood sugar immediately the patients should eat or drink something that has sugar in it, such as orange juice, milk, or a hard candy. It is need to find out the causes of

Laboratory diagnosis of hypoglycemia is very important in medical praxis especially in pediatric, internal medicine (hepatology, renal failure, and cardiology) neuropsychiatry

Glucose is the name of the simple sugar found in plant and animal tissues. It is made within plants as a product of photosynthesis. Although glucose can be produced within the human body, most of it is supplied to people by dietary carbohydrate intake principally as starch. Once consumed and digested, glucose will either be used immediately or stored as glycogen

Glucose is the major energy source for human body and is derived primarily from dietary carbohydrates (grains, starchy vegetables, and legumes), from body stores of carbohydrates (glycogen) and from the synthesis of glucose from protein and glycerol

The glucose level in blood is kept within narrow range through a variety of influences. While there is some variation in blood glucose as circumstance changes (feeding, prolonged

High blood glucose due to diabetes mellitus is the most commonly encountered disorder of carbohydrate metabolism. Low blood glucose is an uncommon cause of serious diseases. There are numerous rare conditions that cause hypoglycemia in neonatal period and early

fasting), levels above or below the normal range usually indicate disease.

6. Hypoglycemia as the complication of treatment of diabetes mellitus.

a fuel, since glucose is the sole energy source for these organs.

for future use, (Caraway & Watts,1986; Mayer,1975).

moiety of triglycerides (gluconeogenesis) (King, 2011).

the major causes of low blood sugar. 4. Infection-related hypoglycemia (in older adults)

5. The adverse medication reactions

and personality changes.

hypoglycemia.

disorders and so on.

cells of Langerhance islets in the pancreas. Its secretion is stimulated by hypoglycemia. The hormone glucagon, epinephrine, norepinephrine, growth hormone, and cortisol exert childhood. In adults, low blood glucose in the fasting state is almost always due to a serious underlying condition (Caraway & Watts,1986; King, 2011; Mayer, 1975; Service,1992)**.** 

### **2. Digestion and absorption of carbohydrates**

Carbohydrates are important components of the diet. The carbohydrates that we ingest range from simple monosaccharides (glucose, fructose and galactose) to disaccharides (lactose, sucrose) and complex polysaccharides, starch and glycogen. Most carbohydrates are digested by salivary and pancreatic amylases, and are further broken down into monosaccharides by enzymes in the brush border membrane (BBM) of enterocytes. Maltase, lactase and sucrase-isomaltase are disaccharidases involved in the hydrolysis of nutritionally important disaccharides, maltose, lactose, saccharose. Once monosaccharides are presented to the BBM, mature enterocytes, expressing nutrient transporters, transport the sugars into the enterocytes, (Drozdowski & Thomson, 2006).

The resultant glucose and other simple carbohydrates, galactose (from lactose) and fructose (from succrose) are transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells. Both fructose and galactose are readily converted to glucose by hepatocytes Absorption of glucose and galactose occurs by an active carrier-mediated transfer process. Fructose is absorbed by facilitated diffusion (Harper,1975; King, 2011; www.deo.ucsf.edu/type1/understanding-diabetes).

Fructose and galactose are phosphorylated by specific enzymes, fructokinase and galactokinase, presented only in the liver, and converted to glucose. Glucose is transported from the liver via the bloodstream to be used by all the body cells as the most important source of energy. Glucose, as unique sugar in systemic blood circulation, leaves the blood, enters cells through specific transport proteins and has one principal fate: it is phosphorylated by ATP to form glucose-6-phosphate by hexokinase in all human body cells or by the action of glucokinase in hepatocytes. This step is notable because glucose-6 phosphate cannot diffuse through the membrane out of the cells (Haris, 1997; Harper,1979; King,2011; Tietz,1986; Voet & Voet, 2004a).

#### **3. Glycemia - Physiological regulation**

Maintenance of blood glucose homeostasis is fundamentally important for health. The plasma glucose level is tightly controlled throughout life in the normal individual, in spite of intermittent food ingestion and periods of fasting, as the net balance between the rates of glucose production and utilization. The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose utilization. The amount of plasma glucose level in healthy humans is usually maintained within a range of 4.4 to 5.8 mmol/L , 80 to 110 mg/dL), (Carraway & Watts,1986; King, 2011; Mayes,1975; Service, 1992; Voet & Voet, 2004a).

#### **4. Intermediary metabolism of carbohydrates**

#### **4.1 Glycogen synthesis**

Glycogen is the storage form of glucose and serves as a tissue reserve for the body's glucose needs. Glycogen synthesis occurs in virtually all animal tissues, but it is especially prominent in the liver and skeletal muscles. In the liver, glycogen serves as a reservoir of

Hypoglycemia as a Pathological Result in Medical Praxis 113

In the proceses of glycogen catabolism or glycogenolysis, glycogen, stored in the liver and muscles, is converted first to glucose-1-phosphate and then into glucose-6-phosphate. (Mayes,1975; Voet &;Voet, 2004b). Three enzymes participate in glycogenolysis: glycogen phosphorylase, oligo-1,4-1,4–glucan transferase or trisaccharide transferase, and (1-6) glucosidase or - amylase. Glycogen phosphorylase catalyzes phosphorolytic cleavage of the -1,4 glycosidic linkages of glycogen (using inorganic phosphate), releasing glucose-1 phosphate as reaction product and limit dextrin. After extensive phosphorylase action on glycogen, the molecule contains four glucose residues in -1,4-glucosidic bond attached by

These structures can be further degraded by the action of a debranching enzyme, which carries out two distinct reactions. The first of these, known as oligo-a1,4-a-1,4) glucan transferase activity or trisaccharide transferase, removes a trisaccharide unit from limit branch and transfers this group to the end of another nearby glycogen chain, with resynthesis of the -1,4 bond. This leaves a single glucose residue in a-(1,6) linkage to the main chain*.* The -1,6-glucosidase or -amylase activity of the debranching enzyme then catalyzes hydrolysis of the (1,6) linkage, leaving a polysaccharide chain with one branch fewer and yielding free glucose. This is a minor fraction of free glucose released from glycogen (Fig 3), since that the major product of glycogen breakdown by phosphorylase activity is glucose-1-phosphate. Phosphoglucomutase catalyzes the reaction: glucose-1-

Glucose-6-phosphate is the first step of the glycolysis pathway if glycogen is the carbohydrate source of further energy needed. If energy is not immediately needed, the glucose-6-phosphate is converted to glucose, by the action of the enzyme glucose-6 phosphatase (mainly in liver), for distribution to various cells by blood, such as brain,

The reactions involved in tissue glycogen synthesis and degradation are carefully controlled and regulated by hormones. The primary hormone responsible for conversion of glucose to glycogen is insulin. Opposite effects to glycogen metabolism have its antagonists: glucagon, adrenaline, cortisol, growth hormone which facilitate glycogenolysis in liver and muscles.

Fig. 2. Glycogenesis

**4.2 Glycogen breakdown (glycogenolysis)** 

a(1,6)-link to the glycogen molecule.

phosphate glucose-6-phosphate.

erythrocytes, adipocytes, etc.

glucose, readily converted into blood glucose for distribution to other tissues, whereas in muscles glycogen is broken down via glycolysis to provide energy for muscle contraction. In human body, glycogen is synthesized and stored when glucose levels are high and is broken down during starvation or periods of high glucose demand.

Glycogen is a highly branched polymeric structure containing glucose as the basic monomer (Mayes, 1975; Voet & Voet, 2004b)*.* It is composed of polymers of -1-4 linked glucose, interrupted by 1-6 linked branch point every 4-10 residues (Fig 1).

Fig. 1. Glycogen structure

Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis. Glycogen synthase will only add glucose units from UDP-glucose onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by alpha(1,4) bonds. The enzyme, glycogenin, initiates glycogenynthesis (oregonstate.edu/.../summer09/lecture/glycogennotes.html; Voet & Voet, 2004b).

The enzyme glycogen synthase then catalyzes elongation of glycogen chains initiated by glycogenin to a chane of 9 – 11 glucose molecule. Glycogen synthase catalyzes transfer of the glucose moiety of UDP-glucose to the hydroxyl at C4 of the terminal residue of a glycogen chain to form an (1-4)-glycosidic linkage (Fig 2) (Mayes,1975; King, 2011; Voet & Voet, 2004b; www.uic.edu/.../glycogen%20metab/Glycogen%20biochemistry.htm).

A branching enzyme forms the branching points in glycogen. The branches arise from -(1**-** 6) linkages which occur every 8 to 12 residues. Glycogen branches are formed by amylo-(1,4- 1,6)-transglycosylase**,** also known as branching enzyme*.* The branching enzyme transfers a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with an -(1-6) linkage. In the presence of glycogenin, glycogen synthase, branching enzyme and UDP glucose (active glucose) form glycogen as a highly branched polymeric structure, containing glucose as the basic monomer (Figure 2).

Glycogen is synthesized and stored mainly in the liver and the muscles as well as in the cytoplasm of all human body cells as granules named "residual bodies", (Voet & Voet, 2004b).

Fig. 2. Glycogenesis

glucose, readily converted into blood glucose for distribution to other tissues, whereas in muscles glycogen is broken down via glycolysis to provide energy for muscle contraction. In human body, glycogen is synthesized and stored when glucose levels are high and is broken

Glycogen is a highly branched polymeric structure containing glucose as the basic monomer (Mayes, 1975; Voet & Voet, 2004b)*.* It is composed of polymers of -1-4 linked glucose,

Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis. Glycogen synthase will only add glucose units from UDP-glucose onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by alpha(1,4) bonds. The enzyme, glycogenin, initiates glycogenynthesis (oregonstate.edu/.../summer09/lecture/glycogennotes.html; Voet &

The enzyme glycogen synthase then catalyzes elongation of glycogen chains initiated by glycogenin to a chane of 9 – 11 glucose molecule. Glycogen synthase catalyzes transfer of the glucose moiety of UDP-glucose to the hydroxyl at C4 of the terminal residue of a glycogen chain to form an (1-4)-glycosidic linkage (Fig 2) (Mayes,1975; King, 2011; Voet &

A branching enzyme forms the branching points in glycogen. The branches arise from -(1**-** 6) linkages which occur every 8 to 12 residues. Glycogen branches are formed by amylo-(1,4- 1,6)-transglycosylase**,** also known as branching enzyme*.* The branching enzyme transfers a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with an -(1-6) linkage. In the presence of glycogenin, glycogen synthase, branching enzyme and UDP glucose (active glucose) form glycogen as a highly

Glycogen is synthesized and stored mainly in the liver and the muscles as well as in the cytoplasm of all human body cells as granules named "residual bodies", (Voet & Voet, 2004b).

Voet, 2004b; www.uic.edu/.../glycogen%20metab/Glycogen%20biochemistry.htm).

branched polymeric structure, containing glucose as the basic monomer (Figure 2).

down during starvation or periods of high glucose demand.

Fig. 1. Glycogen structure

Voet, 2004b).

interrupted by 1-6 linked branch point every 4-10 residues (Fig 1).

#### **4.2 Glycogen breakdown (glycogenolysis)**

In the proceses of glycogen catabolism or glycogenolysis, glycogen, stored in the liver and muscles, is converted first to glucose-1-phosphate and then into glucose-6-phosphate. (Mayes,1975; Voet &;Voet, 2004b). Three enzymes participate in glycogenolysis: glycogen phosphorylase, oligo-1,4-1,4–glucan transferase or trisaccharide transferase, and (1-6) glucosidase or - amylase. Glycogen phosphorylase catalyzes phosphorolytic cleavage of the -1,4 glycosidic linkages of glycogen (using inorganic phosphate), releasing glucose-1 phosphate as reaction product and limit dextrin. After extensive phosphorylase action on glycogen, the molecule contains four glucose residues in -1,4-glucosidic bond attached by a(1,6)-link to the glycogen molecule.

These structures can be further degraded by the action of a debranching enzyme, which carries out two distinct reactions. The first of these, known as oligo-a1,4-a-1,4) glucan transferase activity or trisaccharide transferase, removes a trisaccharide unit from limit branch and transfers this group to the end of another nearby glycogen chain, with resynthesis of the -1,4 bond. This leaves a single glucose residue in a-(1,6) linkage to the main chain*.* The -1,6-glucosidase or -amylase activity of the debranching enzyme then catalyzes hydrolysis of the (1,6) linkage, leaving a polysaccharide chain with one branch fewer and yielding free glucose. This is a minor fraction of free glucose released from glycogen (Fig 3), since that the major product of glycogen breakdown by phosphorylase activity is glucose-1-phosphate. Phosphoglucomutase catalyzes the reaction: glucose-1 phosphate glucose-6-phosphate.

Glucose-6-phosphate is the first step of the glycolysis pathway if glycogen is the carbohydrate source of further energy needed. If energy is not immediately needed, the glucose-6-phosphate is converted to glucose, by the action of the enzyme glucose-6 phosphatase (mainly in liver), for distribution to various cells by blood, such as brain, erythrocytes, adipocytes, etc.

The reactions involved in tissue glycogen synthesis and degradation are carefully controlled and regulated by hormones. The primary hormone responsible for conversion of glucose to glycogen is insulin. Opposite effects to glycogen metabolism have its antagonists: glucagon, adrenaline, cortisol, growth hormone which facilitate glycogenolysis in liver and muscles.

Hypoglycemia as a Pathological Result in Medical Praxis 115

ATP depletion in cells, or low blood glucose level, lead to the activation of glycogenolysis and the enhancement of glucose degradation through glycolysis. Glycolysis is a central metabolic pathway of glucose metabolism, starting with glucose-6-phosphate, produced by glycogenolysis or gluconeogenesis. Glucose-6-phosphate could also be synthesized directly from blood-derived glucose by the action of hexokinase in all human body cells or by the

Glycolysis is the anaerobic catabolism of glucose. It occurs in cytosol of virtually all cells. The glycolytic pathway converts a molecule of glucose into 2 molecules of pyruvic acid and captures 2 molecules of ATP. If glycolysis proceeds in aerobic conditions 2 molecules of pyruvic acid enter mitochondria, transforms into acetyl-CoA which is oxidized by the citric acid cycle. One cycle provides 12 mol ATP per one molecule of pyruvate. Aerobic conditions provide a mechanism for converting NADH back to NAD+ which is essential for glycolysis

**4.3 Glycolysis** 

to operate (Fig 4).

action of glucokinase in hepatocytes.

Fig. 4. Glycolysis and Gluconeogenesis

The principal enzymes of glycogen metabolism are glycogen synthase and glycogen phosphorylase, reciprocally regulated by allosteric effectors and covalent modification (through phosphorylation or dephosphorylation). Glycogen synthase is active when high blood glucose leads to intracellular glucose-6-P increase. Glycogen phosphorylase is active in the presence of high level of cyclic adenosine monophosphate (cAMP ) which suggests that the cells need chemical energy in the form of ATP.

Fig. 3. Glycogenolysis

Glucagon, synthesized by pancreatic -cells, and epinephrine (adrenaline), synthesized by adrenal medulla, regulate glycogen metabolism by covalent modification (phosphorylation and dephosphorylation) through cAMP cascades. Both hormones are produced in response to low blood glucose level. Glucagon activates cAMP formation in liver, while adrenaline activates its formation in muscle. Phosphorylation of the enzyme, via cAMP cascade, induced by adrenaline, results in further activation of glycogen phosphorylase. These regulatory processes ensure release of phosphorylated glucose from glycogen, for entry into glycolysis to provide ATP needed for muscle contraction.

Insulin, produced in response to high blood glucose, antagonizes effects of the cAMP cascade induced by glucagon and adrenaline. It is the only hormone inducing cAMP decrease (Mayes, 1975; Voet & Voet, 2004b).

#### **4.3 Glycolysis**

114 Type 1 Diabetes Complications

The principal enzymes of glycogen metabolism are glycogen synthase and glycogen phosphorylase, reciprocally regulated by allosteric effectors and covalent modification (through phosphorylation or dephosphorylation). Glycogen synthase is active when high blood glucose leads to intracellular glucose-6-P increase. Glycogen phosphorylase is active in the presence of high level of cyclic adenosine monophosphate (cAMP ) which suggests

Glucagon, synthesized by pancreatic -cells, and epinephrine (adrenaline), synthesized by adrenal medulla, regulate glycogen metabolism by covalent modification (phosphorylation and dephosphorylation) through cAMP cascades. Both hormones are produced in response to low blood glucose level. Glucagon activates cAMP formation in liver, while adrenaline activates its formation in muscle. Phosphorylation of the enzyme, via cAMP cascade, induced by adrenaline, results in further activation of glycogen phosphorylase. These regulatory processes ensure release of phosphorylated glucose from glycogen, for entry into

Insulin, produced in response to high blood glucose, antagonizes effects of the cAMP cascade induced by glucagon and adrenaline. It is the only hormone inducing cAMP

glycolysis to provide ATP needed for muscle contraction.

decrease (Mayes, 1975; Voet & Voet, 2004b).

that the cells need chemical energy in the form of ATP.

Fig. 3. Glycogenolysis

ATP depletion in cells, or low blood glucose level, lead to the activation of glycogenolysis and the enhancement of glucose degradation through glycolysis. Glycolysis is a central metabolic pathway of glucose metabolism, starting with glucose-6-phosphate, produced by glycogenolysis or gluconeogenesis. Glucose-6-phosphate could also be synthesized directly from blood-derived glucose by the action of hexokinase in all human body cells or by the action of glucokinase in hepatocytes.

Glycolysis is the anaerobic catabolism of glucose. It occurs in cytosol of virtually all cells. The glycolytic pathway converts a molecule of glucose into 2 molecules of pyruvic acid and captures 2 molecules of ATP. If glycolysis proceeds in aerobic conditions 2 molecules of pyruvic acid enter mitochondria, transforms into acetyl-CoA which is oxidized by the citric acid cycle. One cycle provides 12 mol ATP per one molecule of pyruvate. Aerobic conditions provide a mechanism for converting NADH back to NAD+ which is essential for glycolysis to operate (Fig 4).

Fig. 4. Glycolysis and Gluconeogenesis

Hypoglycemia as a Pathological Result in Medical Praxis 117

Lactate, formed by the oxidation of glucose in skeletal muscles and by erythrocytes through the proceses of anaerobic glycolysis, is transported to the liver and kidney, where it re-forms glucose, which again become available via the circulation for oxidation in the tissues. This

It has been noted that of the amino acids transported from muscles to the liver during starvation or under the action of cortisol, alanine predominate. Glucose-alanine cycle represents a cycling glucose from the liver to the muscles and alanine from muscles to liver, effecting a net transfer of amino nitrogen from muscle to liver and free energy from liver to muscle. At the level of muscles, pyruvate, formed by glycolysis, transforms to alanine by the action of alanine transaminase (ALT) or glutamate pyruvat transaminase (GPT). The reaction is freely reversible; at the level of hepatocytes alanine transfers to pyruvate by the

Glycerol, necessary for the synthesis of triacylglicerols and glycerophosholipids is derived, initially, from the blood glucose since free glycerol cannot be utilised readily for the synthesis of these lipids in tissues. Instead of free glycerol, adipose tissue uses -glycero phosphate or "active glycerol" produced during degradation of glucose by glycolysis.

process is known as the Cory cycle or lactic acid cycle (Fig 5).

Fig. 5. Cory cycle , glucose-alanine cycle and glycerol-glucose cycle

**4.4.1 Cory cycle** 

**4.4.2 Glucose-alanine cycle** 

action of the same anzyme (Fig 5).

Under anaerobic conditions 2 molecules of pyruvate, under the action of lactate dehydrogenaze, and by using NADH2, convert to 2 molecules of lactate. The reaction is freely reversible (Haris, 1997; Mayes, 1975; Voet & Voet, 2004b; users.rcn.com/.../I/ IntermediaryMetabolism.html).

#### **4.4 Gluconeogenesis**

If glucose is not obtained in the diet, during fasting , the body must produce new glucose from noncarbohydrate precursors by the proces of gluconeogenesis. The term gluconeogenesis means the generation (*genesis*) of new (*neo*) glucose.

The production of glucose from other metabolites is necessary to maintain the glucose level in the blood as a fuel source by the brain, erythrocytes, kidney medulla and testes, since glucose is the sole energy source for these organs. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-CoA. The adipose tissue needs glucose which is also necessary for the synthesis of triacylglycerls and glycerophospholipids. The main precursors for gluconeogenesis are lactate and alanine from muscle, glycerol from adipose tissue, and glucogenic amino acids from the proteolysis in peripheral tissues and proteins from the diet. The most of the amino acids, as well as their -keto acids, are TCA cycle intermediates. In addition, the gluconeogenetic processes are used to clear the intermediary products of metabolism of other tissues from the blood, e.g. lactate, produced by muscles and erythrocytes, and glycerol, which is continuously produced by adipose tissue.The principal organs responsible for gluconeogenesis are the liver and kidneys, which account for about 90% and 10% of the body's gluconeogenic activity, respectively. Interestingly, the mammalian organs that consume the most glucose, namely, brain and muscle, carry out very little glucose synthesis (Gerich et al, 2001; King, 2011; Mayes,1975; Voet & Voet, 2004b; Woerle & Stumvoll, 2001).

Gluconeogenesis is similar but not the exact reverse of glycolysis; some of the steps are the identical in reverse direction and three of them are new ones (Fig 4). In glycolysis energy barriers obstruct a simple reversal of glycolysis: reactions catalyzed by pyruvate kinase, phospho-fructokinase and hexokinase. These barriers are circumvented by new, special enzymes of gluconeogenesis: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructoso-1,6-diphosphatase and glucose-6-phosphatase. The conversion of lactate to glucose begins with the oxidation of lactate, by the action of lactate dehydrogenase, to pyruvate. In the presence of ATP, pyruvate carboxylase and CO2 convert pyruvat to oxaloacetate. The enzyme, phosphoenolpyruvate carboxykinase (PEPCK) transfers oxaloacetate to phosphoenolpyruvate in the presence of GTP and by elimination of CO2. Thus, with the help of these two enzymes, and lactate dehydrogenase, lactate can be converted to oxaloacetate. The pyruvate and oxaloacetate are the intermediary products of catabolic pathway of many glycolytic amino acids. The next steps of reversal glycolysis continue just to formation of fructose-1,6-diphosphate, the substrate for fructose-1, 6-diphosphatase. Fructose-6-phosphate, formed by elimination of inorganic phosphate, converts to glucose-6 phosphate (G6P). The energy required for the hepatic synthesis of glucose from lactate is derived from the oxidation of fatty acids. In the liver and kidney, G6P can be dephosphorylated to glucose by the enzyme glucose 6-phosphatase. This is the final step in the gluconeogenesis pathway.

#### **4.4.1 Cory cycle**

116 Type 1 Diabetes Complications

Under anaerobic conditions 2 molecules of pyruvate, under the action of lactate dehydrogenaze, and by using NADH2, convert to 2 molecules of lactate. The reaction is freely reversible (Haris, 1997; Mayes, 1975; Voet & Voet, 2004b; users.rcn.com/.../I/

If glucose is not obtained in the diet, during fasting , the body must produce new glucose from noncarbohydrate precursors by the proces of gluconeogenesis. The term

The production of glucose from other metabolites is necessary to maintain the glucose level in the blood as a fuel source by the brain, erythrocytes, kidney medulla and testes, since glucose is the sole energy source for these organs. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-CoA. The adipose tissue needs glucose which is also necessary for the synthesis of triacylglycerls and glycerophospholipids. The main precursors for gluconeogenesis are lactate and alanine from muscle, glycerol from adipose tissue, and glucogenic amino acids from the proteolysis in peripheral tissues and proteins from the diet. The most of the amino acids, as well as their -keto acids, are TCA cycle intermediates. In addition, the gluconeogenetic processes are used to clear the intermediary products of metabolism of other tissues from the blood, e.g. lactate, produced by muscles and erythrocytes, and glycerol, which is continuously produced by adipose tissue.The principal organs responsible for gluconeogenesis are the liver and kidneys, which account for about 90% and 10% of the body's gluconeogenic activity, respectively. Interestingly, the mammalian organs that consume the most glucose, namely, brain and muscle, carry out very little glucose synthesis (Gerich et al, 2001; King, 2011; Mayes,1975; Voet & Voet, 2004b; Woerle

Gluconeogenesis is similar but not the exact reverse of glycolysis; some of the steps are the identical in reverse direction and three of them are new ones (Fig 4). In glycolysis energy barriers obstruct a simple reversal of glycolysis: reactions catalyzed by pyruvate kinase, phospho-fructokinase and hexokinase. These barriers are circumvented by new, special enzymes of gluconeogenesis: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructoso-1,6-diphosphatase and glucose-6-phosphatase. The conversion of lactate to glucose begins with the oxidation of lactate, by the action of lactate dehydrogenase, to pyruvate. In the presence of ATP, pyruvate carboxylase and CO2 convert pyruvat to oxaloacetate. The enzyme, phosphoenolpyruvate carboxykinase (PEPCK) transfers oxaloacetate to phosphoenolpyruvate in the presence of GTP and by elimination of CO2. Thus, with the help of these two enzymes, and lactate dehydrogenase, lactate can be converted to oxaloacetate. The pyruvate and oxaloacetate are the intermediary products of catabolic pathway of many glycolytic amino acids. The next steps of reversal glycolysis continue just to formation of fructose-1,6-diphosphate, the substrate for fructose-1, 6-diphosphatase. Fructose-6-phosphate, formed by elimination of inorganic phosphate, converts to glucose-6 phosphate (G6P). The energy required for the hepatic synthesis of glucose from lactate is derived from the oxidation of fatty acids. In the liver and kidney, G6P can be dephosphorylated to glucose by the enzyme glucose 6-phosphatase. This is the final step in

gluconeogenesis means the generation (*genesis*) of new (*neo*) glucose.

IntermediaryMetabolism.html).

**4.4 Gluconeogenesis** 

& Stumvoll, 2001).

the gluconeogenesis pathway.

Lactate, formed by the oxidation of glucose in skeletal muscles and by erythrocytes through the proceses of anaerobic glycolysis, is transported to the liver and kidney, where it re-forms glucose, which again become available via the circulation for oxidation in the tissues. This process is known as the Cory cycle or lactic acid cycle (Fig 5).

#### **4.4.2 Glucose-alanine cycle**

It has been noted that of the amino acids transported from muscles to the liver during starvation or under the action of cortisol, alanine predominate. Glucose-alanine cycle represents a cycling glucose from the liver to the muscles and alanine from muscles to liver, effecting a net transfer of amino nitrogen from muscle to liver and free energy from liver to muscle. At the level of muscles, pyruvate, formed by glycolysis, transforms to alanine by the action of alanine transaminase (ALT) or glutamate pyruvat transaminase (GPT). The reaction is freely reversible; at the level of hepatocytes alanine transfers to pyruvate by the action of the same anzyme (Fig 5).

Glycerol, necessary for the synthesis of triacylglicerols and glycerophosholipids is derived, initially, from the blood glucose since free glycerol cannot be utilised readily for the synthesis of these lipids in tissues. Instead of free glycerol, adipose tissue uses -glycero phosphate or "active glycerol" produced during degradation of glucose by glycolysis.

Fig. 5. Cory cycle , glucose-alanine cycle and glycerol-glucose cycle

Hypoglycemia as a Pathological Result in Medical Praxis 119

Insulin has a hypoglycemic effect. Secretion of insulin is a response to increased glucose level in the blood. In addition to the direct effects of hyperglycemia in enhancing the uptake of glucose into both the liver and peripheral tissues, the hormon insulin plays a central role in the regulation of the blood glucose concentration. Similarly, as blood glucose falls, the

Glucagon, as a direct antagonist of insulin, has a hyperglycemic effect. Secretion of glucagon is a response to decreased glucose level in the blood (Chattoraj & Watts,1986; Ginsberg,

Insulin is a small protein consisting of an alpha chain of 21 amino acids linked by two disulfide (S—S) bridges to a beta chain of 30 amino acids. The precursor of insulin is a proinsulin, which contains C peptide (conective peptide). The conversion of proinsulin to insulin requires biologic proteolysis (Ginsberg,1990; Bowen, 2010; Harper, 1975; Chattoraj &

The stimulus for insulin secretion is a high blood glucose. Insulin is produced by β cells of Langerhans islets in pancreas and is secreted into the blood as a direct response to hyperglycemuia. Beta cells have channels in their plasma membrane that serve as glucose detectors. When blood glucose levels rise (after a meal), insulin is secreted from the pancreas into the pancreatic vein, which empties into the portal vein system, so that insulin traverses the liver before it enters the systemic blood supply. Insulin acts to rapidly lower blood glucose concentration in several ways. It stimulates the active transport of glucose across plasma membranes through glucose transporter (GLUT 4) of muscle and adipose tissue. The liver, brain and red blood cells do not require insulin for glucose uptake. Insulin is an anabolic hormone. It promotes anabolic processes in these cells, such as increasing the rate of glycogenesis, lipidogenesis and proteins synthesis. The cellular uptake of glucose from the blood have the net effect of lowering the high blood glucose levels into the normal range. Insulin stimulates cells in most tissues of the body to preferentially use glucose as their metabolic fuel*.* It increases cellular glucose utilization by inducing the synthesis of several important glycolytic enzymes, namely, hexokinase, glucokinase, phosphofructokinase, and pyruvate kinase. In addition, insulin inhibit gluconeogenesis in liver. All of these physiological effects of insulin serve to lower blood glucose levels. In each case, insulin triggers these effects by binding to the insulin receptor - a transmembrane protein embedded in the plasma membrane of the responding cells. When the the glucose concentration in the blood falls, pancreas stops releasing insulin (Ginsberg, 1990 a, 1990b;

Pancreatic beta cells secrete amylin, a peptide of 37 amino acids. All of its actions (inhibition of glucagon secretion, slowing down the stomach emptying, sending a satiety signal to the brain) tend to supplement those of insulin, reducing the level of glucose in the blood (King, 2011; Silvestre et al, 2001; Young, 2005; users.rcn.com/.../I/IntermediaryMetabolism.html).

Glucagon is another 29 amino acid peptide hormone produced by pancreas. It is a hyperglycemic hormone (Bowen, 2007). Glucagon is produced by alpha (α) cells of

amount of insulin secreted by the pancreatic islets goes down.

1990 a, 1990b; Mayes,1975; King, 2011).

**5.2.1 Insulin** 

Watts,1986).

Bowen, 2007; King, 2011).

**5.2.2 Amylin** 

**5.2.3 Glucagon** 

#### **4.4.3 Glycerol - Glucose cycle**

Glycerol, a product of the continual lipolysis, diffuses out of the tissue into the blood. It is converted back to glucose by gluconeogenic mechanisms in the liver and kidney. Thus, a continous cycle exists in which glucose is transported from the liver to adipose tissue and, hence, glycerol is returned to be synthesized into glucose by the liver. Glycerokinase, which requires ATP, catalyzes the conversion of glycerol to -glycero phosphate. Glycerokinase is present in liver and kidney. The enzyme -glycero phosphate dehydrogenase oxidizes glycero phosphate to the dihydroxacetone phosphate, the component of glycolysis, which enters the glycolytic pathway as a substrate for triose phosphate isomerase (Fig 4). Thus, liver is able to convert glycerol to blood glucose by making use of above enzymes - some of the enzyme of glycolysis and specific enzymes of gluconeogenic pathway, fructose-1,6 diphosphatase and glucose-6-phosphatase(Harris & Crabb, 1997; King, 2011; Mayes,1975).

Glucose produced by gluconeogenesis in the liver and kidney is released into the blood and is subsequently absorbed by all human body cells especially brain, heart, muscle, and red blood cells to meet their metabolic needs. In turn, pyruvate, lactate and glycerol produced in these tissues are returned to liver and kidney to be used as gluconeogenic substrates.

#### **5. The physiological regulation of carbohydrate metabolism**

#### **5.1 Glucose homeostasis**

The maintaining of stable levels of blood glucose is one of the most finely regulated of all homeostatic mechanisms and one in which the liver, extrahepatic tissues and several hormones play a part. Even mild disruptions of glucose homeostasis can have adverse consequences. The physiological post absorptive serum glucose concentration in healthy humans range is 4. 4-5.8 mmol/L (80 to 110 mg/dL).

Glycemia is controlled by several physiological processes. It tends to fluctuate to higher levels after meals, due to intestinal absorption of carbohydrates of low molecular weight present in the diet or broken down polysaccharides, such as starch or glycogen. On the other hand, glucose tends to decrease to lower levels induced by cell metabolism, particularly after stress, temperature regulation and physical exercise. Glucose can also be supplied via breakdown of cellular reserves of glycogen. Another input to glycemia levels is gluconeogenesis, whereby glycogen stored in the liver and skeletal muscles are depleted.

*The stability of the glycemia is a reflection of the balance between the rates of whole body glucose production and glucose utilization.* The glucose homeostasis is tightly regulated by the levels of hormones and substrates in blood and by the physiologic functions of body tissues and organs (Carraway & Watts, 1986; Haris, 1997; King,2011).

#### **5.2 Hormonal regulation of glycemia**

The hormones involved in glycemia regulation include insulin (which lowers the blood sugar level) and other hormons which raise blood sugar, namely antagonists of insulin such as glucagon, epinephrine, cortisol, growth hormone, thyreoid hormones (T3 and T4) and many others. The proper functions of these hormones is precise control of glucose concentration in the blood. Insulin and glucagon are two major hormones involved in regulation of blood glucose level. They are both secreted in response to blood sugar levels, but in opposite fashion. At the same time, enhanced insulin secretion induced increased glucagon secretion.

Insulin has a hypoglycemic effect. Secretion of insulin is a response to increased glucose level in the blood. In addition to the direct effects of hyperglycemia in enhancing the uptake of glucose into both the liver and peripheral tissues, the hormon insulin plays a central role in the regulation of the blood glucose concentration. Similarly, as blood glucose falls, the amount of insulin secreted by the pancreatic islets goes down.

Glucagon, as a direct antagonist of insulin, has a hyperglycemic effect. Secretion of glucagon is a response to decreased glucose level in the blood (Chattoraj & Watts,1986; Ginsberg, 1990 a, 1990b; Mayes,1975; King, 2011).

#### **5.2.1 Insulin**

118 Type 1 Diabetes Complications

Glycerol, a product of the continual lipolysis, diffuses out of the tissue into the blood. It is converted back to glucose by gluconeogenic mechanisms in the liver and kidney. Thus, a continous cycle exists in which glucose is transported from the liver to adipose tissue and, hence, glycerol is returned to be synthesized into glucose by the liver. Glycerokinase, which requires ATP, catalyzes the conversion of glycerol to -glycero phosphate. Glycerokinase is present in liver and kidney. The enzyme -glycero phosphate dehydrogenase oxidizes glycero phosphate to the dihydroxacetone phosphate, the component of glycolysis, which enters the glycolytic pathway as a substrate for triose phosphate isomerase (Fig 4). Thus, liver is able to convert glycerol to blood glucose by making use of above enzymes - some of the enzyme of glycolysis and specific enzymes of gluconeogenic pathway, fructose-1,6 diphosphatase and glucose-6-phosphatase(Harris & Crabb, 1997; King, 2011; Mayes,1975). Glucose produced by gluconeogenesis in the liver and kidney is released into the blood and is subsequently absorbed by all human body cells especially brain, heart, muscle, and red blood cells to meet their metabolic needs. In turn, pyruvate, lactate and glycerol produced in these tissues are returned to liver and kidney to be used as gluconeogenic substrates.

The maintaining of stable levels of blood glucose is one of the most finely regulated of all homeostatic mechanisms and one in which the liver, extrahepatic tissues and several hormones play a part. Even mild disruptions of glucose homeostasis can have adverse consequences. The physiological post absorptive serum glucose concentration in healthy

Glycemia is controlled by several physiological processes. It tends to fluctuate to higher levels after meals, due to intestinal absorption of carbohydrates of low molecular weight present in the diet or broken down polysaccharides, such as starch or glycogen. On the other hand, glucose tends to decrease to lower levels induced by cell metabolism, particularly after stress, temperature regulation and physical exercise. Glucose can also be supplied via breakdown of cellular reserves of glycogen. Another input to glycemia levels is gluconeogenesis, whereby glycogen stored in the liver and skeletal muscles are

*The stability of the glycemia is a reflection of the balance between the rates of whole body glucose production and glucose utilization.* The glucose homeostasis is tightly regulated by the levels of hormones and substrates in blood and by the physiologic functions of body tissues and

The hormones involved in glycemia regulation include insulin (which lowers the blood sugar level) and other hormons which raise blood sugar, namely antagonists of insulin such as glucagon, epinephrine, cortisol, growth hormone, thyreoid hormones (T3 and T4) and many others. The proper functions of these hormones is precise control of glucose concentration in the blood. Insulin and glucagon are two major hormones involved in regulation of blood glucose level. They are both secreted in response to blood sugar levels, but in opposite fashion. At the same time, enhanced insulin secretion induced increased

**5. The physiological regulation of carbohydrate metabolism** 

humans range is 4. 4-5.8 mmol/L (80 to 110 mg/dL).

organs (Carraway & Watts, 1986; Haris, 1997; King,2011).

**5.2 Hormonal regulation of glycemia** 

**4.4.3 Glycerol - Glucose cycle** 

**5.1 Glucose homeostasis** 

depleted.

glucagon secretion.

Insulin is a small protein consisting of an alpha chain of 21 amino acids linked by two disulfide (S—S) bridges to a beta chain of 30 amino acids. The precursor of insulin is a proinsulin, which contains C peptide (conective peptide). The conversion of proinsulin to insulin requires biologic proteolysis (Ginsberg,1990; Bowen, 2010; Harper, 1975; Chattoraj & Watts,1986).

The stimulus for insulin secretion is a high blood glucose. Insulin is produced by β cells of Langerhans islets in pancreas and is secreted into the blood as a direct response to hyperglycemuia. Beta cells have channels in their plasma membrane that serve as glucose detectors. When blood glucose levels rise (after a meal), insulin is secreted from the pancreas into the pancreatic vein, which empties into the portal vein system, so that insulin traverses the liver before it enters the systemic blood supply. Insulin acts to rapidly lower blood glucose concentration in several ways. It stimulates the active transport of glucose across plasma membranes through glucose transporter (GLUT 4) of muscle and adipose tissue. The liver, brain and red blood cells do not require insulin for glucose uptake. Insulin is an anabolic hormone. It promotes anabolic processes in these cells, such as increasing the rate of glycogenesis, lipidogenesis and proteins synthesis. The cellular uptake of glucose from the blood have the net effect of lowering the high blood glucose levels into the normal range. Insulin stimulates cells in most tissues of the body to preferentially use glucose as their metabolic fuel*.* It increases cellular glucose utilization by inducing the synthesis of several important glycolytic enzymes, namely, hexokinase, glucokinase, phosphofructokinase, and pyruvate kinase. In addition, insulin inhibit gluconeogenesis in liver. All of these physiological effects of insulin serve to lower blood glucose levels. In each case, insulin triggers these effects by binding to the insulin receptor - a transmembrane protein embedded in the plasma membrane of the responding cells. When the the glucose concentration in the blood falls, pancreas stops releasing insulin (Ginsberg, 1990 a, 1990b; Bowen, 2007; King, 2011).

#### **5.2.2 Amylin**

Pancreatic beta cells secrete amylin, a peptide of 37 amino acids. All of its actions (inhibition of glucagon secretion, slowing down the stomach emptying, sending a satiety signal to the brain) tend to supplement those of insulin, reducing the level of glucose in the blood (King, 2011; Silvestre et al, 2001; Young, 2005; users.rcn.com/.../I/IntermediaryMetabolism.html).

#### **5.2.3 Glucagon**

Glucagon is another 29 amino acid peptide hormone produced by pancreas. It is a hyperglycemic hormone (Bowen, 2007). Glucagon is produced by alpha (α) cells of

Hypoglycemia as a Pathological Result in Medical Praxis 121

induced gluconeogenesis results, primarily, in increased conversion of glycogenic amino acids (from protein breakdown in peripheral tissues) and glycerol (from fat) into glucose

Human growth hormone (GH; also called somatotropin), the protein of 191 amino acids, secreted by somatotrophs of the anterior part of pytuatary gland, regulates overall body and cell growth, carbohydrate, protein and lipid metabolism, and water-electrolyte balance. The GH-secreting cells are stimulated by growth hormone releasing hormone (GHRH) from hypothalamus and inhibited by somatostatin. The release of GH might be regulated not only by hypothalamic GHRH, but also by ghrelin derived from the stomach (Kojima et al., 2005). GH promotes body growth by binding to receptors on the surface of liver cells and stimulates them to release insulin-like growth factor-1 (IGF-1, also known as somatomedin). GH exerts the hyperglycemic effect, stimulating glycogenolysis and lypolysis in peripheral tissues. In liver, GH also stimulates glycogenolysis and glyconeogenesis (Barry, 1992c;

Thyroid hormones are derivatives of the amino acid tyrosine bound covalently to iodine. The two principal thyroid hormones are: triiodothyronin (T3) and thyroxin (T4). Thyroid hormones receptors are intracellular DNA-binding proteins that function as hormoneresponsive transcription factors. The effect of the hormone-receptor complex binding to DNA is to modulate gene expression, either by stimulating or inhibiting transcription of specific genes. It is likely that all cells in the body are targets for thyroid hormones. Thyroid hormones affect oxidative metabolism, especially the metabolism of carbohydrates. Thyroid hormones enhance glucose absorption and the utilization of carbohydrates. They stimulate both the synthesis and disposal of glucose Hypothyroidism or thyroid hormone deficiency leads to decrease in basal metabolic rate and hypoglycemia (Bowen, 2010; Harper, 1975;

**6. Physiological functions of liver, kidneys and brain in carbohydrate** 

them, the most important are liver, kidneys and brain.

**6.1 The role of liver in carbohydrate metabolism** 

Beside hormones, some organs have the important roles in glycemia regulation. Among

The metabolic activities of the liver are essential for providing fuel to the brain, muscle, and other peripheral organs. The liver removes two-thirds of the glucose from the blood and all of the remaining monosaccharides (Lehninger,1977; Cherrington,1999). The absorbed glucose is converted into glucose 6-phosphate by hexokinase and the liver-specific glucokinase, whose Km (Michaelis constant) for glucose is sufficiently higher than the normal circulating concentration of glucose (5mM). The liver plays a unique role in controlling carbohydrate metabolism by maintaining glucose concentrations in a normal range. It possesses the key enzymes for glucose intake (hexokinase and glucokinase) and for

(Ginsberg, 1992c; Chattoraj & Watts, 1986; Gil, 1992; Litwak & Schmidt, 1997a;

users.rcn.com/.../I/IntermediaryMetabolism.html.)

**5.2.6 Growth hormone (GH)** 

Frohman,1992; Litwak.& Schmidt, 1997b).

**5.2.7 Thyroid hormones** 

Zmire et al, 1999).

**metabolism** 

Langerhans islets as proglucagon and proteolytically processed to yield glucagon within alpha cells of the pancreatic islets. Proglucagon is also expressed within the intestinal tract, where it is processed not into glucagon, but to a family of glucagon-like peptides (GLP) (Ginsberg,1990b; Bowen, 2007). Glucagon is secreted in response to hypoglycemia. It is active in liver and adipose tissue*,* but not in other tissues. This peptide hormone travels through the blood to specific receptors on hepatocytes and adipocytes. When the concentration of glucose in blood decreases, α cells of the pancreas begin to release glucagon. Glucagon stimulates hepatocytes to glycogenolysis and gluconeogenesis, resulting in hyperglycemia. It increases the amount of cAMP and stimulates lipolysis, contributing to reduction of the cellular glucose utilization, the increasing of lipolysis in adipose tissue, providing glycerol and free fatty acids which enter oxidation cycle, producing the chemical energy (ATP) to most cells. Glycerol leaves the adipose tissue, and through the blood enters the hepatocytes where it may serve as the substrate in the process of gluconeogenesis (Fig 5).

#### **5.2.4 Epinephrine (adrenaline)**

Epinephrine is a hormone of adrenal medulla, which consists of masses of neurons that are the part of the sympathetic branch of the autonomic nervous system. Instead of releasing their neurotransmitters at a synapse, these neurons release them into the blood. Thus, although part of the nervous system, the adrenal medulla functions as an endocrine gland. It releases catecholamines: adrenaline (epinephrine) and noradrenalin (also called norepinephrine).

Synthesis of both catecholamines begins with the amino acid tyrosine, which is taken up by chromaffin cells. Called the "fight or flight" hormone, adrenaline prepares the organism for mobilization of large amounts of energy and dealing with stress. Together with cortisol and growth hormone they are named "stress hormones". Following release into blood, these hormones bind adrenergic receptors on target cells, where they induce essentially the same effects as direct sympathetic nervous stimulation. Adrenaline acts on liver and muscles. Mechanisms of the actions of adrenaline are the same as the mechanisms of glucagon. Through augmentation of cAMP in the cells, adrenaline initiates the enzyme cascade which leads to the activation of glycogen phosphorylase, leading to rapid breakdown of glycogen, inhibition of glycogen synthesis, stimulation of glycolysis and production of energy*.* In fat cell, it stimulates lipolysis, providing fatty acids as energy source in many tissues. Stimulation of lipolysis contributes to the reduction of the cellular glucose utilization and aids in conservation of dwindling reserves of blood glucose. The stimulation of hepatocytes to glycogenolysis and gluconeogenesis results in regulation of glycemia (Ginsberg,1990a; Chattoraj, & Watts, 1986; www.ncbi.nlm.nih.gov/books/NBK22429/).

#### **5.2.5 Glucocorticoids**

The glucocorticoids (cortisol as the principal one) get their name from their effect of raising the level of blood glucose. Glucocorticoids are a class of steroid hormones, synthesized and secreted from zone fasciculate of adrenal cortex, that exert distinct effects on liver, skeletal muscles, and adipose tissue (Bjelakovic et al,2008,2009). The effects of cortisol are best described as catabolic, because it promotes protein breakdown and decreases protein synthesis in skeletal muscles. However, in the liver, it stimulates gluconeogenesis, inducing increased gene expression of several enzymes of the gluconeogenic pathway. Cortisolinduced gluconeogenesis results, primarily, in increased conversion of glycogenic amino acids (from protein breakdown in peripheral tissues) and glycerol (from fat) into glucose (Ginsberg, 1992c; Chattoraj & Watts, 1986; Gil, 1992; Litwak & Schmidt, 1997a; users.rcn.com/.../I/IntermediaryMetabolism.html.)

#### **5.2.6 Growth hormone (GH)**

120 Type 1 Diabetes Complications

Langerhans islets as proglucagon and proteolytically processed to yield glucagon within alpha cells of the pancreatic islets. Proglucagon is also expressed within the intestinal tract, where it is processed not into glucagon, but to a family of glucagon-like peptides (GLP) (Ginsberg,1990b; Bowen, 2007). Glucagon is secreted in response to hypoglycemia. It is active in liver and adipose tissue*,* but not in other tissues. This peptide hormone travels through the blood to specific receptors on hepatocytes and adipocytes. When the concentration of glucose in blood decreases, α cells of the pancreas begin to release glucagon. Glucagon stimulates hepatocytes to glycogenolysis and gluconeogenesis, resulting in hyperglycemia. It increases the amount of cAMP and stimulates lipolysis, contributing to reduction of the cellular glucose utilization, the increasing of lipolysis in adipose tissue, providing glycerol and free fatty acids which enter oxidation cycle, producing the chemical energy (ATP) to most cells. Glycerol leaves the adipose tissue, and through the blood enters the hepatocytes where it may serve as the substrate in the process

Epinephrine is a hormone of adrenal medulla, which consists of masses of neurons that are the part of the sympathetic branch of the autonomic nervous system. Instead of releasing their neurotransmitters at a synapse, these neurons release them into the blood. Thus, although part of the nervous system, the adrenal medulla functions as an endocrine gland. It releases catecholamines: adrenaline (epinephrine) and noradrenalin (also called

Synthesis of both catecholamines begins with the amino acid tyrosine, which is taken up by chromaffin cells. Called the "fight or flight" hormone, adrenaline prepares the organism for mobilization of large amounts of energy and dealing with stress. Together with cortisol and growth hormone they are named "stress hormones". Following release into blood, these hormones bind adrenergic receptors on target cells, where they induce essentially the same effects as direct sympathetic nervous stimulation. Adrenaline acts on liver and muscles. Mechanisms of the actions of adrenaline are the same as the mechanisms of glucagon. Through augmentation of cAMP in the cells, adrenaline initiates the enzyme cascade which leads to the activation of glycogen phosphorylase, leading to rapid breakdown of glycogen, inhibition of glycogen synthesis, stimulation of glycolysis and production of energy*.* In fat cell, it stimulates lipolysis, providing fatty acids as energy source in many tissues. Stimulation of lipolysis contributes to the reduction of the cellular glucose utilization and aids in conservation of dwindling reserves of blood glucose. The stimulation of hepatocytes to glycogenolysis and gluconeogenesis results in regulation of glycemia (Ginsberg,1990a;

The glucocorticoids (cortisol as the principal one) get their name from their effect of raising the level of blood glucose. Glucocorticoids are a class of steroid hormones, synthesized and secreted from zone fasciculate of adrenal cortex, that exert distinct effects on liver, skeletal muscles, and adipose tissue (Bjelakovic et al,2008,2009). The effects of cortisol are best described as catabolic, because it promotes protein breakdown and decreases protein synthesis in skeletal muscles. However, in the liver, it stimulates gluconeogenesis, inducing increased gene expression of several enzymes of the gluconeogenic pathway. Cortisol-

Chattoraj, & Watts, 1986; www.ncbi.nlm.nih.gov/books/NBK22429/).

of gluconeogenesis (Fig 5).

norepinephrine).

**5.2.5 Glucocorticoids** 

**5.2.4 Epinephrine (adrenaline)** 

Human growth hormone (GH; also called somatotropin), the protein of 191 amino acids, secreted by somatotrophs of the anterior part of pytuatary gland, regulates overall body and cell growth, carbohydrate, protein and lipid metabolism, and water-electrolyte balance. The GH-secreting cells are stimulated by growth hormone releasing hormone (GHRH) from hypothalamus and inhibited by somatostatin. The release of GH might be regulated not only by hypothalamic GHRH, but also by ghrelin derived from the stomach (Kojima et al., 2005). GH promotes body growth by binding to receptors on the surface of liver cells and stimulates them to release insulin-like growth factor-1 (IGF-1, also known as somatomedin). GH exerts the hyperglycemic effect, stimulating glycogenolysis and lypolysis in peripheral tissues. In liver, GH also stimulates glycogenolysis and glyconeogenesis (Barry, 1992c; Frohman,1992; Litwak.& Schmidt, 1997b).

#### **5.2.7 Thyroid hormones**

Thyroid hormones are derivatives of the amino acid tyrosine bound covalently to iodine. The two principal thyroid hormones are: triiodothyronin (T3) and thyroxin (T4). Thyroid hormones receptors are intracellular DNA-binding proteins that function as hormoneresponsive transcription factors. The effect of the hormone-receptor complex binding to DNA is to modulate gene expression, either by stimulating or inhibiting transcription of specific genes. It is likely that all cells in the body are targets for thyroid hormones. Thyroid hormones affect oxidative metabolism, especially the metabolism of carbohydrates. Thyroid hormones enhance glucose absorption and the utilization of carbohydrates. They stimulate both the synthesis and disposal of glucose Hypothyroidism or thyroid hormone deficiency leads to decrease in basal metabolic rate and hypoglycemia (Bowen, 2010; Harper, 1975; Zmire et al, 1999).

#### **6. Physiological functions of liver, kidneys and brain in carbohydrate metabolism**

Beside hormones, some organs have the important roles in glycemia regulation. Among them, the most important are liver, kidneys and brain.

#### **6.1 The role of liver in carbohydrate metabolism**

The metabolic activities of the liver are essential for providing fuel to the brain, muscle, and other peripheral organs. The liver removes two-thirds of the glucose from the blood and all of the remaining monosaccharides (Lehninger,1977; Cherrington,1999). The absorbed glucose is converted into glucose 6-phosphate by hexokinase and the liver-specific glucokinase, whose Km (Michaelis constant) for glucose is sufficiently higher than the normal circulating concentration of glucose (5mM). The liver plays a unique role in controlling carbohydrate metabolism by maintaining glucose concentrations in a normal range. It possesses the key enzymes for glucose intake (hexokinase and glucokinase) and for

Hypoglycemia as a Pathological Result in Medical Praxis 123

are concentrated in hypothalamic area. Hypothalamus is the site of afferent and efferent stimuli between special nuclei and - cells and cells of pancreas, and it regulates induction/inhibition of glucose output from the liver. Insulin gets across the blood-brain barrier, links to special hypothalamic receptors, regulating peripheral glucose (the hypothalamus-pancreas) (Halmos & Suba, 2011). In addition, the hypothalamus can affect metabolic functions by neuroendocrine connections: the hypothalamus-pancreas axis (the control of insulin and glucagone release), the hypothalamus-adrenal axis (the control of the release of adrenaline and noradrenaline) and the hypothalamus-pituitary axis (release of glucocorticoids and thyroid hormones through adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) control, respectively, which modulate glucose

Recently, evidence is accumulating demonstrating that gastrointestinal hormones (peptides) are involved in regulating glucose metabolism through humoral gut–brain axis. Some of them are: ghrelin, neuropeptide Y (NPY), cholecystokinin - CCK, gastric inhibitory polypeptide - GIP, glucagon-like peptide (GLP) etc. (Kojima & Kangawa, 2005; King, 2011;

Generally, hypoglycemia is defined as a serum glucose level below (3.8 mmol/L or, 70 mg/dL). Hypoglycemia is a rare disorder, considered as pathophysiological state rather than a disease. Just as pain and fever require identification of the underlying condition, hypoglycemia warrants diagnosis of the primary disorder causing the low plasma glucose

The symptoms of hypoglycemia are not specific. For this reason it is necessary to demonstrate a low plasma glucose concentration concomitant with symptoms and subsequent relief of symptoms by correction of the hypoglycemia, i.e., Whipple's tirade. This triade can be considered to be the basis for a patient's symptoms, regardless of the cause of hypoglycemia (Service,1992). Whipple's triad considers: 1) symptoms consistent with hypoglycemia, 2) a low plasma glucose concentration, and 3) relief of symptoms after the plasma glucose level is raised. Hypoglycemia most often affects those at the extremes of age, such as infants and the elderly, but may happen at any age. Given the survival value of maintenance of the plasma glucose concentration, it is not surprising that very effective physiological mechanisms prevent or rapidly correct hypoglycemia

Hypoglycemic symptoms are related to the brain and the sympathetic nervous system. The central nervous system requires glucose as the preferred energy substrate. Though the brain accounts for only about 10% of body weight, it uses more than 30% of blood glucose. Hypoglycemic symptoms are mediated through both central and peripheral nervous systems. Once plasma glucose concentration fall belows 3.8 mmol/L or 70 mg/dL, a sequence of events begins to maintain glucose delivery to the brain and prevent

The first of all events is the stimulation of the autonomic nervous system and, after that, release of neuroendocrine hormones (counter-regulatory or anti-insulin hormones). Peripheral autonomic symptoms (adrenergic), including sweating, irritability, tremulousness, anxiety, tachycardia, and hunger, serve as an early warning system and

Korner & Leibel, 2003; Neary et al, 2004 ; Young, 2005).

**7. Neuro-endocrine defence to hypoglycemia** 

metabolism.

concentration.

have evolved.

hypoglycemia.

releasing of glucose from hepatocytes (glucose-6-phosphatase). The liver has a great capability for synthesis and storing of glycogen (glycogenesis), and, in opposite direction, for glycogen breakdown (glycogenolysis). Also, the liver is the place for gluconeogenesis*.* (King.2011, Nordlie et al, 1999; Radziuk & Pye, 2001).

Glucose-6-phosphatase liberates free glucose molecules from hepatocytes into blood, catalyzing the following reaction: glucose-6-phosphate + H2O glucose + Pi.The substrate for this enzyme is glucose-6-phosphate, the product of glycogenolysis or the end product of gluconeogenesis (Berg, 2002; Radziuk & Pye, 2001; Raddatz & Ramadori, 2007; Yamashita et al, 2001; Berg, 2002) .

#### **6.2 The role of kidney in carbohydrate metabolism**

Kidney may play a significant role in carbohydrate metabolism under both physiological and pathological conditions due to renal gluconeogenesis (King, 2011; Gerich et al., 2001). Although the liver is the major site of glucose homeostasis, the kidney plays a vital role in the overall process of regulating the level of blood glucose. Glucose is continually filtered by the glomeruli but is ordinarily returned completely to the blood by the enzymatic reabsorptive system of the renal tubules. The reabsorption of glucose is a process which is similar to that responsible for the absorption of this sugar from the intestine. The capacity of tubular system to reabsorb glucose is limited by the capacity of enzymatically systems of the tubule cells to a rate of about 350 mg/ minute, representing as tubular maximum for glucose (Tmg). Due to that capacity of the kidney, the definitive urine doesn't contain glucose. When the blood levels of glucose are elevated, the glomerular filtrate may contain more glucose then can be reabsorbed; the excess passes into urine to produce glycosuria. In normal individuals, glucosuria occures when the venous blood sugar excesss 9.5-10 mmol/L (170- 180 mg/100 ml). This level of the venous blood sugar is termed the renal threshold for glucose (Mayes,1975; Woerle & Stumvoll, 2001).

#### **6.3 The role of brain in carbohydrate metabolism**

Glucose is the major energy source for maintenance of brain metabolism and function*,*  except during prolonged starvation. However, the brain has limited glucose reserves and needs a continuous supply of glucose. Endogenous glucose provides more than 90% of energy needed for brain function (Cryer, 1997; Gerich et al, 2001; Halmos & Suba,2011; King, 2011). Since the brain cannot synthesize glucose or store more than a few minutes' supply as glycogen, it is critically dependent on a continuous supply of glucose from the circulation. Fatty acids do not serve as fuel for the brain, because they are bound to albumins in plasma and so do not traverse the blood-brain barrier. In prolonged starvation, ketone bodies, generated by the liver, partly replace glucose as fuel for the brain, (Cahill, 2006).

Glucose is transported into brain cells by the glucose transporter GLUT3. This transporter has a low KM for glucose (1.6 mM). Thus, the brain is usually provided with a constant supply of glucose. At normal (or elevated) arterial glucose concentrations, the rate of bloodto-brain glucose transport exceeds the rate of brain glucose metabolism. However, as arterial glucose levels fall below the physiological range, blood-to-brain glucose transport becomes limiting to brain glucose metabolism, and ultimately survival.

Nowadays it is hypothesised that the brain, in particular the hypothalamus, has a great role in carbohydrate metabolism and glucose homeostasis. The brain is an insulin-sensitive organ. Brain-insulin action is required for intact glucose homeostasis. Receptors for insulin

releasing of glucose from hepatocytes (glucose-6-phosphatase). The liver has a great capability for synthesis and storing of glycogen (glycogenesis), and, in opposite direction, for glycogen breakdown (glycogenolysis). Also, the liver is the place for gluconeogenesis*.*

Glucose-6-phosphatase liberates free glucose molecules from hepatocytes into blood, catalyzing the following reaction: glucose-6-phosphate + H2O glucose + Pi.The substrate for this enzyme is glucose-6-phosphate, the product of glycogenolysis or the end product of gluconeogenesis (Berg, 2002; Radziuk & Pye, 2001; Raddatz & Ramadori, 2007; Yamashita et

Kidney may play a significant role in carbohydrate metabolism under both physiological and pathological conditions due to renal gluconeogenesis (King, 2011; Gerich et al., 2001). Although the liver is the major site of glucose homeostasis, the kidney plays a vital role in the overall process of regulating the level of blood glucose. Glucose is continually filtered by the glomeruli but is ordinarily returned completely to the blood by the enzymatic reabsorptive system of the renal tubules. The reabsorption of glucose is a process which is similar to that responsible for the absorption of this sugar from the intestine. The capacity of tubular system to reabsorb glucose is limited by the capacity of enzymatically systems of the tubule cells to a rate of about 350 mg/ minute, representing as tubular maximum for glucose (Tmg). Due to that capacity of the kidney, the definitive urine doesn't contain glucose. When the blood levels of glucose are elevated, the glomerular filtrate may contain more glucose then can be reabsorbed; the excess passes into urine to produce glycosuria. In normal individuals, glucosuria occures when the venous blood sugar excesss 9.5-10 mmol/L (170- 180 mg/100 ml). This level of the venous blood sugar is termed the renal threshold for

Glucose is the major energy source for maintenance of brain metabolism and function*,*  except during prolonged starvation. However, the brain has limited glucose reserves and needs a continuous supply of glucose. Endogenous glucose provides more than 90% of energy needed for brain function (Cryer, 1997; Gerich et al, 2001; Halmos & Suba,2011; King, 2011). Since the brain cannot synthesize glucose or store more than a few minutes' supply as glycogen, it is critically dependent on a continuous supply of glucose from the circulation. Fatty acids do not serve as fuel for the brain, because they are bound to albumins in plasma and so do not traverse the blood-brain barrier. In prolonged starvation, ketone bodies,

Glucose is transported into brain cells by the glucose transporter GLUT3. This transporter has a low KM for glucose (1.6 mM). Thus, the brain is usually provided with a constant supply of glucose. At normal (or elevated) arterial glucose concentrations, the rate of bloodto-brain glucose transport exceeds the rate of brain glucose metabolism. However, as arterial glucose levels fall below the physiological range, blood-to-brain glucose transport becomes

Nowadays it is hypothesised that the brain, in particular the hypothalamus, has a great role in carbohydrate metabolism and glucose homeostasis. The brain is an insulin-sensitive organ. Brain-insulin action is required for intact glucose homeostasis. Receptors for insulin

generated by the liver, partly replace glucose as fuel for the brain, (Cahill, 2006).

limiting to brain glucose metabolism, and ultimately survival.

(King.2011, Nordlie et al, 1999; Radziuk & Pye, 2001).

**6.2 The role of kidney in carbohydrate metabolism** 

glucose (Mayes,1975; Woerle & Stumvoll, 2001).

**6.3 The role of brain in carbohydrate metabolism** 

al, 2001; Berg, 2002) .

are concentrated in hypothalamic area. Hypothalamus is the site of afferent and efferent stimuli between special nuclei and - cells and cells of pancreas, and it regulates induction/inhibition of glucose output from the liver. Insulin gets across the blood-brain barrier, links to special hypothalamic receptors, regulating peripheral glucose (the hypothalamus-pancreas) (Halmos & Suba, 2011). In addition, the hypothalamus can affect metabolic functions by neuroendocrine connections: the hypothalamus-pancreas axis (the control of insulin and glucagone release), the hypothalamus-adrenal axis (the control of the release of adrenaline and noradrenaline) and the hypothalamus-pituitary axis (release of glucocorticoids and thyroid hormones through adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) control, respectively, which modulate glucose metabolism.

Recently, evidence is accumulating demonstrating that gastrointestinal hormones (peptides) are involved in regulating glucose metabolism through humoral gut–brain axis. Some of them are: ghrelin, neuropeptide Y (NPY), cholecystokinin - CCK, gastric inhibitory polypeptide - GIP, glucagon-like peptide (GLP) etc. (Kojima & Kangawa, 2005; King, 2011; Korner & Leibel, 2003; Neary et al, 2004 ; Young, 2005).

#### **7. Neuro-endocrine defence to hypoglycemia**

Generally, hypoglycemia is defined as a serum glucose level below (3.8 mmol/L or, 70 mg/dL). Hypoglycemia is a rare disorder, considered as pathophysiological state rather than a disease. Just as pain and fever require identification of the underlying condition, hypoglycemia warrants diagnosis of the primary disorder causing the low plasma glucose concentration.

The symptoms of hypoglycemia are not specific. For this reason it is necessary to demonstrate a low plasma glucose concentration concomitant with symptoms and subsequent relief of symptoms by correction of the hypoglycemia, i.e., Whipple's tirade. This triade can be considered to be the basis for a patient's symptoms, regardless of the cause of hypoglycemia (Service,1992). Whipple's triad considers: 1) symptoms consistent with hypoglycemia, 2) a low plasma glucose concentration, and 3) relief of symptoms after the plasma glucose level is raised. Hypoglycemia most often affects those at the extremes of age, such as infants and the elderly, but may happen at any age. Given the survival value of maintenance of the plasma glucose concentration, it is not surprising that very effective physiological mechanisms prevent or rapidly correct hypoglycemia have evolved.

Hypoglycemic symptoms are related to the brain and the sympathetic nervous system. The central nervous system requires glucose as the preferred energy substrate. Though the brain accounts for only about 10% of body weight, it uses more than 30% of blood glucose. Hypoglycemic symptoms are mediated through both central and peripheral nervous systems. Once plasma glucose concentration fall belows 3.8 mmol/L or 70 mg/dL, a sequence of events begins to maintain glucose delivery to the brain and prevent hypoglycemia.

The first of all events is the stimulation of the autonomic nervous system and, after that, release of neuroendocrine hormones (counter-regulatory or anti-insulin hormones). Peripheral autonomic symptoms (adrenergic), including sweating, irritability, tremulousness, anxiety, tachycardia, and hunger, serve as an early warning system and

Hypoglycemia as a Pathological Result in Medical Praxis 125

Enzyme sucrase decomposes disaccharide sacharose to glucose and fructose molecules. There is a number of reports of an inherited deficiency of the disaccharidases, sucrase and isomalatase, occurring within the mucosa of the small intestine. Symptoms occur in early childhood following ingestion of sucrose. The symptoms are the same as those described in lactase deficiency except that they are evoked by the ingestion of table sugar *(*Gary,1978 ;

Glucose Galactose Malabsorption (GGM) is a genetic disorder caused by a defect in glucose and galactose transport across the intestinal brush border membrane. Normally, lactose in milk is broken down into glucose and galactose by lactase, an ectoenzyme on the brush border, and the hexoses, having nearly identical chemical structure, are transported into the cell by the Na+-glucose cotransporter SGLT1 (Fig 6). The mutations causing the defect in sugar transport have been identified (Gary, 1978; Wright, 1998; Wright et al, 2002; Wright et

Fig. 6. Sugar transport across intestinal apithelium (Wright et al., 2004, modified)

Glucose-galactose malabsorption is an autosomal recessive disorder in which affected individuals inherit two defective copies of the SGLT1 gene, located on chromosome 22. This disease presents in newborn infants as a life-threatening diarrhea. The diarrhea ceases within 1 h of removing oral intake of lactose, glucose and galactose, but promptly returns with the introduction of one or more of the offending sugars into the diet. We conclude that mutations in the SGLT1 gene are the cause of glucose-galactose malabsorption, and sugar transport is impaired mainly because the mutant proteins are either truncated or are not

**8.1.1.3 Sucrase deficiency** 

Sinclair,1979; Tietz et al,1986 )

al, 2004; Wright, 2003).

**8.1.1.4 Glucose galactose malabsorption** 

preceed the central neuroglycopenic symptoms due to cerebral glucose deprivation (e.g., confusion, paralysis, seizures, and coma) (Zammitt & Frier, 2005).

A hierarchical hormonal response exists in response to decreasing blood glucose levels. As blood glucose drops, pancreatic β-cell reduce insulin secretion. If blood glucose drops further, the pancreatic α-cell secrete glucagon and the adrenal medulla release adrenaline. Both, glucagon and adrenaline, act rapidly to increase glucose availability and therefore are the two major counter-regulatory hormones. Cortisol and growth hormone are also released, but they are unable to prevent prolonged hypoglycemia withouth the preliminary actions of glucagone and adrenaline. In sensing hypoglycemia, the nutritionally deprived brain also stimulates the sympathetic nervous system, leading to neurogenic symptoms.Decreased levels of glucose lead to deficient cerebral glucose availability i.e., neuroglycopenia, that can manifest as confusion, headache, difficulty with concentration. If the symptoms are overlooked, there can be irreversible brain damage. Eventually, the patient may go into coma and death. The adrenergic symptoms often precede the neuroglycopenic symptoms and, thus, provide an early warning system for the patient, (Cryer,1997; Heijboer et., 2006).

#### **8. Hypoglycemia as a result of inherited or acquired disorder of carbohydrates metabolism**

#### **8.1 Hypoglicemia and inborn errors of carbohydrate metabolism**

Hypoglycemia is not a disease by itself, but its presence is an indication of a problematic health condition. As a relatively frequent common event in the pediatric newborn period (childhood), hypoglicemia may be a consequence of some inborn errors of carbohydrate metabolism (Service,1992; Sinclair,1979; Caraway & Watts, 1986).

#### **8.1.1 Defects in digestion & absorption of carbohydrates**

#### **8.1.1.1 Inherited lactase deficiency (alactasia)**

Enzyme lactase is necessary to digest lactose to glucose and galactose in the small intestine. Lactase deficiency is a rare congenital disorder in which infants are born without lactase*.* If lactase is deficient,undigested lactose enters the large intestine, where it is fermented by colonic bacteria, producing lactic acid and gases (hydrogen, methane, carbon dioxide).The gas produced creates the uncomfortable feeling of gut distention and the annoying problem of flatulence. The lactic acid produced by the microorganisms is osmotically active and draws water into the intestine, as does any undigested lactose, resulting in diarrhea. As children are weaned and milk becomes less prominent in their diets, lactase activity normally declines to about 5 to 10% of the level at birth (Gary,1978; Sinclair,1979; Tietz et al,1986). The simplest treatment is to avoid the consumption of products containing much lactose. Alternatively, the enzyme lactase can be ingested with milk products.

#### **8.1.1.2 Lactose intolerance**

Lactose intolerance is an inability to digest significant amounts of lactose due to an absence of the enzyme lactase in adult intestines. The symptoms of this disorder, which include diarrhea and general discomfort, can be relieved by eliminating milk from the diet (Maxton et al, 1990; Tietz et al,1986).

#### **8.1.1.3 Sucrase deficiency**

124 Type 1 Diabetes Complications

preceed the central neuroglycopenic symptoms due to cerebral glucose deprivation (e.g.,

A hierarchical hormonal response exists in response to decreasing blood glucose levels. As blood glucose drops, pancreatic β-cell reduce insulin secretion. If blood glucose drops further, the pancreatic α-cell secrete glucagon and the adrenal medulla release adrenaline. Both, glucagon and adrenaline, act rapidly to increase glucose availability and therefore are the two major counter-regulatory hormones. Cortisol and growth hormone are also released, but they are unable to prevent prolonged hypoglycemia withouth the preliminary actions of glucagone and adrenaline. In sensing hypoglycemia, the nutritionally deprived brain also stimulates the sympathetic nervous system, leading to neurogenic symptoms.Decreased levels of glucose lead to deficient cerebral glucose availability i.e., neuroglycopenia, that can manifest as confusion, headache, difficulty with concentration. If the symptoms are overlooked, there can be irreversible brain damage. Eventually, the patient may go into coma and death. The adrenergic symptoms often precede the neuroglycopenic symptoms and, thus, provide an early warning system for the patient,

confusion, paralysis, seizures, and coma) (Zammitt & Frier, 2005).

**8. Hypoglycemia as a result of inherited or acquired disorder of** 

Hypoglycemia is not a disease by itself, but its presence is an indication of a problematic health condition. As a relatively frequent common event in the pediatric newborn period (childhood), hypoglicemia may be a consequence of some inborn errors of carbohydrate

Enzyme lactase is necessary to digest lactose to glucose and galactose in the small intestine. Lactase deficiency is a rare congenital disorder in which infants are born without lactase*.* If lactase is deficient,undigested lactose enters the large intestine, where it is fermented by colonic bacteria, producing lactic acid and gases (hydrogen, methane, carbon dioxide).The gas produced creates the uncomfortable feeling of gut distention and the annoying problem of flatulence. The lactic acid produced by the microorganisms is osmotically active and draws water into the intestine, as does any undigested lactose, resulting in diarrhea. As children are weaned and milk becomes less prominent in their diets, lactase activity normally declines to about 5 to 10% of the level at birth (Gary,1978; Sinclair,1979; Tietz et al,1986). The simplest treatment is to avoid the consumption of products containing much lactose. Alternatively, the enzyme lactase can be ingested with

Lactose intolerance is an inability to digest significant amounts of lactose due to an absence of the enzyme lactase in adult intestines. The symptoms of this disorder, which include diarrhea and general discomfort, can be relieved by eliminating milk from the diet (Maxton

**8.1 Hypoglicemia and inborn errors of carbohydrate metabolism** 

metabolism (Service,1992; Sinclair,1979; Caraway & Watts, 1986).

**8.1.1 Defects in digestion & absorption of carbohydrates** 

**8.1.1.1 Inherited lactase deficiency (alactasia)** 

(Cryer,1997; Heijboer et., 2006).

**carbohydrates metabolism** 

milk products.

**8.1.1.2 Lactose intolerance** 

et al, 1990; Tietz et al,1986).

Enzyme sucrase decomposes disaccharide sacharose to glucose and fructose molecules. There is a number of reports of an inherited deficiency of the disaccharidases, sucrase and isomalatase, occurring within the mucosa of the small intestine. Symptoms occur in early childhood following ingestion of sucrose. The symptoms are the same as those described in lactase deficiency except that they are evoked by the ingestion of table sugar *(*Gary,1978 ; Sinclair,1979; Tietz et al,1986 )

#### **8.1.1.4 Glucose galactose malabsorption**

Glucose Galactose Malabsorption (GGM) is a genetic disorder caused by a defect in glucose and galactose transport across the intestinal brush border membrane. Normally, lactose in milk is broken down into glucose and galactose by lactase, an ectoenzyme on the brush border, and the hexoses, having nearly identical chemical structure, are transported into the cell by the Na+-glucose cotransporter SGLT1 (Fig 6). The mutations causing the defect in sugar transport have been identified (Gary, 1978; Wright, 1998; Wright et al, 2002; Wright et al, 2004; Wright, 2003).

Fig. 6. Sugar transport across intestinal apithelium (Wright et al., 2004, modified)

Glucose-galactose malabsorption is an autosomal recessive disorder in which affected individuals inherit two defective copies of the SGLT1 gene, located on chromosome 22. This disease presents in newborn infants as a life-threatening diarrhea. The diarrhea ceases within 1 h of removing oral intake of lactose, glucose and galactose, but promptly returns with the introduction of one or more of the offending sugars into the diet. We conclude that mutations in the SGLT1 gene are the cause of glucose-galactose malabsorption, and sugar transport is impaired mainly because the mutant proteins are either truncated or are not

Hypoglycemia as a Pathological Result in Medical Praxis 127

still form UDP-galactose from glucose. This explains the normal growth and development of affected children in spite of the galactose-free diets which are used to control the symptoms

In adults, the toxicity of dietary galactose appears to be less severe, due, in part, to the metabolism by alternative metabolic pathway of galactose-1-P by UDP-glucose pyrophosphorylase**,** which apparently can accept galactose-1-P in place of glucose-1-P. The levels of this enzyme may increase in the liver of galactosemic individuals, in order to

Uridine diphosphate galactose-4-epimerase (UDP-galactose-4-epimerase, GALE) converts UDP-galactose to UDP-glucose. Reaction is freely reversible (Fig 7). In this manner, glucose, as a unique monosaccharide in systemic blood circulation in healthy subects , can be converted to galactose in many different tissues of human body if UDP-galactose-4-epimerase is present. Galactosemia due to epimerase deficiency is the rarest and most poorly understood form. In most patients with GALE (or epimerase) deficiency, the defect presenting with clinical features is similar to classic galactosemia. The treatment for children with generalised GALE

Since galactose is an essential component of galactoproteins and galactolipids, theoretically, in generalised GALE deficiency, no endogenous production of galactose is possible, with a

The most common treatment is to remove galactose (and lactose) from the diet. The enigma of galactosemia is that, although elimination of galactose from the diet prevents liver disease and cataract development, the majority of patients still suffer from central nervous system malfunction, most commonly a delayed acquisition of language skills. Females will also

Fructose is monosacharide found in honey and in numerous vegetables and fruits. Disacharide sucrose consists of one molecule fructose attached to a molecule of glucose. It should be mentioned that fructose constitutes the main sugar of seminal fluid. Three inherited abnormalities in fructose metabolism have been identified: essential fructosuria, hereditary fructose intolerance and hereditary fructose-1,6-bisphosphatase deficiency

After absorption by the proces of facilitated diffusion, fructose enters hepatocytes , by the portal blood. A specific kinase, fructokinase, in liver and kidney catalyzes the phosphorylation of fructose to fructose 1-phosphate*.* Fructose 1-phosphate is cleaved to Dglyceraldehyde and dihydroxyacetone phosphate by aldolase B, an enzyme found in the liver. D-glyceraldehyde enters glycolysis via phosphorylation to glyceraldehyde 3 phosphate catalyzed by triokinase. The two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, may either be degraded by glycolysis or may be substrates for aldolase A , the enzyme which forms fructose1,6-diphosphate and hence

gluconeogenesis, which is the fate of much of the fructose metabolized in the liver.

Fructose ATP ructose 1 P ADP

 

*fructokinase F*

*aldolaseB*

Fructose 1 P dihydroxyacetone P glyceraldehyde

accommodate the metabolism of galactose, later, after 10 years. **8.1.2.3 Uridine diphosphate galactose 4-epimerase deficiency** 

deficiency, as with GALT deficiency is the restriction of dietary galactose.

resulting deficiency in galactolipids and galacto-proteoglycanes production.

display ovarian failure (Sarkar et al, 2010; Segal, 1989; Walter et al, 1999).

**8.1.3 The inherited abnormalities in fructose metabolism** 

(Baerlocher et al,1978; Gitzelmann et al., 1989; Froesch, 1978).

of the disease (Harper, 1975).

targeted properly to the cell membrane. The glucose and galactose, if left untransported, draw water out of the body into the intestinal lumen, resulting in diarrhea.

Although no cure exists for GGM, patients can control their symptoms (diarrhea) by removing lactose, sucrose and glucose from their diets. Infants showing a prenatal diagnosis of GGM will thrive on a fructose-based replacement formula and will later continue their "normal" physical development on a fructose-based solid diet. Older children and adults with severe GGM can also manage their symptoms on a fructose-based diet and may show improved glucose tolerance and even clinical remission as they age (Wright et al., 2001; Wright,1998; Gary, 1978).

#### **8.1.2 Galactosemia**

Galactose is found in the disaccharide lactose, the principal milk sugar, made from galactose and glucose. It will be recalled that galactose is required in the body, not only in the formation of milk lactose during lactogenesis in lactating mammary glands, but also as a constituent of the glucosphyngolipids (cerebrosides, globosides, gangliosides) and for the synthesis of mucopolysaccharides (MPS) or glucosaminoglycanes (GAG). Glucosaminoglycanes are linked to core proteins forming proteoglycanes. Proteoglycanes and glucosphyngolipids perform numerous vital functions within the body, some of which still remain to be studied (King, 2011; Segal, 1989).

The disruption of galactose metabolism is referred to as galactosemia. Galactosemia can result from deficiencies of three different enzymes: galactose-1-phosphate uridyl transferase (GALT), galactokinase (GALK) and uridine diphosphate galactose 4-epimerase (GALE).

#### **8.1.2.1 Galactokinase deficiency**

Galactokinase is the first enzyme in the pathway of galactose metabolism, converting galactose to galactose-1-P. The only consequence of galactokinase deficiency is the development of cataract.

#### **8.1.2.2 Deficiency of galactose-1-phosphate uridyl transferase (GALT)**

The most common and severe form of galactosemia, called classic galactosemia or Galactosemia Type 1, is an inherited deficiency of GALT, the enzyme that converts galactose-1-phosphate (galactose-1-P) to uridine diphosphate galactose (UDPgalactose). Absence or deficiency of GALT prevents the conversion of galactose into glucose in liver. People with absent or deficient GALT have intolerance to galactose.

When an infant or neonate is given milk the blood galactose level is markedly elevated (galactosemia), and galactose is found in urine (galactosuria). These can cause severe damage to eyes, kidneys, liver and brain. Afflicted infants fail to thrive. They vomit or have diarrhea after consuming milk and enlargement of liver and jaundice are common, sometimes progressing to cirrhosis. Cataracts will form, and lethargy and retarded mental development are also common. A cataract, the clouding of the normally clear lens of the eye, is a consequence of the accumulation of galactose in the lens of the eye; in the presence of high galactose amount and of aldose reductase, galactose is reduced to galactitol. The absence of the transferase in red blood cells is a definitive diagnostic criterion (Mayatepek et al, 2010).

These problems can be prevented by removing galactose and lactose from the diet. In classic galactosemia conversion of UDP-galactose to UDP-glucose is blocked. The epimerase reaction is, however, present in adequate amounts, so that the galactosemic individual can

targeted properly to the cell membrane. The glucose and galactose, if left untransported,

Although no cure exists for GGM, patients can control their symptoms (diarrhea) by removing lactose, sucrose and glucose from their diets. Infants showing a prenatal diagnosis of GGM will thrive on a fructose-based replacement formula and will later continue their "normal" physical development on a fructose-based solid diet. Older children and adults with severe GGM can also manage their symptoms on a fructose-based diet and may show improved glucose tolerance and even clinical remission as they age (Wright et al., 2001;

Galactose is found in the disaccharide lactose, the principal milk sugar, made from galactose and glucose. It will be recalled that galactose is required in the body, not only in the formation of milk lactose during lactogenesis in lactating mammary glands, but also as a constituent of the glucosphyngolipids (cerebrosides, globosides, gangliosides) and for the synthesis of mucopolysaccharides (MPS) or glucosaminoglycanes (GAG). Glucosaminoglycanes are linked to core proteins forming proteoglycanes. Proteoglycanes and glucosphyngolipids perform numerous vital functions within the body, some of which

The disruption of galactose metabolism is referred to as galactosemia. Galactosemia can result from deficiencies of three different enzymes: galactose-1-phosphate uridyl transferase (GALT), galactokinase (GALK) and uridine diphosphate galactose 4-epimerase (GALE).

Galactokinase is the first enzyme in the pathway of galactose metabolism, converting galactose to galactose-1-P. The only consequence of galactokinase deficiency is the

The most common and severe form of galactosemia, called classic galactosemia or Galactosemia Type 1, is an inherited deficiency of GALT, the enzyme that converts galactose-1-phosphate (galactose-1-P) to uridine diphosphate galactose (UDPgalactose). Absence or deficiency of GALT prevents the conversion of galactose into glucose in liver.

When an infant or neonate is given milk the blood galactose level is markedly elevated (galactosemia), and galactose is found in urine (galactosuria). These can cause severe damage to eyes, kidneys, liver and brain. Afflicted infants fail to thrive. They vomit or have diarrhea after consuming milk and enlargement of liver and jaundice are common, sometimes progressing to cirrhosis. Cataracts will form, and lethargy and retarded mental development are also common. A cataract, the clouding of the normally clear lens of the eye, is a consequence of the accumulation of galactose in the lens of the eye; in the presence of high galactose amount and of aldose reductase, galactose is reduced to galactitol. The absence of the transferase in red blood cells is a definitive diagnostic criterion (Mayatepek et

These problems can be prevented by removing galactose and lactose from the diet. In classic galactosemia conversion of UDP-galactose to UDP-glucose is blocked. The epimerase reaction is, however, present in adequate amounts, so that the galactosemic individual can

**8.1.2.2 Deficiency of galactose-1-phosphate uridyl transferase (GALT)** 

People with absent or deficient GALT have intolerance to galactose.

draw water out of the body into the intestinal lumen, resulting in diarrhea.

Wright,1998; Gary, 1978).

still remain to be studied (King, 2011; Segal, 1989).

**8.1.2.1 Galactokinase deficiency** 

development of cataract.

al, 2010).

**8.1.2 Galactosemia** 

still form UDP-galactose from glucose. This explains the normal growth and development of affected children in spite of the galactose-free diets which are used to control the symptoms of the disease (Harper, 1975).

In adults, the toxicity of dietary galactose appears to be less severe, due, in part, to the metabolism by alternative metabolic pathway of galactose-1-P by UDP-glucose pyrophosphorylase**,** which apparently can accept galactose-1-P in place of glucose-1-P. The levels of this enzyme may increase in the liver of galactosemic individuals, in order to accommodate the metabolism of galactose, later, after 10 years.

#### **8.1.2.3 Uridine diphosphate galactose 4-epimerase deficiency**

Uridine diphosphate galactose-4-epimerase (UDP-galactose-4-epimerase, GALE) converts UDP-galactose to UDP-glucose. Reaction is freely reversible (Fig 7). In this manner, glucose, as a unique monosaccharide in systemic blood circulation in healthy subects , can be converted to galactose in many different tissues of human body if UDP-galactose-4-epimerase is present.

Galactosemia due to epimerase deficiency is the rarest and most poorly understood form. In most patients with GALE (or epimerase) deficiency, the defect presenting with clinical features is similar to classic galactosemia. The treatment for children with generalised GALE deficiency, as with GALT deficiency is the restriction of dietary galactose.

Since galactose is an essential component of galactoproteins and galactolipids, theoretically, in generalised GALE deficiency, no endogenous production of galactose is possible, with a resulting deficiency in galactolipids and galacto-proteoglycanes production.

The most common treatment is to remove galactose (and lactose) from the diet. The enigma of galactosemia is that, although elimination of galactose from the diet prevents liver disease and cataract development, the majority of patients still suffer from central nervous system malfunction, most commonly a delayed acquisition of language skills. Females will also display ovarian failure (Sarkar et al, 2010; Segal, 1989; Walter et al, 1999).

#### **8.1.3 The inherited abnormalities in fructose metabolism**

Fructose is monosacharide found in honey and in numerous vegetables and fruits. Disacharide sucrose consists of one molecule fructose attached to a molecule of glucose. It should be mentioned that fructose constitutes the main sugar of seminal fluid. Three inherited abnormalities in fructose metabolism have been identified: essential fructosuria, hereditary fructose intolerance and hereditary fructose-1,6-bisphosphatase deficiency (Baerlocher et al,1978; Gitzelmann et al., 1989; Froesch, 1978).

After absorption by the proces of facilitated diffusion, fructose enters hepatocytes , by the portal blood. A specific kinase, fructokinase, in liver and kidney catalyzes the phosphorylation of fructose to fructose 1-phosphate*.* Fructose 1-phosphate is cleaved to Dglyceraldehyde and dihydroxyacetone phosphate by aldolase B, an enzyme found in the liver. D-glyceraldehyde enters glycolysis via phosphorylation to glyceraldehyde 3 phosphate catalyzed by triokinase. The two triose phosphates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, may either be degraded by glycolysis or may be substrates for aldolase A , the enzyme which forms fructose1,6-diphosphate and hence gluconeogenesis, which is the fate of much of the fructose metabolized in the liver.

> Fructose ATP ructose 1 P ADP Fructose 1 P dihydroxyacetone P glyceraldehyde *fructokinase F aldolaseB*

Hypoglycemia as a Pathological Result in Medical Praxis 129

Glycogen storage disease type I (glucose-6-phosphatase deficiency; von Gierke disease; Hepato-renal glycogenoses). GSD type I (or von Gierke disease) is an autosomal recessive disorder that is caused by deficient G6Pase activity. Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and kidneys, plays a critical role in blood glucose homoeostasis, providing glucose during starvation. One of the important functions of the liver and, to a lesser extent, of the kidney cortex is to provide glucose during conditions of starvation. Glucose is formed from gluconeogenic precursors in both tissues, and in the liver also from glycogen. Both glycogenolysis and gluconeogenesis result in the formation of glucose 6-phosphate, which has to be hydrolysed by G6Pase before being liberated as

Glucose-6-phosphatase is the enzyme that catalyzes the last step of glycogenolysis and gluconeogenesis in liver and kidneys, i.e. the hydrolysis of glucose 6-phosphate to free glucose and inorganic phosphate. Its genetic deficiency is characterized by the association of hepatomegaly and nephromegaly due to the accumulation of large amounts of glycogen in

GSD type Ia is the most frequent form of glycogenosis, accounting for about 80% of the cases. It is caused by a lack of G6Pase activity, which is easily demonstrated by measuring the activity of this enzyme in a liver biopsy specimen. The deficiency of G6Pase activity is

On the basis of the imunological studies, by using the antibodies against to several of five components of glucose-6-phosphatase, GSD type I has been subcategorised into types a, b, and c, with type a as the most common, but all types have similar clinical manifestation as hypoglycemia and hepatomegaly due to the deposition of glycogen with normal structure. Fasting-induced hypoglycemia may be extreme, and with combination with lactic acidosis. Although severely affected patients may suffer brain damage in early infancy, this is not usually the case, and the most patients have normal intelligence. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-

**8.1.4.1 Glycogen storage disease type I** 

glucose into the circulation.

these organs, with hypoglycaemia and lactic acidosis.

caused by mutations in the gene encoding this enzyme.

Fig. 7. The glucose-6-phosphatase system

#### **8.1.3.1 Essential fructosuria**

Essential fructosuria is a benign metabolic disorder caused by the lack of fructokinase, which is normally present in liver and kidney cortex. The disorder is asymptomatic and it may go undiagnosed.

#### **8.1.3.2 Hereditary fructose intolerance (aldolase B deficiency)**

Hereditary fructose intolerance is a potentially lethal disorder resulting from a lack of aldolase B which decomposes fructose-1-phosphate to dihydroxyaceton-phosphate plus glycerine aldehyde. The disorder is characterized by severe hypoglycemia and vomiting following fructose or sacharose intake. Prolonged intake of fructose by infants with this defect leads to vomiting, poor feeding*,* jaundice, hepatomegaly, hemorrhage and eventually death. The hypoglycemia that results, following fructose uptake, is caused by fructose-1 phosphate inhibition of glycogenolysis, by interfering with the phosphorylase reaction and inhibition of gluconeogenesis at the deficient aldolase step. Patients remain symptom free on a diet devoid of fructose and sucrose(Baerlocher et al, 1978; Gitzelman at al, 1989; Odièvre et al, 1978).

#### **8.1.3.3 Hereditary fructose-1,6-bisphosphatase deficiency**

This disordered is characterized by hypoglycemia, ketosis and lactic acidosis and oftern lethal course in newborn infants. Due to enzyme defect gluconeogenesis is severely impaired. Gluconeogenis precursors, such as amino acids, lactate and ketones, accumulate as soon as liver glycogen stores are depleted (Asberg et al, 2010; Baerlocher et al, 2010; Froesch, 1978; Mortensen, 2006; Song, 2010; Zaidi, 2009).

#### **8.1.4 The glycogen storage diseases (GSDs)**

Glycogen is the storage form of glucose and is present in virtually all living cells, although the liver is primary organ for storage and subsequent release of glucose into the circulation. Glycogen biosynthesis from glucose (glycogenesis), along with the release of glucose from glycogen by the process of glycogenolysis, is highly regulated process that aids in the maintaing of normal blood glucose concentration during fasting.

During the last century, patients who have deficient activity in virtually every enzyme important in the normal synthesis, degradation or regulation of glycogen have been identified. Most of them are inherited in an autosomal recessive manner. Several inborn errors of glycogen metabolism have been described, and they result from mutations in genes that code for proteins involved in various steps of glycogen synthesis, degradation, or regulation. Glycogen storage diseases are characterized by an abnormal tissue concentration (>70 mg per gram of liver or 15 mg per gram of muscle tissue of normal or abnormal structure of glycogen**).** Hypoglycemia is the main biochemical consequence of GSD type I and some of the other GSDs. The basis of dietary therapy is nutritional manipulation to prevent hypoglycemia and improve metabolic dysfunction (Heller et al, 2008; Mayatepek et al, 2010).

All glycogenosis may divide in two groups: hepatic and muscular forms of glycogenosis. The various hepatic enzyme deficiencies are expressed primarily as hypoglycemia and hepatomegaly. In glycogenosis type I, III and VI there is limitation in the output of glucose from hepatic tissue, and hypoglycemia is an prominent laboratory sing. (Goldberg & Slonim, 1993; Heller et al, 2008; Howell, 1978; Wolfsdorf.& Weinstein, 2003).

#### **8.1.4.1 Glycogen storage disease type I**

128 Type 1 Diabetes Complications

Essential fructosuria is a benign metabolic disorder caused by the lack of fructokinase, which is normally present in liver and kidney cortex. The disorder is asymptomatic and it

Hereditary fructose intolerance is a potentially lethal disorder resulting from a lack of aldolase B which decomposes fructose-1-phosphate to dihydroxyaceton-phosphate plus glycerine aldehyde. The disorder is characterized by severe hypoglycemia and vomiting following fructose or sacharose intake. Prolonged intake of fructose by infants with this defect leads to vomiting, poor feeding*,* jaundice, hepatomegaly, hemorrhage and eventually death. The hypoglycemia that results, following fructose uptake, is caused by fructose-1 phosphate inhibition of glycogenolysis, by interfering with the phosphorylase reaction and inhibition of gluconeogenesis at the deficient aldolase step. Patients remain symptom free on a diet devoid of fructose and sucrose(Baerlocher et al, 1978; Gitzelman at al, 1989;

This disordered is characterized by hypoglycemia, ketosis and lactic acidosis and oftern lethal course in newborn infants. Due to enzyme defect gluconeogenesis is severely impaired. Gluconeogenis precursors, such as amino acids, lactate and ketones, accumulate as soon as liver glycogen stores are depleted (Asberg et al, 2010; Baerlocher et al, 2010;

Glycogen is the storage form of glucose and is present in virtually all living cells, although the liver is primary organ for storage and subsequent release of glucose into the circulation. Glycogen biosynthesis from glucose (glycogenesis), along with the release of glucose from glycogen by the process of glycogenolysis, is highly regulated process that aids in the

During the last century, patients who have deficient activity in virtually every enzyme important in the normal synthesis, degradation or regulation of glycogen have been identified. Most of them are inherited in an autosomal recessive manner. Several inborn errors of glycogen metabolism have been described, and they result from mutations in genes that code for proteins involved in various steps of glycogen synthesis, degradation, or regulation. Glycogen storage diseases are characterized by an abnormal tissue concentration (>70 mg per gram of liver or 15 mg per gram of muscle tissue of normal or abnormal structure of glycogen**).** Hypoglycemia is the main biochemical consequence of GSD type I and some of the other GSDs. The basis of dietary therapy is nutritional manipulation to prevent hypoglycemia and improve metabolic dysfunction (Heller et al, 2008; Mayatepek et

All glycogenosis may divide in two groups: hepatic and muscular forms of glycogenosis. The various hepatic enzyme deficiencies are expressed primarily as hypoglycemia and hepatomegaly. In glycogenosis type I, III and VI there is limitation in the output of glucose from hepatic tissue, and hypoglycemia is an prominent laboratory sing. (Goldberg &

Slonim, 1993; Heller et al, 2008; Howell, 1978; Wolfsdorf.& Weinstein, 2003).

**8.1.3.2 Hereditary fructose intolerance (aldolase B deficiency)** 

**8.1.3.3 Hereditary fructose-1,6-bisphosphatase deficiency** 

Froesch, 1978; Mortensen, 2006; Song, 2010; Zaidi, 2009).

maintaing of normal blood glucose concentration during fasting.

**8.1.4 The glycogen storage diseases (GSDs)** 

**8.1.3.1 Essential fructosuria** 

may go undiagnosed.

Odièvre et al, 1978).

al, 2010).

Glycogen storage disease type I (glucose-6-phosphatase deficiency; von Gierke disease; Hepato-renal glycogenoses). GSD type I (or von Gierke disease) is an autosomal recessive disorder that is caused by deficient G6Pase activity. Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and kidneys, plays a critical role in blood glucose homoeostasis, providing glucose during starvation. One of the important functions of the liver and, to a lesser extent, of the kidney cortex is to provide glucose during conditions of starvation. Glucose is formed from gluconeogenic precursors in both tissues, and in the liver also from glycogen. Both glycogenolysis and gluconeogenesis result in the formation of glucose 6-phosphate, which has to be hydrolysed by G6Pase before being liberated as glucose into the circulation.

Glucose-6-phosphatase is the enzyme that catalyzes the last step of glycogenolysis and gluconeogenesis in liver and kidneys, i.e. the hydrolysis of glucose 6-phosphate to free glucose and inorganic phosphate. Its genetic deficiency is characterized by the association of hepatomegaly and nephromegaly due to the accumulation of large amounts of glycogen in these organs, with hypoglycaemia and lactic acidosis.

GSD type Ia is the most frequent form of glycogenosis, accounting for about 80% of the cases. It is caused by a lack of G6Pase activity, which is easily demonstrated by measuring the activity of this enzyme in a liver biopsy specimen. The deficiency of G6Pase activity is caused by mutations in the gene encoding this enzyme.

Fig. 7. The glucose-6-phosphatase system

On the basis of the imunological studies, by using the antibodies against to several of five components of glucose-6-phosphatase, GSD type I has been subcategorised into types a, b, and c, with type a as the most common, but all types have similar clinical manifestation as hypoglycemia and hepatomegaly due to the deposition of glycogen with normal structure. Fasting-induced hypoglycemia may be extreme, and with combination with lactic acidosis. Although severely affected patients may suffer brain damage in early infancy, this is not usually the case, and the most patients have normal intelligence. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-

Hypoglycemia as a Pathological Result in Medical Praxis 131

A key role of gluconeogenesis is in the maintenance of blood sugar. Deficiency of any enzyme participating in gluconeogenesis can lead to hypoglycemia with lactic acidosis. Inborn deficiencies are known of each of the four enzymes of the glycolytic-gluconeogenic pathway that ensure a unidirectional flux from pyruvate to glucose: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-

Pyruvate carboxylase, a member of the biotin-dependent enzyme family, catalyses the ATPdependent carboxylation of pyruvate to oxaloacetate. Pyruvate carboxylase is an important enzyme in gluconeogenesis. Deficiency of pyruvate carboxylase can lead to hypoglycemia with lactic acidosis. Pyruvate carboxylase deficiency is a rare disorder that can cause developmental delay and failure to thrive starting in the neonatal or early infantile period. PCD results in malfunction of the citric acid cycle and gluconeogenesis, thereby depriving the body of energy. Based on the severity of the clinical presentation and the biochemical disturbances, two clinical forms have been described. The milder form A presents in infancy with delayed neurological development, chronic lactic acidemia and a normal lactate to pyruvate ratio. Longer survival, but with severe clinical sequelae, is common in the mild form A. The complex form B presents neonatally or in early infancy with severe metabolic acidosis, lactic acidosis, ketosis, and hepatomegaly (Jitrapakdee &. Wallace, 1999; Ahmad

Phosphoenolpyruvate carboxykinase is an important enzyme in gluconeogenesis. It is found in both cytosol and mitochondria of the liver cells. Deficiency of the enzyme, a rare inherited disorder, can cause severe, persistent neonatal hypoglycaemia and liver impairment

Fructose-1,6-diphosphatase (F1,6DPase) catalyzes the conversion of fructose-1,6 diphosphate (F1,6DP) to fructose-6-phosphate. The F1,6BPase reaction is a major point of control of gluconeogenesis. Fructose-1,6-diphosphatase (FDPase) deficiency is an autosomal recessive disorder caused by a mutation of the FDP1 gene and results in impaired

Patients with FDPase deficiency typically present in the newborn period with symptoms or signs related to hypoglycemia and metabolic acidosis following ingestion of fructose. Patients lacking FDPase accumulate intrahepatocellular fructose-1,6-bisphosphate (FDP) which inhibits gluconeogenesis and, if intracellular phosphate stores are depleted, inhibits glycogenolysis. The inability to convert lactic acid or glycerol into glucose leads to hypoglycemia and lactic acidosis (Asberg et al, 2010; Hellerud, 2010; Song, 2011; Zaidi, 2009; Mortensen, 2006). The accumulation of fructose 1,6-diphosphate (FDP) inhibits

Glycerol kinase (GK) catalyzes the phosphorylation of glycerol to glycerol 3-phosphate (G3P) which is important in the formation of triacylglycerol (TAG) and fat storage. GK is at the interface of fat and carbohydrate metabolism. GK deficiency (GKD) is an X-linked

**8.1.5 Disorders of gluconeogenesis** 

phosphatase (van den Berghe, 1996).

et al., 1999).

**8.1.5.1 Pyruvate carboxylase deficiency (PCD)** 

**8.1.5.2 Phosphoenolpyruvate carboxykinase deficiency** 

**8.1.5.3 Fructose-1,6-diphosphatase (FDPase) deficiency** 

phosphorylase a in liver, the first enzyme of glycogenoslysis.

**8.1.5.4 Glycero kinase deficiency** 

gluconeogenesis ( Froesch, 1978; Gitzelmann et al, 1989; Hellerud, 2010).

(Hommes et al, 1976; Vidnes & Sovik, 1976).

CoA. It is likely that the nervous system is able to metabolise such substance as ketones and lactic acid.

Glucose-6-phosphatase consists of a hydrolase, whose catalytic site faces the lumen of the organelle and of translocases required for the transport of glucose 6-phosphate, Pi and glucose (Figure 7). Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum by a specific transporter before being hydrolysed by glucose-6-phosphatase, a transmembrane protein with its catalytic site oriented towards the lumen of the endoplasmic reticulum. Glycogen storage disease type Ia (GSD Ia) is due to a defect in glucose-6-phosphatase catalytic site and glycogen storage disease type Ib, to a defect in the glucose 6-phosphate transporter (Schaftingen and Gerin, 2002).

#### **8.1.4.2 Debranching enzyme deficiency (type III glycogen storage disease; limit dextrinosis; Cori's disease)**

Type III glycogen storage disease (amylo-1, 6-glucosidase (debrancher) deficiency) most often affects only liver, but may affect muscles as well. In this form of glycogen storage disease, a glycogen accumulates which has a structure resembling the limit dextrin produced by degradation of of glycogen by phosphorylase a , which is free of debrancher (amylo-1, 6-glucosidase) activity. Early in life, hepatomegaly and growth retardation may be striking. In contrast to patients with Type I glycogenosis, moderate enlargement of the spleen is sometime seen. Glycogen of abnormal structure frequently accumulates in muscle and heart, as well as in the liver. In the older patients it may cause a chronic progressive myopathy and cardiomegaly. With muscle involvement, the serum creatine phosphokinase (CPK) activity is elevated, and patients are usually classified as having type III b diseases. There is no renal enlargement in this disease. Generally, the clinical course of this disease is milder than that of Type I glycogenoses.

#### **8.1.4.3 Type VI glycogenosis (hepatic phosphorylase deficiency; Hers' disease)**

Large group of patients with hepatic forms of the glycogen storage diseases have increased hepatic glycogen (with normal structure ) and a reduction to about 25 percent normal of hepatic phosphorylase activity. Patients with increased liver glycogen and profound reduction in liver phosphorylase activity (and normal activating system) continued to be observed. Hypoglycemia is present (Wolfsdorf & Weinstein, 2003; Heller et al, 2008).

#### **8.1.4.4 Glycogen synthase deficiency (type 0 glycogen storage disease; GSD0)**

Type 0 glycogen storage disease (GSD0) is caused by deficiency of the hepatic isoform of glycogen synthase (Weinstein et al, 2006). Although GSD0 has been classified as a glycogen storage disease, this is a misnomer. In contrast to all other types of glycogenoses, which are characterized by increased glycogen storage, deficiency of glycogen synthase causes a marked decrease in liver glycogen content. GSD0 is the only GSD not associated with hepatomegaly and hypoglycemia typically is milder than in the other types of GSD (Wolfsdorf & Weinstein, 2003; Weinstein, 2006).

Patients with GSD0 have fasting ketotic hypoglycemia. Most children are cognitively and developmentally normal. Until recently, the definitive diagnosis of GSD0 depended on the demonstration of decreased hepatic glycogen on a liver biopsy. The need for an invasive procedure may be one reason that this condition has been infrequently diagnosed. Mutation analysis of the GYS2 gene (12p12.2) is a non-invasive method for making this diagnosis in patients suspected to have this disorder.

#### **8.1.5 Disorders of gluconeogenesis**

130 Type 1 Diabetes Complications

CoA. It is likely that the nervous system is able to metabolise such substance as ketones and

Glucose-6-phosphatase consists of a hydrolase, whose catalytic site faces the lumen of the organelle and of translocases required for the transport of glucose 6-phosphate, Pi and glucose (Figure 7). Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum by a specific transporter before being hydrolysed by glucose-6-phosphatase, a transmembrane protein with its catalytic site oriented towards the lumen of the endoplasmic reticulum. Glycogen storage disease type Ia (GSD Ia) is due to a defect in glucose-6-phosphatase catalytic site and glycogen storage disease type Ib, to a defect in the

Type III glycogen storage disease (amylo-1, 6-glucosidase (debrancher) deficiency) most often affects only liver, but may affect muscles as well. In this form of glycogen storage disease, a glycogen accumulates which has a structure resembling the limit dextrin produced by degradation of of glycogen by phosphorylase a , which is free of debrancher (amylo-1, 6-glucosidase) activity. Early in life, hepatomegaly and growth retardation may be striking. In contrast to patients with Type I glycogenosis, moderate enlargement of the spleen is sometime seen. Glycogen of abnormal structure frequently accumulates in muscle and heart, as well as in the liver. In the older patients it may cause a chronic progressive myopathy and cardiomegaly. With muscle involvement, the serum creatine phosphokinase (CPK) activity is elevated, and patients are usually classified as having type III b diseases. There is no renal enlargement in this disease. Generally, the clinical course of this disease is

glucose 6-phosphate transporter (Schaftingen and Gerin, 2002).

**8.1.4.2 Debranching enzyme deficiency (type III glycogen storage disease; limit** 

**8.1.4.3 Type VI glycogenosis (hepatic phosphorylase deficiency; Hers' disease)** 

observed. Hypoglycemia is present (Wolfsdorf & Weinstein, 2003; Heller et al, 2008). **8.1.4.4 Glycogen synthase deficiency (type 0 glycogen storage disease; GSD0)** 

Large group of patients with hepatic forms of the glycogen storage diseases have increased hepatic glycogen (with normal structure ) and a reduction to about 25 percent normal of hepatic phosphorylase activity. Patients with increased liver glycogen and profound reduction in liver phosphorylase activity (and normal activating system) continued to be

Type 0 glycogen storage disease (GSD0) is caused by deficiency of the hepatic isoform of glycogen synthase (Weinstein et al, 2006). Although GSD0 has been classified as a glycogen storage disease, this is a misnomer. In contrast to all other types of glycogenoses, which are characterized by increased glycogen storage, deficiency of glycogen synthase causes a marked decrease in liver glycogen content. GSD0 is the only GSD not associated with hepatomegaly and hypoglycemia typically is milder than in the other types of GSD

Patients with GSD0 have fasting ketotic hypoglycemia. Most children are cognitively and developmentally normal. Until recently, the definitive diagnosis of GSD0 depended on the demonstration of decreased hepatic glycogen on a liver biopsy. The need for an invasive procedure may be one reason that this condition has been infrequently diagnosed. Mutation analysis of the GYS2 gene (12p12.2) is a non-invasive method for making this diagnosis in

lactic acid.

**dextrinosis; Cori's disease)** 

milder than that of Type I glycogenoses.

(Wolfsdorf & Weinstein, 2003; Weinstein, 2006).

patients suspected to have this disorder.

A key role of gluconeogenesis is in the maintenance of blood sugar. Deficiency of any enzyme participating in gluconeogenesis can lead to hypoglycemia with lactic acidosis. Inborn deficiencies are known of each of the four enzymes of the glycolytic-gluconeogenic pathway that ensure a unidirectional flux from pyruvate to glucose: pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6 phosphatase (van den Berghe, 1996).

#### **8.1.5.1 Pyruvate carboxylase deficiency (PCD)**

Pyruvate carboxylase, a member of the biotin-dependent enzyme family, catalyses the ATPdependent carboxylation of pyruvate to oxaloacetate. Pyruvate carboxylase is an important enzyme in gluconeogenesis. Deficiency of pyruvate carboxylase can lead to hypoglycemia with lactic acidosis. Pyruvate carboxylase deficiency is a rare disorder that can cause developmental delay and failure to thrive starting in the neonatal or early infantile period. PCD results in malfunction of the citric acid cycle and gluconeogenesis, thereby depriving the body of energy. Based on the severity of the clinical presentation and the biochemical disturbances, two clinical forms have been described. The milder form A presents in infancy with delayed neurological development, chronic lactic acidemia and a normal lactate to pyruvate ratio. Longer survival, but with severe clinical sequelae, is common in the mild form A. The complex form B presents neonatally or in early infancy with severe metabolic acidosis, lactic acidosis, ketosis, and hepatomegaly (Jitrapakdee &. Wallace, 1999; Ahmad et al., 1999).

#### **8.1.5.2 Phosphoenolpyruvate carboxykinase deficiency**

Phosphoenolpyruvate carboxykinase is an important enzyme in gluconeogenesis. It is found in both cytosol and mitochondria of the liver cells. Deficiency of the enzyme, a rare inherited disorder, can cause severe, persistent neonatal hypoglycaemia and liver impairment (Hommes et al, 1976; Vidnes & Sovik, 1976).

#### **8.1.5.3 Fructose-1,6-diphosphatase (FDPase) deficiency**

Fructose-1,6-diphosphatase (F1,6DPase) catalyzes the conversion of fructose-1,6 diphosphate (F1,6DP) to fructose-6-phosphate. The F1,6BPase reaction is a major point of control of gluconeogenesis. Fructose-1,6-diphosphatase (FDPase) deficiency is an autosomal recessive disorder caused by a mutation of the FDP1 gene and results in impaired gluconeogenesis ( Froesch, 1978; Gitzelmann et al, 1989; Hellerud, 2010).

Patients with FDPase deficiency typically present in the newborn period with symptoms or signs related to hypoglycemia and metabolic acidosis following ingestion of fructose. Patients lacking FDPase accumulate intrahepatocellular fructose-1,6-bisphosphate (FDP) which inhibits gluconeogenesis and, if intracellular phosphate stores are depleted, inhibits glycogenolysis. The inability to convert lactic acid or glycerol into glucose leads to hypoglycemia and lactic acidosis (Asberg et al, 2010; Hellerud, 2010; Song, 2011; Zaidi, 2009; Mortensen, 2006). The accumulation of fructose 1,6-diphosphate (FDP) inhibits phosphorylase a in liver, the first enzyme of glycogenoslysis.

#### **8.1.5.4 Glycero kinase deficiency**

Glycerol kinase (GK) catalyzes the phosphorylation of glycerol to glycerol 3-phosphate (G3P) which is important in the formation of triacylglycerol (TAG) and fat storage. GK is at the interface of fat and carbohydrate metabolism. GK deficiency (GKD) is an X-linked

Hypoglycemia as a Pathological Result in Medical Praxis 133

Symptomatic hypoglycemia is uncommon in liver diseases because glucose homeostasis can be maintained with as little as 20 per cent of healthy parenchymal cells, but biochemical hypoglycemia has been reported in a wide variety of aquired hepatic diseases. The hypoglycemia of Reye's syndrome and sepsis, as well as alcohol hypoglycemia, are considered to be the consequence of hepatic disturbance. Acute viral hepatitis results in serious impairment in hepatic glycogen synthesis and gluconeogenesis and frequently gives rise to fasting hypoglycemia (Felig et al., 1970). Glycogen stores rapidly disappear as liver disease (including

Reye syndrome is a fatal disease, most commonly occuring following some virus infections (influenza A, influenza B, herpes, varicella zoster and several other common viral infections). Epidemiologic evidence suggests that aspirin plays a potentiating role in the pathogenesis of this syndrome. The hepatic dysfunction appears to be the primary error and the direct result of a mitochondrial disturbance that causes secondary metabolic derangement, (hyperamoniemia, hypoprotrombinemia without hyperbilirubinemia),

Hypoglycaemia in patients with hepatocellular carcinoma usually occurs during the terminal stage of the illness, but there are patients with hepatocellular carcinoma who develop hypoglycaemia early in the course of their illness. Hypoglycemia occurs predominantly as a paraneoplastic manifestation of hepatocellular carcinoma, (Sorlini et al.,

Ethanol is a potent hypoglycemic agent, causing decreased endogenous glucose production and glycogenolysis. The volume of alcohol intake is correlated with the degree of resulting hypoglycemia (Raghavan et al., 2007; Smeeks, 2008). Ethanol-induced hypoglycemia arises from inhibition of gluconeogenesis as a result of the increase in the NADH-NAD ratio, which suppresses the conversion of lactate to pyruvate, glycerophosphate to dihydroxyacetone phosphate, and glutamate to -ketoglutarate and several tricarboxylic cycle reactions. Ethanol reduces rates of hepatic glucose production, supresses plasma insulin concentration, increases plasma lactate concentration, beta hydroxybutirate, glycerol and free fatty acids, and increases lactate-pyruvate and beta-hydroxybutirate-acetoacetate ratios. Hypoglycemia usually develops within 6 to 36 hours of the ingestion, of even moderate amounts of ethanol by persons chronically malnouriched or by healthy persons who have missed one of the meals. Healthy children are especially susceptible to ethanol hypoglycemia. Blood ethanol levels may not be elevated when the patient is hypoglycemic. Healthy children are especially susceptible to ethanol hypoglycemia. Blood ethanol levels may not be elevated when the patient is

Kidneys play a significant role in carbohydrate metabolism under both physiological and pathological conditions due to renal gluconeogenesis. Hypoglycemia in patients with renal failure may be due to inadequate gluconeogenic substrate availability. It seems that disturbances in renal gluconeogenesis together with lower degradation of insulin played the key role in creating hypoglycaemia in patients with renal diseases. Hypoglycemia should be suspected in any patient with renal failure who exhibits any change in mental or neurologic status (Arem, 1989; Rutsky,1978). Also, kidney disease is a frequent cause of adverse medication reactions due to the problems in excretion of certain medications and, therefore,

cirrhosis due to alcoholism) progresses, causing recurrent hypoglycemia (Service, 1992).

**9.1.1 Hypoglycemia in liver disorders** 

including hypoglycemia**.** 

2010; Thipaporn et al., 2005; Young, 2007).

hypoglycemic( Arky, 1989; Badawy, 1977; Service, 1992).

causing hypoglycemia in older adults (Gerich, 2001)**.** 

**9.1.2 Hypoglycemia in kidney disorders** 

inborn error of metabolism that is characterized biochemically by hyperglycerolemia and glyceroluria and is due to mutations within or deletions of the GK gene on Xp21 (Rahib et al, 2007). Isolated GKD can occur in patients with or without symptoms, mainly due to disturbed energy homeostasis associated with hyperketotic hypoglycemia (Sjarif et al, 2004). The greater importance of glycerol as a gluconeogenetic substrate in children than in adults may explain the episodes in young patients with GKD, often elicited by catabolic stress (Hellerud et al, 2004).

#### **8.1.6 Leucine-sensitive hypoglycemia**

This type of hypoglycemia was reported in 1956 by Cochrane who described children who became hypoglycemic on a casein-rich diet and whose symptoms worsened on feeding on high protein and low carbohydrate diet. The one-third of all infants with unexplained hypoglycemia may be sensitive to leucin. Presentation is most often in the first year of life.A common symptom is the development of convulsion after milk-feeding, (MacMullen et al., 2001) .

Leucine produces hypoglycemia by causing the release of insulin from the pancreas islet cells. The hypoglycemia found in maple syrup urine diseases is probably caused by high circulating levels of leucin. Two-thirds of the infants who have had leucine-sensitive hypoglycemia have subsequently mental retardation and neurological disorders (Roe &, Kogut, 1982; Sinclair, 1979).

#### **8.1.7 Hyperinsulinism/hyperammonemia (HI/HA) syndrome**

Hyperinsulinism is the most common cause of hypoglycemia in early infancy. Congenital hyperinsulinism, is usually caused by genetic defects in beta-cell regulation, including a syndrome of hyperinsulinism plus hyperammonemia (Kelly et al., 2001; Kogut, 1982; Stanley, 1997).

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptomatic, persistent elevations of plasma ammonium levels. The disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. These data confirm the importance of allosteric regulation of GDH, as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for the regulation of GDH activity by both GTP and ATP (MacMullen et al, 2001; Kogut,1982).

#### **9. Hypoglycemia as results of acquired disorders of carbohydrates metabolism**

#### **9.1 Liver and kidney disorders**

Although hypoglycemia is usually linked with diabetes, there are various types of conditions, which are generally rare, that can cause it even in those who do not have diabetes. Any disorder or abnormality in the functioning of the liver can disturb the process of blood-sugar regulation, resulting in hypoglycemia. On the other hand, disorders in kidney can cause problems in excretion of certain medications. Hence, kidney disorders can be one of the major causes of low blood sugar.

#### **9.1.1 Hypoglycemia in liver disorders**

132 Type 1 Diabetes Complications

inborn error of metabolism that is characterized biochemically by hyperglycerolemia and glyceroluria and is due to mutations within or deletions of the GK gene on Xp21 (Rahib et al, 2007). Isolated GKD can occur in patients with or without symptoms, mainly due to disturbed energy homeostasis associated with hyperketotic hypoglycemia (Sjarif et al, 2004). The greater importance of glycerol as a gluconeogenetic substrate in children than in adults may explain the episodes in young patients with GKD, often elicited by catabolic stress

This type of hypoglycemia was reported in 1956 by Cochrane who described children who became hypoglycemic on a casein-rich diet and whose symptoms worsened on feeding on high protein and low carbohydrate diet. The one-third of all infants with unexplained hypoglycemia may be sensitive to leucin. Presentation is most often in the first year of life.A common symptom is the development of convulsion after milk-feeding, (MacMullen et al.,

Leucine produces hypoglycemia by causing the release of insulin from the pancreas islet cells. The hypoglycemia found in maple syrup urine diseases is probably caused by high circulating levels of leucin. Two-thirds of the infants who have had leucine-sensitive hypoglycemia have subsequently mental retardation and neurological disorders (Roe &,

Hyperinsulinism is the most common cause of hypoglycemia in early infancy. Congenital hyperinsulinism, is usually caused by genetic defects in beta-cell regulation, including a syndrome of hyperinsulinism plus hyperammonemia (Kelly et al., 2001; Kogut, 1982;

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptomatic, persistent elevations of plasma ammonium levels. The disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. These data confirm the importance of allosteric regulation of GDH, as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for the regulation of GDH activity by both GTP and ATP (MacMullen et al, 2001;

Although hypoglycemia is usually linked with diabetes, there are various types of conditions, which are generally rare, that can cause it even in those who do not have diabetes. Any disorder or abnormality in the functioning of the liver can disturb the process of blood-sugar regulation, resulting in hypoglycemia. On the other hand, disorders in kidney can cause problems in excretion of certain medications. Hence, kidney disorders can

**9. Hypoglycemia as results of acquired disorders of carbohydrates** 

**8.1.7 Hyperinsulinism/hyperammonemia (HI/HA) syndrome** 

(Hellerud et al, 2004).

Kogut, 1982; Sinclair, 1979).

Stanley, 1997).

Kogut,1982).

**metabolism** 

**9.1 Liver and kidney disorders** 

be one of the major causes of low blood sugar.

2001) .

**8.1.6 Leucine-sensitive hypoglycemia** 

Symptomatic hypoglycemia is uncommon in liver diseases because glucose homeostasis can be maintained with as little as 20 per cent of healthy parenchymal cells, but biochemical hypoglycemia has been reported in a wide variety of aquired hepatic diseases. The hypoglycemia of Reye's syndrome and sepsis, as well as alcohol hypoglycemia, are considered to be the consequence of hepatic disturbance. Acute viral hepatitis results in serious impairment in hepatic glycogen synthesis and gluconeogenesis and frequently gives rise to fasting hypoglycemia (Felig et al., 1970). Glycogen stores rapidly disappear as liver disease (including cirrhosis due to alcoholism) progresses, causing recurrent hypoglycemia (Service, 1992).

Reye syndrome is a fatal disease, most commonly occuring following some virus infections (influenza A, influenza B, herpes, varicella zoster and several other common viral infections). Epidemiologic evidence suggests that aspirin plays a potentiating role in the pathogenesis of this syndrome. The hepatic dysfunction appears to be the primary error and the direct result of a mitochondrial disturbance that causes secondary metabolic derangement, (hyperamoniemia, hypoprotrombinemia without hyperbilirubinemia), including hypoglycemia**.** 

Hypoglycaemia in patients with hepatocellular carcinoma usually occurs during the terminal stage of the illness, but there are patients with hepatocellular carcinoma who develop hypoglycaemia early in the course of their illness. Hypoglycemia occurs predominantly as a paraneoplastic manifestation of hepatocellular carcinoma, (Sorlini et al., 2010; Thipaporn et al., 2005; Young, 2007).

Ethanol is a potent hypoglycemic agent, causing decreased endogenous glucose production and glycogenolysis. The volume of alcohol intake is correlated with the degree of resulting hypoglycemia (Raghavan et al., 2007; Smeeks, 2008). Ethanol-induced hypoglycemia arises from inhibition of gluconeogenesis as a result of the increase in the NADH-NAD ratio, which suppresses the conversion of lactate to pyruvate, glycerophosphate to dihydroxyacetone phosphate, and glutamate to -ketoglutarate and several tricarboxylic cycle reactions. Ethanol reduces rates of hepatic glucose production, supresses plasma insulin concentration, increases plasma lactate concentration, beta hydroxybutirate, glycerol and free fatty acids, and increases lactate-pyruvate and beta-hydroxybutirate-acetoacetate ratios. Hypoglycemia usually develops within 6 to 36 hours of the ingestion, of even moderate amounts of ethanol by persons chronically malnouriched or by healthy persons who have missed one of the meals. Healthy children are especially susceptible to ethanol hypoglycemia. Blood ethanol levels may not be elevated when the patient is hypoglycemic. Healthy children are especially susceptible to ethanol hypoglycemia. Blood ethanol levels may not be elevated when the patient is hypoglycemic( Arky, 1989; Badawy, 1977; Service, 1992).

#### **9.1.2 Hypoglycemia in kidney disorders**

Kidneys play a significant role in carbohydrate metabolism under both physiological and pathological conditions due to renal gluconeogenesis. Hypoglycemia in patients with renal failure may be due to inadequate gluconeogenic substrate availability. It seems that disturbances in renal gluconeogenesis together with lower degradation of insulin played the key role in creating hypoglycaemia in patients with renal diseases. Hypoglycemia should be suspected in any patient with renal failure who exhibits any change in mental or neurologic status (Arem, 1989; Rutsky,1978). Also, kidney disease is a frequent cause of adverse medication reactions due to the problems in excretion of certain medications and, therefore, causing hypoglycemia in older adults (Gerich, 2001)**.** 

Hypoglycemia as a Pathological Result in Medical Praxis 135

Predictors of hypoglycemia in patients with type 2 diabetes include treatment with insulin and duration of insulin treatment, a history of previous hypoglycemia. Primary risk factors for hypoglycemia in decreasing importance have been reported as age over 64, current insulin treatment, sulfonylurea treatment, polypharmacy, renal impairment and previous

**Antibiotics.** Pentamidine used in treating opportunistic infections associated with immunosuppression (e.g. Pneumocystis pneumonia) and protozoan parasites, causes severe hypoglycemia by increasing insulin secretion. Isoniazid causes hypoglycemia through

**Sulfonamides and Fluorquinolones** have been known to cause significant, life-threatening

**Cardiac medications.** Beta-blockers inhibit glycogenolysis and are most likely to be associated with hypoglycemia in older adults. Isolated reports indicate that angiotensinconverting enzyme inhibitors can cause hypoglycemia by increasing insulin sensitivity. **Salicylates.** It has been determined more recently that salicylates, such as aspirin, decrease serum glucose by reversing or inhibiting the process of insulin resistance related to

**Psychotropic medications.** It should be avoided haloperidol in older adults with a history of hypoglycemia, or sulfonylurea or insulin use, due to the risk of severe hypoglycemic interactions. Tricyclic antidepressants, chlorpromazine, MAO inhibitors, and lithium also

**Quinolines.** Quinines, used as an anti-malarial and anti-arrhythmic agents, have strong hypoglycemic properties, increasing insulin secretion as the sulfonylureas do (Service, 1992).

Hypoglycemia is defined as a serum glucose level below (3.8 mmol/L or, 70 mg/dL). The plasma glucose level is tightly controlled throughout life in the normal individual. The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose utilization. The physiological post absorptive serum glucose concentration in healthy humans range is 4.4-5.*8* mmol/L (80 to 110 mg/dL). Variation in blood glucose levels above or below the normal range usually indicate to serious diseases. Even mild disruptions of glucose homeostasis can have adverse

Hypoglycemia most often affects those at the extremes of age, such as infants and the elderly, but may happen at any age, in neonatal period and early childhood As a relatively frequent common event in the pediatric newborn period (childhood), hypoglicemia may be a consequence of some inborn errors of carbohydrate metabolism. In adults, hypoglycemia is a result of acquired disorders, primarily due to disturbance of physiological function of some organs (liver, kidney, CNS) or manifests disorders of some endocrine glands,

The symptoms of hypoglycemia are not specific and are related to disturbance of the brain and the sympathetic nervous system. The stimulation of the autonomic nervous system produces sweating, pale skin, irritability, anxiety, weakness, hunger, nausea, serving as an early warning system and preceding the neuroglycopenic symptoms due to cerebral glucose deprivation, e.g. headache, confusion, inability to concentrate or pay attention, mental

confusion, difficulty in thinking*,* changes in vision, lethargy, sleepiness, stupor.

hypoglycemic episodes (Miller et al., 2001).

cytotoxic hepatic damage (Service, 1992).

generalized inflammatory responses.

involved in carbohydrate metabolism .

**10. Conclusion** 

consequences.

hypoglycemia by increasing insulin secretion.

have been reported to cause severe hypoglycemia.

#### **9.2 Hormonal disturbances and hypoglycemia**

**Adrenocortical insufficiency** or **Adrenocortical hypofunction** is defined as the deficient production of glucocorticoids or mineralocorticoids, or both. Hypoglycemia is common in adrenocortical insufficiency. Primary adrenocortical insufficiency (Addison's disease) is due to destruction of the adrenal cortex, whereas in secondary adrenocortical insufficiency impaired cortisol production is due to deficient ACTH production. Spontaneous hypoglycemia has been reported to be a frequent finding in isolated ACTH deficiency. The cause for primary adrenocortical insufficiency is autoimmune destruction or tuberculosis of the adrenal cortex.

**Hypoglycemia in hypopituitarism** is common in children under 6 years of age but less so beyond that age. Asymptomatic hypoglycemia has been observed in isolated growth hormone deficiency after prolonged fasting (Tyrrell, 1992).

**Insulinoma**. Insulin-producing tumors of pancreas can cause severe hypoglycemia; among these are islet cell adenoma and carcinoma (insulinoma). Insulinoma is uncommon in persons less than 20 years of age and is rare in those less than 5 years of age. Of the patients with insulinoma, approxymately 87 per cent have single benign tumors. These tumors are most common in women (60%), with median age of diagnosis 50 years (Service, 1992).

#### **9.3 Reactive hypoglycemia**

**The post-prandial hypoglycemia** occurs immediately following meals, with no known causes (idiopathic reactive hypoglycemia, RH) (Krinsley & Grover, 2007).

**Alimentary hypoglycemia**, another form of RH related to prior upper GI surgery (Guettier, 2006), results from rapid glucose absorption into the intestine and increased insulin secretion after every meal. The food stimulated hypoglycemia usually cause symptomes mediated by the autonomic nervous system - sweating, shakiness, anxiety, palpitations, and weakness, and rarely those of impairment of central nervous system function. The food deprived hypoglycemia, on the other hand, usually result in impariment of central nervous system functions - reduced intellectual capacity, confusion, irritability, abnormal behavior, convulsions and coma (Service,1992).

**Infections.** Hypoglycemia often occurs during or following acute infections in older adults. Infection-related hypoglycemia increases the risk of death and morbidity among persons over age 70 Secretion of glucagon, epinephrine, and growth hormone during hypoglycemia diminishes significantly after age 65, reducing autonomic warning symptoms in older adults.

**Sepsis** as a cause of hypoglycemia should be readily apparent. The mechanism for hypoglycemia with sepsis is not well defined. Depleted glycogen stores, impaired gluconeogenesis, and increased peripheral utilization of glucose may all be contributing factors. Laboratory testing can confirm the suspicion of hepatic dysfunction (Rattarasarn, 1997).

#### **9.3.1 Hypoglycemia due to drug medications**

**Insulin treatment of Diabetes mellitus.** Hypoglycaemia is a serious, frequent and recurrent complication of treatment of diabetes mellitus with insulin, which may become a direct danger to the patient's life. Hypoglycaemia represents the limiting factor to obtain good glycemic control. Dysregulation of counteracting mechanisms and autonomic nervous system neuropathy contribute to a strong increase in the incidence of hypoglycaemia in type 1 diabetic patients, but also in long lasting type 2 diabetic patients (Cryer, 2001, 2008).

Predictors of hypoglycemia in patients with type 2 diabetes include treatment with insulin and duration of insulin treatment, a history of previous hypoglycemia. Primary risk factors for hypoglycemia in decreasing importance have been reported as age over 64, current insulin treatment, sulfonylurea treatment, polypharmacy, renal impairment and previous hypoglycemic episodes (Miller et al., 2001).

**Antibiotics.** Pentamidine used in treating opportunistic infections associated with immunosuppression (e.g. Pneumocystis pneumonia) and protozoan parasites, causes severe hypoglycemia by increasing insulin secretion. Isoniazid causes hypoglycemia through cytotoxic hepatic damage (Service, 1992).

**Sulfonamides and Fluorquinolones** have been known to cause significant, life-threatening hypoglycemia by increasing insulin secretion.

**Cardiac medications.** Beta-blockers inhibit glycogenolysis and are most likely to be associated with hypoglycemia in older adults. Isolated reports indicate that angiotensinconverting enzyme inhibitors can cause hypoglycemia by increasing insulin sensitivity.

**Salicylates.** It has been determined more recently that salicylates, such as aspirin, decrease serum glucose by reversing or inhibiting the process of insulin resistance related to generalized inflammatory responses.

**Psychotropic medications.** It should be avoided haloperidol in older adults with a history of hypoglycemia, or sulfonylurea or insulin use, due to the risk of severe hypoglycemic interactions. Tricyclic antidepressants, chlorpromazine, MAO inhibitors, and lithium also have been reported to cause severe hypoglycemia.

**Quinolines.** Quinines, used as an anti-malarial and anti-arrhythmic agents, have strong hypoglycemic properties, increasing insulin secretion as the sulfonylureas do (Service, 1992).

#### **10. Conclusion**

134 Type 1 Diabetes Complications

**Adrenocortical insufficiency** or **Adrenocortical hypofunction** is defined as the deficient production of glucocorticoids or mineralocorticoids, or both. Hypoglycemia is common in adrenocortical insufficiency. Primary adrenocortical insufficiency (Addison's disease) is due to destruction of the adrenal cortex, whereas in secondary adrenocortical insufficiency impaired cortisol production is due to deficient ACTH production. Spontaneous hypoglycemia has been reported to be a frequent finding in isolated ACTH deficiency. The cause for primary adrenocortical insufficiency is autoimmune destruction or tuberculosis of

**Hypoglycemia in hypopituitarism** is common in children under 6 years of age but less so beyond that age. Asymptomatic hypoglycemia has been observed in isolated growth

**Insulinoma**. Insulin-producing tumors of pancreas can cause severe hypoglycemia; among these are islet cell adenoma and carcinoma (insulinoma). Insulinoma is uncommon in persons less than 20 years of age and is rare in those less than 5 years of age. Of the patients with insulinoma, approxymately 87 per cent have single benign tumors. These tumors are most common in women (60%), with median age of diagnosis 50 years (Service, 1992).

**The post-prandial hypoglycemia** occurs immediately following meals, with no known

**Alimentary hypoglycemia**, another form of RH related to prior upper GI surgery (Guettier, 2006), results from rapid glucose absorption into the intestine and increased insulin secretion after every meal. The food stimulated hypoglycemia usually cause symptomes mediated by the autonomic nervous system - sweating, shakiness, anxiety, palpitations, and weakness, and rarely those of impairment of central nervous system function. The food deprived hypoglycemia, on the other hand, usually result in impariment of central nervous system functions - reduced intellectual capacity, confusion, irritability, abnormal behavior,

**Infections.** Hypoglycemia often occurs during or following acute infections in older adults. Infection-related hypoglycemia increases the risk of death and morbidity among persons over age 70 Secretion of glucagon, epinephrine, and growth hormone during hypoglycemia diminishes significantly after age 65, reducing autonomic warning symptoms in older adults. **Sepsis** as a cause of hypoglycemia should be readily apparent. The mechanism for hypoglycemia with sepsis is not well defined. Depleted glycogen stores, impaired gluconeogenesis, and increased peripheral utilization of glucose may all be contributing factors.

**Insulin treatment of Diabetes mellitus.** Hypoglycaemia is a serious, frequent and recurrent complication of treatment of diabetes mellitus with insulin, which may become a direct danger to the patient's life. Hypoglycaemia represents the limiting factor to obtain good glycemic control. Dysregulation of counteracting mechanisms and autonomic nervous system neuropathy contribute to a strong increase in the incidence of hypoglycaemia in type 1 diabetic patients, but also in long lasting type 2 diabetic patients (Cryer, 2001, 2008).

Laboratory testing can confirm the suspicion of hepatic dysfunction (Rattarasarn, 1997).

causes (idiopathic reactive hypoglycemia, RH) (Krinsley & Grover, 2007).

**9.2 Hormonal disturbances and hypoglycemia** 

hormone deficiency after prolonged fasting (Tyrrell, 1992).

the adrenal cortex.

**9.3 Reactive hypoglycemia** 

convulsions and coma (Service,1992).

**9.3.1 Hypoglycemia due to drug medications** 

Hypoglycemia is defined as a serum glucose level below (3.8 mmol/L or, 70 mg/dL). The plasma glucose level is tightly controlled throughout life in the normal individual. The stability of the plasma glucose level is a reflection of the balance between the rates of whole body glucose production and glucose utilization. The physiological post absorptive serum glucose concentration in healthy humans range is 4.4-5.*8* mmol/L (80 to 110 mg/dL). Variation in blood glucose levels above or below the normal range usually indicate to serious diseases. Even mild disruptions of glucose homeostasis can have adverse consequences.

Hypoglycemia most often affects those at the extremes of age, such as infants and the elderly, but may happen at any age, in neonatal period and early childhood As a relatively frequent common event in the pediatric newborn period (childhood), hypoglicemia may be a consequence of some inborn errors of carbohydrate metabolism. In adults, hypoglycemia is a result of acquired disorders, primarily due to disturbance of physiological function of some organs (liver, kidney, CNS) or manifests disorders of some endocrine glands, involved in carbohydrate metabolism .

The symptoms of hypoglycemia are not specific and are related to disturbance of the brain and the sympathetic nervous system. The stimulation of the autonomic nervous system produces sweating, pale skin, irritability, anxiety, weakness, hunger, nausea, serving as an early warning system and preceding the neuroglycopenic symptoms due to cerebral glucose deprivation, e.g. headache, confusion, inability to concentrate or pay attention, mental confusion, difficulty in thinking*,* changes in vision, lethargy, sleepiness, stupor.

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**6** 

*Spain* 

**Autoimmune Associated Diseases in Pediatric** 

**According to HLA-DQ Genetic Polymorphism** 

Diabetes mellitus type 1 is the most common endocrine metabolic disorder in childhood and adolescence. In this condition there is an absolute insulin deficiency secondary to progressive destruction of pancreatic beta cells, causing severe alterations in the metabolism

The most obvious alteration is chronic hyperglycemia, which is essential to diagnose the disease, and moreover, is the main responsible of many vascular and neurological

In the development of diabetes mellitus type 1 involving both genetic and environmental factors. The traditional concept is that environmental factors may act as triggers of the immune response against β of Langerhans cell phenotype in a genetically predisposed to the

Autoimmune diseases are syndromes caused by activation of T cells or B or both, without evidence of other causes such as infection or cancer. When dendritic cells expressing selfantigens in the context of HLA molecules stimulate peripheral T cells, they do so that they remain alive but anergic, no response until they contact a dendritic cell with multiple

Although many autoinmumnes diseases characterized by abnormal production of pathogenic autoantibodies, most of it is caused by an overreaction, combined T and B cells. In animal models of type 1 diabetes mellitus have demonstrated the high expression of MAdCAM-1 and GlyCAM-1 on HEV (high endothelial venules) of the inflamed pancreatic islets and the treatment of animals with inhibitors of the L-function selectin and α4 integrin,

The American Diabetes Association divides the type 1 diabetes mellitus in two subgroups: 1A is the result of autoimmune destruction of beta cells and the 1B subtype, which do not immunomarkers indicating a destructive autoimmune process of beta cellspancreas. However develop insulin deficiency by unidentified mechanisms and are prone to ketosis. It

Genetic studies have shown that it is an inherited disease with polygenic trait. Genome wide studies have indicated the presence of at least 20 chromosomal regions that may contribute to genetic predisposition to type 1 diabetes mellitus. The most important genes that

has been a predominance of African Americans and Asians in the 1B subtype.

of all essential elements (carbohydrates, lipids and proteins).

complications that diabetic patients may develop long-term.

moleculescostimulatory expressing microbial antigens.

blockeddevelopment of type 1 diabetes mellitus.

development of diabetes mellitus type 1.

**1. Introduction** 

**Patients with Type 1 Diabetes Mellitus** 

Miguel Ángel García Cabezas and Bárbara Fernández Valle

*Servicio de Pediatría. Hospital General de Ciudad Real,* 


### **Autoimmune Associated Diseases in Pediatric Patients with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism**

Miguel Ángel García Cabezas and Bárbara Fernández Valle *Servicio de Pediatría. Hospital General de Ciudad Real, Spain* 

#### **1. Introduction**

142 Type 1 Diabetes Complications

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Diabetes mellitus type 1 is the most common endocrine metabolic disorder in childhood and adolescence. In this condition there is an absolute insulin deficiency secondary to progressive destruction of pancreatic beta cells, causing severe alterations in the metabolism of all essential elements (carbohydrates, lipids and proteins).

The most obvious alteration is chronic hyperglycemia, which is essential to diagnose the disease, and moreover, is the main responsible of many vascular and neurological complications that diabetic patients may develop long-term.

In the development of diabetes mellitus type 1 involving both genetic and environmental factors. The traditional concept is that environmental factors may act as triggers of the immune response against β of Langerhans cell phenotype in a genetically predisposed to the development of diabetes mellitus type 1.

Autoimmune diseases are syndromes caused by activation of T cells or B or both, without evidence of other causes such as infection or cancer. When dendritic cells expressing selfantigens in the context of HLA molecules stimulate peripheral T cells, they do so that they remain alive but anergic, no response until they contact a dendritic cell with multiple moleculescostimulatory expressing microbial antigens.

Although many autoinmumnes diseases characterized by abnormal production of pathogenic autoantibodies, most of it is caused by an overreaction, combined T and B cells. In animal models of type 1 diabetes mellitus have demonstrated the high expression of MAdCAM-1 and GlyCAM-1 on HEV (high endothelial venules) of the inflamed pancreatic islets and the treatment of animals with inhibitors of the L-function selectin and α4 integrin, blockeddevelopment of type 1 diabetes mellitus.

The American Diabetes Association divides the type 1 diabetes mellitus in two subgroups: 1A is the result of autoimmune destruction of beta cells and the 1B subtype, which do not immunomarkers indicating a destructive autoimmune process of beta cellspancreas. However develop insulin deficiency by unidentified mechanisms and are prone to ketosis. It has been a predominance of African Americans and Asians in the 1B subtype.

Genetic studies have shown that it is an inherited disease with polygenic trait. Genome wide studies have indicated the presence of at least 20 chromosomal regions that may contribute to genetic predisposition to type 1 diabetes mellitus. The most important genes that

Autoimmune Associated Diseases in Pediatric Patients

exposure to cow's milk and nitrosoureas.

resistance to the destruction process.

children without diabetes.

TSH.

hypothyroidism.

pursue and establish treatment as soon as detected.

<5%).

with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 145

Autoantibodies against islet cells (ICA) is a combination of several different antibodies directed against islet molecules such as GAD, insulin, IA-2/ICA-512 and islet ganglioside and serve as a marker of the autoimmune process of type 1 diabetes mellitus. The determination of the ICA may be useful to classify as type 1 diabetes mellitus and nondiabetic individuals identify risk. The ICA is present in most (> 75%) of individuals newly diagnosed with type 1 diabetes mellitus in a significant minority of diabetics newly diagnosed type 2 (5-10%) and sometimes, in pregnant women with gestational diabetes (

In 3-4% of first-degree relatives of individuals with type 1 diabetes mellitus ICA exist. Along with the presence of a disorder of insulin secretion in the proof of intravenous glucose tolerance predict a 50% higher risk of developing type 1 diabetes mellitus in the next 5 years. Without this disorder of insulin secretion, the presence of ICA predicts a five-year risk <25%. After this it follows that the risk of a first degree relative of type 1 diabetes mellitus is low. Today no approved treatment to prevent development of type 1 diabetes mellitus, so the detection of ICA in non-diabetic population has not been established as screening. There is another theory that talks about environmental factors as triggers of the autoimmune process in genetically vulnerable patients, but it is difficult to find an environmental trigger, since the event may precede by several years the development of the disease. Among the hypothetical environmental triggers include viruses (coxsackie and rubella), proteins early

A major advance would get delay or prevent diabetes, there have been some intervention in animal models, whose main objective has been the immune system (immunosuppression, selective deletion of T cell subsets, immune tolerance induction to proteinsisland), while others avoid the death of islet cells by blocking the cytotoxic cytokines or increasing islet

Type 1 diabetes mellitus is often associated with autoimmune diseases. Thus, there has been an increased prevalence of autoantibodies related to celiac disease and many other autoantibodies against endocrine and nonendocrine organs. Not infrequently, these diseases manifest themselves associated paucisymptomatic and are diagnosed late. Despite this apparent relationship between type 1 diabetes mellitus and these autoimmune diseases has not been shown that the degree of glycemic control influence the likelihood of developing or subsequent developments. We must not forget that many of these clinical situations produce per se a decrease in the expectancy and quality of life of patients, another reason to actively

Autoantibodies can be found in up to 25% of children and adolescents with diabetes, but only 3-5% have hypothyroidism. Hyperthyroidism is less common, but more often than

Autoimmune hypothyroidism may be associated with goitre (Hashimoto's thyroiditis or goiter), or in later stages of the disease, minimal residual thyroid tissue (atrophic thyroiditis). Because the autoimmune process gradually reduces thyroid function, there is a compensatory phase during which thyroid hormone levels are maintained by an elevated

Although some patients may have mild symptoms, this phase is called subclinical or mild hypothyroidism. Later, T4 levels fall and TSH levels increase even more, the symptoms become more obvious at this stage (usually TSH> 10 mU/L) is called clinical

influence susceptibility to type 1 diabetes are located in the HLA complex located on the short arm of chromosome 6 (6p21.3).

The HLA-DQ is the locus that confers the major genetic susceptibility to develop type 1 diabetes in humans. DQ molecules have two chains, alpha and beta, encoded by the DQA and DQB genes. Susceptibility to type 1 diabetes mellitus has been described by combining alpha-beta chains with no amino acid aspartic acid at position 57 of beta chain (DQB1\*0302 linked to DR4 and DQB1\*0201 linked to DR3) and the presence of the amino acid arginine at position 52 of the alpha chain (DQA1\*0301 linked to DR4 and DQA1\*0501 linked to DR3).

Based on these findings have been described both genotypes DQA/DQB disease associated with DQA1\*0501/DQB1\*0201 and DQA1\*0301/DQB1\*0302, which is more specific than DR3/DR4 genotype.

Although other types of islet cells and α cells (glucagon producing), delta cells (somatostatin producing) cells or PP (pancreatic polypeptide-producing) are functionally and embryologically similar to beta cells and express the most same. These proteins, inexplicably are free of autoimmune process.

From the pathological standpoint, the cells of the pancreatic islets are infiltrated by lymphocytes (a process called insulitis). After the destruction of beta cells, the inflammatory process forwards, the islets are atrophic and disappear immunomarkers.

Insulitis studies in humans and in animal models of type 1 diabetes mellitus (NOD mouse and BB rat) have identified the following abnormalities in both humoral branch as in the immune system cells:


The beta cells appear to be particularly vulnerable to the toxic effect of some cytokines (tumor necrosis factor), interferon gamma and interleukin 1. Precise mechanisms are unknown the death of beta cells, but may involve formation of nitric oxide metabolites, apoptosis and direct cytotoxic effects of CD8+ T cells. It is believed that the destruction process does not involve autoantibodies against islet cells, since these antibodies do not react in general to the surface of islet cells and are capable of transferring diabetes mellitus in animals.

Among islet molecules that are targets of the autoimmune process are insulin, glutamic acid decarboxylase (glutamic acid decarboxylase, GAD), the biosynthetic enzyme of the neurotransmitter gamma aminobutyric acid (gamma-aminobutyric acid,GABA), ICA-512/IA-2 (with homology to tyrosine phosphatases, and fogrina (protein in secretory granules of insulin). Other less precisely defined autoantigens are islet ganglioside and carboxypeptidase H. Except none of the insulin-specific autoantigens are beta cells, which makes us wonder how these are destroyed selectively.

Current theories favor the onset of an autoimmune process directed against beta cell molecule, which then spreads to other islet molecules as the autoimmune process destroys the beta cells and creates a series of secondary autoantigens. Beta cells of individuals with type 1 diabetes mellitus do not differ from the beta cells of normal people, because the transplanted islets are destroyed by the recurrence of autoimmune process of type 1 diabetes mellitus.

influence susceptibility to type 1 diabetes are located in the HLA complex located on the

The HLA-DQ is the locus that confers the major genetic susceptibility to develop type 1 diabetes in humans. DQ molecules have two chains, alpha and beta, encoded by the DQA and DQB genes. Susceptibility to type 1 diabetes mellitus has been described by combining alpha-beta chains with no amino acid aspartic acid at position 57 of beta chain (DQB1\*0302 linked to DR4 and DQB1\*0201 linked to DR3) and the presence of the amino acid arginine at position 52 of the alpha chain (DQA1\*0301 linked to DR4 and DQA1\*0501 linked to DR3). Based on these findings have been described both genotypes DQA/DQB disease associated with DQA1\*0501/DQB1\*0201 and DQA1\*0301/DQB1\*0302, which is more specific than

Although other types of islet cells and α cells (glucagon producing), delta cells (somatostatin producing) cells or PP (pancreatic polypeptide-producing) are functionally and embryologically similar to beta cells and express the most same. These proteins, inexplicably

From the pathological standpoint, the cells of the pancreatic islets are infiltrated by lymphocytes (a process called insulitis). After the destruction of beta cells, the inflammatory

Insulitis studies in humans and in animal models of type 1 diabetes mellitus (NOD mouse and BB rat) have identified the following abnormalities in both humoral branch as in the

2. Activated lymphocytesin islets, peripancreatic lymph nodes and widespread

The beta cells appear to be particularly vulnerable to the toxic effect of some cytokines (tumor necrosis factor), interferon gamma and interleukin 1. Precise mechanisms are unknown the death of beta cells, but may involve formation of nitric oxide metabolites, apoptosis and direct cytotoxic effects of CD8+ T cells. It is believed that the destruction process does not involve autoantibodies against islet cells, since these antibodies do not react in general to the surface of islet cells and are capable of transferring diabetes mellitus

Among islet molecules that are targets of the autoimmune process are insulin, glutamic acid decarboxylase (glutamic acid decarboxylase, GAD), the biosynthetic enzyme of the neurotransmitter gamma aminobutyric acid (gamma-aminobutyric acid,GABA), ICA-512/IA-2 (with homology to tyrosine phosphatases, and fogrina (protein in secretory granules of insulin). Other less precisely defined autoantigens are islet ganglioside and carboxypeptidase H. Except none of the insulin-specific autoantigens are beta cells, which

Current theories favor the onset of an autoimmune process directed against beta cell molecule, which then spreads to other islet molecules as the autoimmune process destroys the beta cells and creates a series of secondary autoantigens. Beta cells of individuals with type 1 diabetes mellitus do not differ from the beta cells of normal people, because the transplanted islets are destroyed by the recurrence of autoimmune process of type 1

process forwards, the islets are atrophic and disappear immunomarkers.

3. T lymphocytes that proliferate when stimulated with islet proteins.

short arm of chromosome 6 (6p21.3).

DR3/DR4 genotype.

immune system cells:

circulation.

in animals.

diabetes mellitus.

are free of autoimmune process.

1. Autoantibodies against cell islet.

4. Release of cytokines within the insulitis.

makes us wonder how these are destroyed selectively.

Autoantibodies against islet cells (ICA) is a combination of several different antibodies directed against islet molecules such as GAD, insulin, IA-2/ICA-512 and islet ganglioside and serve as a marker of the autoimmune process of type 1 diabetes mellitus. The determination of the ICA may be useful to classify as type 1 diabetes mellitus and nondiabetic individuals identify risk. The ICA is present in most (> 75%) of individuals newly diagnosed with type 1 diabetes mellitus in a significant minority of diabetics newly diagnosed type 2 (5-10%) and sometimes, in pregnant women with gestational diabetes ( <5%).

In 3-4% of first-degree relatives of individuals with type 1 diabetes mellitus ICA exist. Along with the presence of a disorder of insulin secretion in the proof of intravenous glucose tolerance predict a 50% higher risk of developing type 1 diabetes mellitus in the next 5 years. Without this disorder of insulin secretion, the presence of ICA predicts a five-year risk <25%. After this it follows that the risk of a first degree relative of type 1 diabetes mellitus is low. Today no approved treatment to prevent development of type 1 diabetes mellitus, so the detection of ICA in non-diabetic population has not been established as screening.

There is another theory that talks about environmental factors as triggers of the autoimmune process in genetically vulnerable patients, but it is difficult to find an environmental trigger, since the event may precede by several years the development of the disease. Among the hypothetical environmental triggers include viruses (coxsackie and rubella), proteins early exposure to cow's milk and nitrosoureas.

A major advance would get delay or prevent diabetes, there have been some intervention in animal models, whose main objective has been the immune system (immunosuppression, selective deletion of T cell subsets, immune tolerance induction to proteinsisland), while others avoid the death of islet cells by blocking the cytotoxic cytokines or increasing islet resistance to the destruction process.

Type 1 diabetes mellitus is often associated with autoimmune diseases. Thus, there has been an increased prevalence of autoantibodies related to celiac disease and many other autoantibodies against endocrine and nonendocrine organs. Not infrequently, these diseases manifest themselves associated paucisymptomatic and are diagnosed late. Despite this apparent relationship between type 1 diabetes mellitus and these autoimmune diseases has not been shown that the degree of glycemic control influence the likelihood of developing or subsequent developments. We must not forget that many of these clinical situations produce per se a decrease in the expectancy and quality of life of patients, another reason to actively pursue and establish treatment as soon as detected.

Autoantibodies can be found in up to 25% of children and adolescents with diabetes, but only 3-5% have hypothyroidism. Hyperthyroidism is less common, but more often than children without diabetes.

Autoimmune hypothyroidism may be associated with goitre (Hashimoto's thyroiditis or goiter), or in later stages of the disease, minimal residual thyroid tissue (atrophic thyroiditis). Because the autoimmune process gradually reduces thyroid function, there is a compensatory phase during which thyroid hormone levels are maintained by an elevated TSH.

Although some patients may have mild symptoms, this phase is called subclinical or mild hypothyroidism. Later, T4 levels fall and TSH levels increase even more, the symptoms become more obvious at this stage (usually TSH> 10 mU/L) is called clinical hypothyroidism.

Autoimmune Associated Diseases in Pediatric Patients

recurrence of symptoms when gluten is reintroduced in the diet .

make a systemic disease of varying severity.

all people who express DQ2 have celiac disease.

hypoglycemia and reduced daily insulin requirements.

children and adolescents with type 1 diabetes mellitus.

or lack of nutrients.

of ACTH to its receptors.

prevalence in childhood.

detected.

HLA DR3.

with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 147

villous atrophy, causing a chronic enteropathy with a broad range of manifestations, which

Adherence to a gluten-free diet is followed by clinical and histological improvement in these patients, with normalization of long-term intestinal architecture, and the property of the

The presence of an immune component in the etiology of the disease was suspected for three reasons. First, no serum IgA antigliadin antibodies and endomysial, although it is unclear whether primary or secondary to tissue injury. Endomysial antibody has a sensitivity of 90 and specificity 95%, and its antigen is tissue transglutaminase. Secondly, treatment with prednisolone for four weeks in a celiac patient who continues to eat gluten induces remission and duodenal epithelium gives a more normal level. Finally, the gliadin peptides interact with gliadin-specific T cells, which in turn can act as mediators of tissue injury or cause the release of one or more cytokines that are responsible for tissue injury. In celiac disease are also implicated genetic factors, its incidence varies widely among different population groups (high in Caucasians and low in color and eastern race) and is 10% in first degree relatives of patients with celiac disease. In addition, about 95% of celiac patients express the allele of the human leukocyte antigen DQ2, whereas only a minority of

Not forget that the diagnosis of celiac disease is made by biopsy of the small intestine. Is performed on patients with symptoms and laboratory findings suggestive of malabsorption

Is often asymptomatic, but can cause gastrointestinal symptoms, short stature and anemia. This condition is also associated with an increased number of hypoglycemic episodes and a progressive decrease in insulin requirements in the year prior to diagnosis. It is also recommended measuring endomysial or tissue transglutaminase after diagnosis and every 2-3 years in post, in asymptomatic patients or whenever there is clinical suspicion, given the possibility of seroconversion over time in some patientsin which antibodies were initially

Addison's disease, another autoimmune disease is present up to 2% of type 1 diabetes presenting autoantibodies against the enzyme 21-hydroxylase and the enzyme cleavage of the side chain, but it ignores the importance of these antincuerpos in the pathogenesis ofadrenal insufficiency. Some antibodies cause adrenal insufficiency by blocking the binding

The appearance of two or more of these autoimmune endocrine same person characterized in a polyglandular autoimmune syndrome type II (thyroid, parathyroid and gonadal tissue), this syndrome has yet mutated gene on chromosome 6 and is associated with alleles B8 and

The presence of adrenal insufficiency is rare, so it is recommended not look systematically. In addition to the classic symptoms for adrenal insufficiency are at risk of frequent

The 15-20% of adults have diabetes autoimmune gastropathy presenting autoantibodies to gastric parietal cells, and 50% have clinical or pathological signs of atrophic gastritis. Yet there are no recommendations regarding the detection of these antibodies, given the lower

With respect to autoimmune diseases of the skin, vitiligo has been found up to 7% of

In Hashimoto's thyroiditis, there is a marked lymphocytic infiltration of the thyroid with germinal center formation, atrophy of thyroid follicles accompanied by oxyphil metaplasia, absence of colloid and mild or moderate fibrosis. As with most autoimmune disorders, susceptibility to this type of hypothyroidism depends on a combination of genetic and environmental factors and is increased sibling risk of autoimmune hypothyroidism or Graves disease.

Genetic risk factors for this type of hypothyroidism in subjects caucasians are HLA-DR polymorphisms, specifically the HLA-DR3, HLA-DR4 and HLA-DR5. There is also a weak relationship between polymorphism of CTLA-4, a gene regulating T cells and autoimmune hypothyroidism. HLA-DR polymorphisms and CTLA-4 constitute about half of cases hipotiroidsimo autoimmune susceptibility. It is still necessary to identify other contributing loci. A gene located on chromosome 21 could be the cause of the relationship between autoimmune hypothyroidism and Down syndrome.

The lymphocytic infiltrate thyroid autoimmune hypothyroidism is composed of CD4+ T cells and activated CD8+ and B cells. It is believed that the destruction of thyroid cells is mediated in a primary CD8+ T cells cytotoxic, which destroy their targets by perforin that cause cellular necrosis or through granzyme B, which induces apoptosis.

Addition, cytokine production by local T cells, such as tumor necrosis factor, IL-1 and interferon gamma, can return to thyroid cells more susceptible to apoptosis mediated by death receptors such as Fas, which activate their ligands respective T cellIn addition, these cytokines directly disrupt the function of thyroid cells, and induce the expression of other proinflammatory molecules by thyroid cells themselves, such as cytokines, molecules of HLA class I and II, adhesion molecules, CD40 and nitric oxide.

The administration of high concentrations for therapeutic cytokines (notably IFN alpha) is associated with enhancement of autoimmune thyroid disease, possibly by mechanisms similar to those involved in sporadic disease.

The Tg and TPO antibodies are markers of thyroid autoimmunity with clinical utility, but their pathogenic effect is limited to a secondary role in the amplification of a developing immune response. TPO antibodies fix complement and are complex to the complement membrane attack the thyroid gland in case of autoimmune hypothyroidism. However, the transplacental passage of anti-Tg antibodies or anti-TPO has no effect on fetal thyroid gland, indicating that it takes an injury mediated by T cells to initiate autoimmune injury of the gland.

Although it has been associated with the presence of subclinical hypothyroidism with an increased risk of symptomatic hypoglycemia and hipocrecimiento, it is quite common for thyroid dysfunction clinically pass unnoticed, so you must determine the levels of thyrotropic hormone (TSH) annually in those without autoantibodies or more often if there are or the patient has any symptoms.

Celiac disease is 10 times more common in diabetics than in the general population and may affect up to 1-10% of diabetic patients. Also known as celiac sprue or gluten sensitive enteropathy is an autoimmune disorder triggered by ingestion of gliadin fractions present in the gluten and similar proteins of rye and barley in genetically predisposed individuals.

Gluten is the main protein component of wheat, rye and barley. In celiac disease is triggered by an immune reaction that leads to inflammation of the small intestine mediated by T lymphocytes, with the development of hyperplastic crypts, intraepithelial lymphocytes and

In Hashimoto's thyroiditis, there is a marked lymphocytic infiltration of the thyroid with germinal center formation, atrophy of thyroid follicles accompanied by oxyphil metaplasia, absence of colloid and mild or moderate fibrosis. As with most autoimmune disorders, susceptibility to this type of hypothyroidism depends on a combination of genetic and environmental factors and is increased sibling risk of autoimmune hypothyroidism or

Genetic risk factors for this type of hypothyroidism in subjects caucasians are HLA-DR polymorphisms, specifically the HLA-DR3, HLA-DR4 and HLA-DR5. There is also a weak relationship between polymorphism of CTLA-4, a gene regulating T cells and autoimmune hypothyroidism. HLA-DR polymorphisms and CTLA-4 constitute about half of cases hipotiroidsimo autoimmune susceptibility. It is still necessary to identify other contributing loci. A gene located on chromosome 21 could be the cause of the relationship between

The lymphocytic infiltrate thyroid autoimmune hypothyroidism is composed of CD4+ T cells and activated CD8+ and B cells. It is believed that the destruction of thyroid cells is mediated in a primary CD8+ T cells cytotoxic, which destroy their targets by perforin that

Addition, cytokine production by local T cells, such as tumor necrosis factor, IL-1 and interferon gamma, can return to thyroid cells more susceptible to apoptosis mediated by death receptors such as Fas, which activate their ligands respective T cellIn addition, these cytokines directly disrupt the function of thyroid cells, and induce the expression of other proinflammatory molecules by thyroid cells themselves, such as cytokines, molecules of

The administration of high concentrations for therapeutic cytokines (notably IFN alpha) is associated with enhancement of autoimmune thyroid disease, possibly by mechanisms

The Tg and TPO antibodies are markers of thyroid autoimmunity with clinical utility, but their pathogenic effect is limited to a secondary role in the amplification of a developing immune response. TPO antibodies fix complement and are complex to the complement membrane attack the thyroid gland in case of autoimmune hypothyroidism. However, the transplacental passage of anti-Tg antibodies or anti-TPO has no effect on fetal thyroid gland, indicating that it takes an injury mediated by T cells to initiate autoimmune injury of the

Although it has been associated with the presence of subclinical hypothyroidism with an increased risk of symptomatic hypoglycemia and hipocrecimiento, it is quite common for thyroid dysfunction clinically pass unnoticed, so you must determine the levels of thyrotropic hormone (TSH) annually in those without autoantibodies or more often if there

Celiac disease is 10 times more common in diabetics than in the general population and may affect up to 1-10% of diabetic patients. Also known as celiac sprue or gluten sensitive enteropathy is an autoimmune disorder triggered by ingestion of gliadin fractions present in the gluten and similar proteins of rye and barley in genetically predisposed

Gluten is the main protein component of wheat, rye and barley. In celiac disease is triggered by an immune reaction that leads to inflammation of the small intestine mediated by T lymphocytes, with the development of hyperplastic crypts, intraepithelial lymphocytes and

cause cellular necrosis or through granzyme B, which induces apoptosis.

HLA class I and II, adhesion molecules, CD40 and nitric oxide.

similar to those involved in sporadic disease.

are or the patient has any symptoms.

Graves disease.

gland.

individuals.

autoimmune hypothyroidism and Down syndrome.

villous atrophy, causing a chronic enteropathy with a broad range of manifestations, which make a systemic disease of varying severity.

Adherence to a gluten-free diet is followed by clinical and histological improvement in these patients, with normalization of long-term intestinal architecture, and the property of the recurrence of symptoms when gluten is reintroduced in the diet .

The presence of an immune component in the etiology of the disease was suspected for three reasons. First, no serum IgA antigliadin antibodies and endomysial, although it is unclear whether primary or secondary to tissue injury. Endomysial antibody has a sensitivity of 90 and specificity 95%, and its antigen is tissue transglutaminase. Secondly, treatment with prednisolone for four weeks in a celiac patient who continues to eat gluten induces remission and duodenal epithelium gives a more normal level. Finally, the gliadin peptides interact with gliadin-specific T cells, which in turn can act as mediators of tissue injury or cause the release of one or more cytokines that are responsible for tissue injury.

In celiac disease are also implicated genetic factors, its incidence varies widely among different population groups (high in Caucasians and low in color and eastern race) and is 10% in first degree relatives of patients with celiac disease. In addition, about 95% of celiac patients express the allele of the human leukocyte antigen DQ2, whereas only a minority of all people who express DQ2 have celiac disease.

Not forget that the diagnosis of celiac disease is made by biopsy of the small intestine. Is performed on patients with symptoms and laboratory findings suggestive of malabsorption or lack of nutrients.

Is often asymptomatic, but can cause gastrointestinal symptoms, short stature and anemia. This condition is also associated with an increased number of hypoglycemic episodes and a progressive decrease in insulin requirements in the year prior to diagnosis. It is also recommended measuring endomysial or tissue transglutaminase after diagnosis and every 2-3 years in post, in asymptomatic patients or whenever there is clinical suspicion, given the possibility of seroconversion over time in some patientsin which antibodies were initially detected.

Addison's disease, another autoimmune disease is present up to 2% of type 1 diabetes presenting autoantibodies against the enzyme 21-hydroxylase and the enzyme cleavage of the side chain, but it ignores the importance of these antincuerpos in the pathogenesis ofadrenal insufficiency. Some antibodies cause adrenal insufficiency by blocking the binding of ACTH to its receptors.

The appearance of two or more of these autoimmune endocrine same person characterized in a polyglandular autoimmune syndrome type II (thyroid, parathyroid and gonadal tissue), this syndrome has yet mutated gene on chromosome 6 and is associated with alleles B8 and HLA DR3.

The presence of adrenal insufficiency is rare, so it is recommended not look systematically. In addition to the classic symptoms for adrenal insufficiency are at risk of frequent hypoglycemia and reduced daily insulin requirements.

The 15-20% of adults have diabetes autoimmune gastropathy presenting autoantibodies to gastric parietal cells, and 50% have clinical or pathological signs of atrophic gastritis. Yet there are no recommendations regarding the detection of these antibodies, given the lower prevalence in childhood.

With respect to autoimmune diseases of the skin, vitiligo has been found up to 7% of children and adolescents with type 1 diabetes mellitus.

Autoimmune Associated Diseases in Pediatric Patients

and were assigned allele specific amplification.

with 4 viable possibilities, different genetic risk groups.

Fig. 1. HLA-DQ Diabetogenic risk groups

pathology.

an alpha of 0.05%.

**2.1 Results** 

with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 149

The degree of innovation under our study was that the determination of HLA-DQ alleles was performed by molecular biology techniques, to avoid large differences, about 50% errors (58% according to the results of our group), which are generated if only theserological determinations. HLA-DQ alleles were determined by reaction polymerase chain, with allele specific amplification (PCR-SSP). We used specific primers for DQA1 and DQB1 genes (Protrans, Ger.) Amplified products were separated by agarose gel electrophoresis in 2%

Determinations of antithyroid antibodies, and antithyroglobulin antimicrosomal performed by chemiluminescence, IMMULITE 2000 (Dipesa ®). Thyroid hormones: T4 and TSH by chemiluminescent immunoassay technology microparticles, ARCHITECT (Abbott ®). As serologic marker in detecting celiac disease transglutaminase antibodies were analyzed by enzyme immunoassay with recombinant human tissue transglutaminase (Eurospital ®) with a sensitivity around 95% and a specificity above 95% for populationspediatric. Patients with positive markers underwent a biopsy of the duodenum and subsequent intestinal

For the analysis of the data is first created a database with Microsoft Access and have subsequently be exported for statistical analysis by SPSS for Windows, version 12.0. We conducted a 6x4 design. Each patient was studied in 6 different moments of its evolution,

Study possible changes in the variables under study during the monitoring period, if the changes are influenced by risk group HLA-DQ, and if it influenced other control variables such as sex, age, etc. All statistical tests were performed with a significance level of 95% and

According to the HLA-DQ haplotypes obtained, the distribution of diabetogenic risk groups was as follows: group I (HLA-DQA1\*0501/DQB1\*0201): 45 patients, accounting for 34.9%, group II (HLA-DQA1\*0301/DQB1\*0302): 38 patients, representing 29.5%, group III (HLA-DQA1\*0501,\*0301/HLA-DQB1\*0201,\*0302): 38 patients, representing 29.5%, and group IV

(no gene associated with DM1 group): 8 patients, representing 6, 2% (Figure 1).

The objectives of this study are the following: make an epidemiological study of type 1 diabetes mellitus in childhood and adolescence, to study the HLA-DQ genetic group and general parameters in the onset of the disease, and the pursuit of development of autoimmune diseases.

Today diabetes education is fundamental and essential, in consultation diabetes control emphasizes good glycemic control in order to reduce microvascular complications, rare in children because their development is in adulthood. It also reports on the association with microvascular problems such as diabetic retinopathy, microalbuminuria leading to nephropathy, and diabetic neuropathy, as well as on macrovascular problems such as atherosclerosis.

Are known to coexist in these patients, with chronic hyperglycemia in cardiovascular risk otrosfactores. Diabetes education programs and health promotion should report on the harmful effects of some of them, such as smoking, overweight or sedentary. We should not forget the high prevalence of these diseases justifies the systematic implementation of its screening in the units of pediatric endocrinology. The early diagnosis of these can improve the control of type 1 diabetes mellitus.

#### **2. Patient and methods**

This study was carried out at the Department of Pediatrics, General Hospital of Ciudad Real. This work is a descriptive epidemiological study on 129 children and adolescents under age 16 with type 1 diabetes mellitus, studied in this hospital since 1990. With regard to epidemiological studies by our group in the province of Ciudad Real, with an estimated total of 423 patients with DM1, of which 204 are under 16 years, the size of the sample makes it representative of the distribution of type 1 diabetes mellitus population.

The analysis began in January 2003 starting with a retrospective study of patients and performing a 3-year prospective follow-up on these patients and patients who were going to the Department of Pediatrics at the start of his diabetes. The analysis and recruitment was completed in December 2007. This study was approved by the Research and Ethics Committee of the General Hospital of Ciudad Real. Reported and informed consent was obtained from parents or guardians.

In our study we asked whether there is a relationship between the occurrence of autoimmune diseases in pediatric patients with type 1 diabetes mellitus, with the HLA-DQ genetic group. Main objective genetic group analyzed HLA-DQ by molecular biology of our patients. According to these HLA-DQ haplotypes have organized groups I, II and III, considered by the usual bibliography and diabetogenic risk.


As secondary objectives we analyzed the disease onset general parameters such as sex and age. We collected data on whether patients had autoimmune disease associated with type 1 diabetes mellitus, if the onset of the disease has been before or after the debut of type 1 diabetes mellitus and the median time to onset of the disease. These parameters are studied during a follow-up period of 3 years with updates every 6 months. All of these secondary objectives relate them to the diabetogenic risk group assigned to each of our patients.

The degree of innovation under our study was that the determination of HLA-DQ alleles was performed by molecular biology techniques, to avoid large differences, about 50% errors (58% according to the results of our group), which are generated if only theserological determinations. HLA-DQ alleles were determined by reaction polymerase chain, with allele specific amplification (PCR-SSP). We used specific primers for DQA1 and DQB1 genes (Protrans, Ger.) Amplified products were separated by agarose gel electrophoresis in 2% and were assigned allele specific amplification.

Determinations of antithyroid antibodies, and antithyroglobulin antimicrosomal performed by chemiluminescence, IMMULITE 2000 (Dipesa ®). Thyroid hormones: T4 and TSH by chemiluminescent immunoassay technology microparticles, ARCHITECT (Abbott ®).

As serologic marker in detecting celiac disease transglutaminase antibodies were analyzed by enzyme immunoassay with recombinant human tissue transglutaminase (Eurospital ®) with a sensitivity around 95% and a specificity above 95% for populationspediatric. Patients with positive markers underwent a biopsy of the duodenum and subsequent intestinal pathology.

For the analysis of the data is first created a database with Microsoft Access and have subsequently be exported for statistical analysis by SPSS for Windows, version 12.0. We conducted a 6x4 design. Each patient was studied in 6 different moments of its evolution, with 4 viable possibilities, different genetic risk groups.

Study possible changes in the variables under study during the monitoring period, if the changes are influenced by risk group HLA-DQ, and if it influenced other control variables such as sex, age, etc. All statistical tests were performed with a significance level of 95% and an alpha of 0.05%.

#### **2.1 Results**

148 Type 1 Diabetes Complications

The objectives of this study are the following: make an epidemiological study of type 1 diabetes mellitus in childhood and adolescence, to study the HLA-DQ genetic group and general parameters in the onset of the disease, and the pursuit of development of

Today diabetes education is fundamental and essential, in consultation diabetes control emphasizes good glycemic control in order to reduce microvascular complications, rare in children because their development is in adulthood. It also reports on the association with microvascular problems such as diabetic retinopathy, microalbuminuria leading to nephropathy, and diabetic neuropathy, as well as on macrovascular problems such as

Are known to coexist in these patients, with chronic hyperglycemia in cardiovascular risk otrosfactores. Diabetes education programs and health promotion should report on the harmful effects of some of them, such as smoking, overweight or sedentary. We should not forget the high prevalence of these diseases justifies the systematic implementation of its screening in the units of pediatric endocrinology. The early diagnosis of these can improve

This study was carried out at the Department of Pediatrics, General Hospital of Ciudad Real. This work is a descriptive epidemiological study on 129 children and adolescents under age 16 with type 1 diabetes mellitus, studied in this hospital since 1990. With regard to epidemiological studies by our group in the province of Ciudad Real, with an estimated total of 423 patients with DM1, of which 204 are under 16 years, the size of the sample

The analysis began in January 2003 starting with a retrospective study of patients and performing a 3-year prospective follow-up on these patients and patients who were going to the Department of Pediatrics at the start of his diabetes. The analysis and recruitment was completed in December 2007. This study was approved by the Research and Ethics Committee of the General Hospital of Ciudad Real. Reported and informed consent was

In our study we asked whether there is a relationship between the occurrence of autoimmune diseases in pediatric patients with type 1 diabetes mellitus, with the HLA-DQ genetic group. Main objective genetic group analyzed HLA-DQ by molecular biology of our patients. According to these HLA-DQ haplotypes have organized groups I, II and III,

As secondary objectives we analyzed the disease onset general parameters such as sex and age. We collected data on whether patients had autoimmune disease associated with type 1 diabetes mellitus, if the onset of the disease has been before or after the debut of type 1 diabetes mellitus and the median time to onset of the disease. These parameters are studied during a follow-up period of 3 years with updates every 6 months. All of these secondary objectives relate them to the diabetogenic risk group assigned to each of our

makes it representative of the distribution of type 1 diabetes mellitus population.

autoimmune diseases.

atherosclerosis.

the control of type 1 diabetes mellitus.

obtained from parents or guardians.

patients.

 **Group I:** HLA-DQA1\*0501/DQB1\*0201 **Group II:** HLA-DQA1\*0301/DQB1\*0302

considered by the usual bibliography and diabetogenic risk.

**Group IV:** No genetic group associated with DM1

**Group III**: HLA-DQA1\*0501,\*0301/HLA-DQB1\*0201,\*0302

**2. Patient and methods** 

According to the HLA-DQ haplotypes obtained, the distribution of diabetogenic risk groups was as follows: group I (HLA-DQA1\*0501/DQB1\*0201): 45 patients, accounting for 34.9%, group II (HLA-DQA1\*0301/DQB1\*0302): 38 patients, representing 29.5%, group III (HLA-DQA1\*0501,\*0301/HLA-DQB1\*0201,\*0302): 38 patients, representing 29.5%, and group IV (no gene associated with DM1 group): 8 patients, representing 6, 2% (Figure 1).

Fig. 1. HLA-DQ Diabetogenic risk groups

Autoimmune Associated Diseases in Pediatric Patients

significant differences in favor of women (p <0.001).

Fig. 3. Autoimmune thyroiditis by diabetogenic risk groups

Fig. 4. Celiac disease by diabetogenic risk groups

of 34 months.

with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 151

distribution is as follows: 75% (n = 6) are women and 25% (n = 2) are male. We found

Distribution diabetogenic risk groups was as follows: Group I: 2 patients, Group II: 2 patients, Group III: 3 patients, and Group IV: 1 patient. No significant differences were

As for the timing of the debut has been observed that in 75% (n = 6), the debut of celiac disease after debut of type 1 diabetes mellitus, and 25% (n = 2) the onset is earlier. In most cases of celiac disease were asymptomatic at diagnosis and only observed the existence of signs of malabsorption in 1 patient, abdominal distention and diarrhea. The average time between debut and diagnosis was 22 months with a minimum of 6 months and a maximum

found in the diagnosis of celiac disease by diabetogenic risk groups (Figure 4).

The gender distribution of our study population of 129 patients was as follows: males 67 patients representing 51.9% and women 62 patients representing 48.1%, with a ratio child of 1,08.

The distribution of patients by age at onset was as follows: 0 to 4 years 32 patients representing 24.8%, between 5 and 9 years 67 patients, representing 51.9%, between 10 and 14 years 29 patients who account for 22.4% and between 15 and 16 years 1 patientrepresents 0.8%. The mean age of patients, whose mean values (mean ± SD), expressed in years, diabetogenic risk groups were as follows: Group I (8.6 ± 3.4), Group II (7 ± 3), Group III (6.1 ± 2.7) and Group IV (6.8 ± 2.6). In our study found significant differences in age at debut by diabetogenic risk groups, age at onset being significantly lower in group III with group I (Figure 2).

Fig. 2. Distribution of patients by age at onset

The incidence of patients with autoimmune thyroiditis in our series is 15.5%, representing a total of 20 patients. At the time of diagnosis of thyroiditis, 85% were euthyroid autoimmune thyroiditis and 15% had undergone an underactive thyroid. In the euthyroid, 17.6% (n = 3) associated with thyroid hypofunction later.

The sex distribution is as follows: 55% (n = 11) were women and 45% (n = 9) were male. No significant differences were observed in the distribution of autoimmune thyroiditis by sex.

Distribution diabetogenic risk groups was as follows: Group I: 6 patients, Group II: 5 patients, Group III: 8 patients, and Group IV: 1 patient. No significant differences were found in the diagnosis of autoimmune thyroiditis diabetogenic risk groups (Figure 3).

When analyzing patients with autoimmune thyroiditis, it was observed that 76.4% started after the debut of type 1 diabetes mellitus, whereas 23.6% were diagnosed simultaneously with the debut of it. By contrast patients with underactive thyroiditis, 28.6% presented prior to the commencement of type 1 diabetes mellitus, 42.8% thereafter, and 28.6% to debut simultaneously do the same.

In our series we found a case of hyperthyroidism in a 11-year-old was diagnosed with type 1 diabetes three years ago. The frequency of patients with celiac disease associated with type 1 diabetes mellitus in our series is 6.2%, which corresponds to 8 patients. The sex

The gender distribution of our study population of 129 patients was as follows: males 67 patients representing 51.9% and women 62 patients representing 48.1%, with a ratio child of

The distribution of patients by age at onset was as follows: 0 to 4 years 32 patients representing 24.8%, between 5 and 9 years 67 patients, representing 51.9%, between 10 and 14 years 29 patients who account for 22.4% and between 15 and 16 years 1 patientrepresents 0.8%. The mean age of patients, whose mean values (mean ± SD), expressed in years, diabetogenic risk groups were as follows: Group I (8.6 ± 3.4), Group II (7 ± 3), Group III (6.1 ± 2.7) and Group IV (6.8 ± 2.6). In our study found significant differences in age at debut by diabetogenic risk groups, age at onset being significantly lower in group III with group I

The incidence of patients with autoimmune thyroiditis in our series is 15.5%, representing a total of 20 patients. At the time of diagnosis of thyroiditis, 85% were euthyroid autoimmune thyroiditis and 15% had undergone an underactive thyroid. In the euthyroid, 17.6% (n = 3)

The sex distribution is as follows: 55% (n = 11) were women and 45% (n = 9) were male. No significant differences were observed in the distribution of autoimmune thyroiditis by sex. Distribution diabetogenic risk groups was as follows: Group I: 6 patients, Group II: 5 patients, Group III: 8 patients, and Group IV: 1 patient. No significant differences were found in the diagnosis of autoimmune thyroiditis diabetogenic risk groups (Figure 3). When analyzing patients with autoimmune thyroiditis, it was observed that 76.4% started after the debut of type 1 diabetes mellitus, whereas 23.6% were diagnosed simultaneously with the debut of it. By contrast patients with underactive thyroiditis, 28.6% presented prior to the commencement of type 1 diabetes mellitus, 42.8% thereafter, and 28.6% to debut

In our series we found a case of hyperthyroidism in a 11-year-old was diagnosed with type 1 diabetes three years ago. The frequency of patients with celiac disease associated with type 1 diabetes mellitus in our series is 6.2%, which corresponds to 8 patients. The sex

1,08.

(Figure 2).

Fig. 2. Distribution of patients by age at onset

associated with thyroid hypofunction later.

simultaneously do the same.

distribution is as follows: 75% (n = 6) are women and 25% (n = 2) are male. We found significant differences in favor of women (p <0.001).

Fig. 3. Autoimmune thyroiditis by diabetogenic risk groups

Distribution diabetogenic risk groups was as follows: Group I: 2 patients, Group II: 2 patients, Group III: 3 patients, and Group IV: 1 patient. No significant differences were found in the diagnosis of celiac disease by diabetogenic risk groups (Figure 4).

Fig. 4. Celiac disease by diabetogenic risk groups

As for the timing of the debut has been observed that in 75% (n = 6), the debut of celiac disease after debut of type 1 diabetes mellitus, and 25% (n = 2) the onset is earlier. In most cases of celiac disease were asymptomatic at diagnosis and only observed the existence of signs of malabsorption in 1 patient, abdominal distention and diarrhea. The average time between debut and diagnosis was 22 months with a minimum of 6 months and a maximum of 34 months.

Autoimmune Associated Diseases in Pediatric Patients

arthritis.

**3. Conclusion** 

**4. References** 

with Type 1 Diabetes Mellitus According to HLA-DQ Genetic Polymorphism 153

of Saukkonen et al (1996), which are located around the 2 years following the onset of type 1 diabetes mellitus. Other studies such as Maki and colleagues (1995), who observed a lower average interval around 13 months. Our results on the significant association in women are endorsed by other studies in this regard, as published in Spain by Roldan et al (1998). Most patients were asymptomatic at the time of his presentation and noted that there were no signs of frank malnutrition. Were diagnosed by serological screening and subsequent confirmation with intestinal biopsy. Our results are consistent with those of Barrera et al. Not found in our series more partnerships with other autoimmune diseases such as pernicious anemia, Addison's disease, Sjögren syndrome, alopecia areata, and rheumatoid

Among the background approximately 10% of the patients had atopy and bronchial asthma. These data are consistent with those reported by Lopez Medina et al in their series. The presence of vitiligo in our series (0.7%) is lower than that observed in other studies like the

The major autoimmune diseases, autoimmune thyroiditis and celiac disease are more prevalent in our diabetic patients than in the nondiabetic population. Although we found

In conclusion, these data support the recommendation that from the moment of diagnosis of type 1 diabetes mellitus regular determination of thyroid antibodies and celiac disease related. Current recommendations are vague as to what should be the most appropriate timing for this in pediatric patients. We must try to detect such diseases early, but that does not justify excessive and unnecessary repetition of diagnostic tests. The use of standardized monitoring protocols is becoming increasingly necessary to ensure better health care for

Green, A.; Gale, EA. & Patterson, CC for the EURODIAB ACE Study Group (1992) Incidence

Roldan, MB.; Alonso M.; Barrio R.; (1999) Thyroid autoimmunity in children and adolescents with type 1 diabetes mellitus. *Diabetes Nutr Metab* 1999; 12:27–31. Lorini, R; Scaramuzza, A; Vitali, L; d'Annunzio, G; Avanzini, MA; De Giacomo, C et al.

Lindberg, B.; Ericsson, UB.; Ljung, R.; (1997) High prevalence of thyroid autoantibodies at

Holmes GTK. (2002). Screening for coeliac disease in type 1 diabetes. *Arch Dis Child*

Barera, G.; Bonfanti, R.; Viscardi, M et al.(2002) Ocurrence of celiac disease alter onset of

of childhood-onset insulin-dependent diabetes mellitus: the EURODIAB ACE

(1996) Clinical aspects of celiac disease in children with insulin-dependent diabetes

diagnosis of insulin-dependent diabetes mellitus in Sweden children. *J Lab Clin Med*

type 1 diabetes: a six-year prospective longitudinal study. *Pediatrics* 2002; 109: 261-

one made in Italy by Romano et al (1998), showing a prevalence of 9%.

more patients in risk group III, no significant differences with other groups.

mellitus. *J Pediatr Endocrinol Metab* 1996;(Suppl 1):101-11.

children and adolescents with type 1 diabetes mellitus.

study. *Lancet* 1992; 339: 905-909.

1997; 130: 585-589.

2002;87:495-8.

267.

In our series, 2 patients are associated with type 1 diabetes mellitus, celiac disease and autoimmune thyroiditis (1 patient 1 patient euthyroid and hypothyroid).

In 13 patients (10.1%) were type allergic processes associated allergic rhinitis and conjunctivitis, 8 patients (6.2%) had asthma, 8 patients (6.2%) were diagnosed with atopic dermatitis and 1 patient ( 0.7%) of vitiligo.

#### **2.1.1 Discussion**

In type 1 diabetes mellitus there is a polygenic susceptibility. The most important genes that influence human susceptibility to type 1 diabetes mellitus are located in the complex HLA class II. In our series, these data are corroborated, and that these associations are most common. 93.8% of our patients with type 1 diabetes mellitus corresponds to the genetic risk groups as has been described elsewhere, so a 6.2% suffer from type 1 diabetes mellitus without belonging to a group of HLA-DQ risk.

Our results agree with others, as published by the EURODIAB indicating that in most mediterranean countries the male/female ratio is around 1, with a slight male predominance, but with no significant differences between them.

The period of highest incidence in the study is between 5 and 9 years. These findings are consistent with studies in other countries, which show a tendency for the disease much earlier debut. In our study found significant differences in age at debut by diabetogenic risk groups, age at onset being significantly lower in group III with group I. These results indicate that the combination of different molecules in susceptibility, heterozygous in group III, accelerating the destruction of beta cells by promoting an early onset of type 1 diabetes mellitus.

Autoimmune disease most often associated to type 1 diabetes mellitus is an autoimmune thyroid disease. Our results, 15.5% compared to the percentage of subjects who agree thyroiditis associated with many studies, such as in Spain by Roland and cols (1999) or made in Italy by Lorini and cols (1996). However, some international studies show higher prevalence, such as that conducted by Lindberg et al (1997), with a prevalence of 38%.

As in other studies in our series of cases in which thyroiditis manifested clinically, it is in the form of an underactive thyroid. Although thyroid status of the majority of subjects with positive markers is euthyroid.

In our series we found a case of hyperthyroidism in a 11-year-old was diagnosed with type 1 diabetes three years ago. Hyperthyroidism is associated with type 1 diabetes mellitus present in 1% of cases, most often in adults. Other studies indicate that hyperthyroidism is usually diagnosed before or while type 1 diabetes mellitus.

Studies show that thyroiditis is more prevalent in diabetic girls than in boys. However, our results do not indicate such a difference, matching other studies.

The prevalence of celiac disease associated with children with type 1 diabetes mellitus varies between 1-16%. In our series, we found a total of 8 patients under 6.2% of all patients. These results are consistent with those of Vitoria et al. However, some studies show minor incidents, such as by Barera and colleagues (2002) with a prevalence of 3.9%. In some other series have reported higher frequencies, between 8 and 12.3%.

As for the timing of the debut our results coincide with those published by Barera et al, and Holmes et al (2002), in which the majority of patients the diagnosis of celiac disease is posterior to that of type 1 diabetes mellitus. The time elapsed since the debut of the type 1 diabetes mellitus and identification of antibodies in our series are consistent with the results of Saukkonen et al (1996), which are located around the 2 years following the onset of type 1 diabetes mellitus. Other studies such as Maki and colleagues (1995), who observed a lower average interval around 13 months. Our results on the significant association in women are endorsed by other studies in this regard, as published in Spain by Roldan et al (1998).

Most patients were asymptomatic at the time of his presentation and noted that there were no signs of frank malnutrition. Were diagnosed by serological screening and subsequent confirmation with intestinal biopsy. Our results are consistent with those of Barrera et al.

Not found in our series more partnerships with other autoimmune diseases such as pernicious anemia, Addison's disease, Sjögren syndrome, alopecia areata, and rheumatoid arthritis.

Among the background approximately 10% of the patients had atopy and bronchial asthma. These data are consistent with those reported by Lopez Medina et al in their series. The presence of vitiligo in our series (0.7%) is lower than that observed in other studies like the one made in Italy by Romano et al (1998), showing a prevalence of 9%.

#### **3. Conclusion**

152 Type 1 Diabetes Complications

In our series, 2 patients are associated with type 1 diabetes mellitus, celiac disease and

In 13 patients (10.1%) were type allergic processes associated allergic rhinitis and conjunctivitis, 8 patients (6.2%) had asthma, 8 patients (6.2%) were diagnosed with atopic

In type 1 diabetes mellitus there is a polygenic susceptibility. The most important genes that influence human susceptibility to type 1 diabetes mellitus are located in the complex HLA class II. In our series, these data are corroborated, and that these associations are most common. 93.8% of our patients with type 1 diabetes mellitus corresponds to the genetic risk groups as has been described elsewhere, so a 6.2% suffer from type 1 diabetes mellitus

Our results agree with others, as published by the EURODIAB indicating that in most mediterranean countries the male/female ratio is around 1, with a slight male

The period of highest incidence in the study is between 5 and 9 years. These findings are consistent with studies in other countries, which show a tendency for the disease much earlier debut. In our study found significant differences in age at debut by diabetogenic risk groups, age at onset being significantly lower in group III with group I. These results indicate that the combination of different molecules in susceptibility, heterozygous in group III, accelerating the destruction of beta cells by promoting an early onset of type 1 diabetes

Autoimmune disease most often associated to type 1 diabetes mellitus is an autoimmune thyroid disease. Our results, 15.5% compared to the percentage of subjects who agree thyroiditis associated with many studies, such as in Spain by Roland and cols (1999) or made in Italy by Lorini and cols (1996). However, some international studies show higher prevalence, such as that conducted by Lindberg et al (1997), with a prevalence of 38%. As in other studies in our series of cases in which thyroiditis manifested clinically, it is in the form of an underactive thyroid. Although thyroid status of the majority of subjects with

In our series we found a case of hyperthyroidism in a 11-year-old was diagnosed with type 1 diabetes three years ago. Hyperthyroidism is associated with type 1 diabetes mellitus present in 1% of cases, most often in adults. Other studies indicate that hyperthyroidism is

Studies show that thyroiditis is more prevalent in diabetic girls than in boys. However, our

The prevalence of celiac disease associated with children with type 1 diabetes mellitus varies between 1-16%. In our series, we found a total of 8 patients under 6.2% of all patients. These results are consistent with those of Vitoria et al. However, some studies show minor incidents, such as by Barera and colleagues (2002) with a prevalence of 3.9%. In some other

As for the timing of the debut our results coincide with those published by Barera et al, and Holmes et al (2002), in which the majority of patients the diagnosis of celiac disease is posterior to that of type 1 diabetes mellitus. The time elapsed since the debut of the type 1 diabetes mellitus and identification of antibodies in our series are consistent with the results

autoimmune thyroiditis (1 patient 1 patient euthyroid and hypothyroid).

dermatitis and 1 patient ( 0.7%) of vitiligo.

without belonging to a group of HLA-DQ risk.

predominance, but with no significant differences between them.

usually diagnosed before or while type 1 diabetes mellitus.

results do not indicate such a difference, matching other studies.

series have reported higher frequencies, between 8 and 12.3%.

**2.1.1 Discussion** 

mellitus.

positive markers is euthyroid.

The major autoimmune diseases, autoimmune thyroiditis and celiac disease are more prevalent in our diabetic patients than in the nondiabetic population. Although we found more patients in risk group III, no significant differences with other groups.

In conclusion, these data support the recommendation that from the moment of diagnosis of type 1 diabetes mellitus regular determination of thyroid antibodies and celiac disease related. Current recommendations are vague as to what should be the most appropriate timing for this in pediatric patients. We must try to detect such diseases early, but that does not justify excessive and unnecessary repetition of diagnostic tests. The use of standardized monitoring protocols is becoming increasingly necessary to ensure better health care for children and adolescents with type 1 diabetes mellitus.

#### **4. References**


**Part 2** 

**Cardiovascular Complications** 

