**Double Diabetes: The Search for a Treatment Paradigm in Children and Adolescents**

Benjamin U. Nwosu

[50] Fries W, Muja C, Crisafulli C et al. Infliximab and etanercept are equally effective in reducing enterocyte APOPTOSIS in experimental colitis. Int.J.Med.Sci. 2008; 5 (4)

[51] Wullaert A, Heyninck K, Beyaert R. Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes. Biochem.Pharmacol. 2006; 72 (9)

[52] Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF-alpha-induced

[53] Boden G, She P, Mozzoli M et al. Free fatty acids produce insulin resistance and acti‐ vate the proinflammatory nuclear factor-kappaB pathway in rat liver. Diabetes 2005;

[54] Romagnoli M, Gomez-Cabrera MC, Perrelli MG et al. Xanthine oxidase-induced oxi‐ dative stress causes activation of NF-kappaB and inflammation in the liver of type I

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144 Hot Topics in Endocrine and Endocrine-Related Diseases

1090-1101

54 (12) 3458-3465

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53314

## **1. Introduction**

Diabetes mellitus is one of the most prevalent chronic diseases in children. Diabetes mellitus is classified into four major types. Type 1, type 2, gestational, and other specific types. Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. Type 2 diabetes (T2D) results from a combination of insulin resistance and be‐ ta cell insulin secretory defect. The rising prevalence of childhood obesity has made it more difficult to differentiate between these types of diabetes in children. There is a new expres‐ sion of diabetes in children known as double diabetes, or hybrid diabetes. This is a clinical state where both T1D and T2D co-exist in the same individual as shown in Figure 1 below.

Childhood obesity is one of the most serious public health challenges of the 21st century [1]. According to the National Health and Nutrition Examination Survey data, about 16% of children and adolescents in the United States have a body mass index (BMI) (kg/m2 ) ≥95th percentile for age and gender [2]. Body mass index of >95th percentile is classified as over‐ weight by the Center for Disease Control and Prevention [3,4], and as obesity by European criteria [5].

The prevalence of obesity has tripled in the past three decades [6] among male and female adolescents, and across different racial and ethnic groups [6-8]. There has also been a paral‐ lel increase in the prevalence of many obesity-related co-morbid conditions [9] such as T2D, dyslipidemia, hypertension, obstructive sleep apnea, poor quality of life and mortality in adulthood [10-13]. Although obesity is associated primarily with T2D due to insulin resist‐ ance, [14], it may also impact T1D morbidity.

T1D is caused by autoimmune destruction of the beta cells of the pancreas leading to insuli‐ nopenia. It is sub-classified into 2 main categories- type 1A and 1B [15]. In type 1A, individ‐

© 2013 Nwosu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

uals have one or more of the anti-islet cell (including glutamic acid decarboxylase, and insulinoma antigen-2) or anti-insulin antibodies. In type 1B these antibodies are absent, but the clinical and biochemical features are similar to 1A. T2D is characterized by insulin resist‐ ance and absence of diabetes-associated antibodies in serum.

tients with T1D who fail to make the necessary healthy lifestyle changes that are

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Unlike T1D where the *MHC* region of chromosome 6 accounts for approximately 40% of the genetic risk of the disease in concert with other genes [25], and in T2D where genome-wide association studies have identified approximately 50 genetic loci associated with T2D in lean and obese individuals [26-28], there are no distinct genes that are unique to double diabetes. However, it is believed that the major genes that are independently associated with suscept‐ ibility to either T1D (e.g., the MHC and cytotoxic T lymphocyte-associated antigen-4 (*CTLA-4*) [29] or T2D (e.g., the genes encoding adiponectin (*APM1*) and transcription factor 7-like 2 (*TCF7L2*) [30] can serve as genetic determinants for double diabetes, such that the frequency of the major T1D genetic susceptibility gene (*MHC*) is reduced, whereas the ex‐

Apart from the principal genetic determinants of T1D and T2D, there are a number of genes that could potentially lead to an outcome of double diabetes by influencing the pathogenetic processes operating in both T1D and T2D [32]. One of such genes resulting from a genetic variance in insulin receptor substrate 1 (*IRS-1*) plays an important role in insulin resistance, a key component of T2D, and also in β cell apoptosis which is associated with T1D [33]. High mobility group A1 (HMGA1) protein, a product of the Hmga1 gene has been identi‐ fied as a crucial effector in the control of glucose homeostasis, such that impaired HMGA1 function may contribute to the development of specific forms of diabetes [34]. HMGA1-defi‐ cient indiviuduals have reduced insulin receptor expression, reduced insulin signaling and

The epidemic of childhood obesity has led to increased diagnosis of metabolic syndrome and T2D in all children including those with existing T1D [35]. Obese or overweight chil‐ dren have been reported to develop T1D at younger ages than children of normal weight [35]. The SEARCH for Diabetes in Youth Study [36] reported an obesity prevalence rate of 12.6% in US youth with T1D. The study also reported a higher prevalence of overweight sta‐ tus (BMI 85th – 95th percentile) among youth with T1D than in those without diabetes (22.1% vs. 16.1%) (P<0.05). Some children with T1D have either a first- or second-degree rel‐ ative with T2D [1]. Furthermore, weight gain is prevalent in adolescents with T1D after at‐

Therefore, several enviromental factors could lead to the development of double diabetes by their influence on the disease processes of T1D and T2D. Many of the major genetic factors involved in the etiopathogenesis of T2D appear to promote the development of the disease through their influence on obesity and feeding behavior [38]. There is evidence that rapid growth and obesity in early childhood might increase the risk of T1D [35,39]. The strong en‐ vironmental basis for this obesity pandemic and influence on feeding behavior was recently

tainment of adult height, which might further impair insulin sensitivity [37].

recommended for maintenance of normal weight.

pression of the genes associated with T2D is enhanced [31].

decreased insulin secretion similar to the phenotype of T2D [34].

**3.2. Environmental and behavioral factors**

**3.1. Genetic factors**

A new subset of diabetes, called double diabetes is becoming increasingly prevalent as a re‐ sult of the epidemic of childhood obesity [16-18]. In double diabetes, elements of both T1D and T2D co-exist. In this condition, individuals with T1D have insensitivity to insulin that is most often associated with obesity; and individuals with T2D have antibodies against the pancreatic beta cells [14] (Figure 1). Unlike T1D and T2D, there is no consensus on the thera‐ peutic modalities for double diabetes.

The incidence of both T1D and T2D is rising in children and adolescents [14]. Data from the EURODIAB study indicate that the overall prevalence of T1D among young people under 15 years is increasing by greater than 3% each year, and by more than 6% a year in children aged up to four years [19].Analysis of the 2002 to 2003 data from SEARCH for Diabetes in Youth, a multicenter study funded by the Centers for Disease Control and Prevention and the National Institutes of Health to examine T1D and T2D among children and adolescents in the United States, showed that annually, about 15,000 youth in the United States are new‐ ly diagnosed with T1D, and about 3,700 youth with T2D. The reported rate of new cases among youth was 19 per 100,000 each year for T1D, and 5.3 per 100,000 for T2D [20].

## **2. Prevalence**

The prevalence of double diabetes is unknown [16]. However, reports show that about 25% of children with T1D are either overweight or obese [21]. Other reports show that about 35% of children and adolescents with T2D have at least one diabetes-associated antibody [22]. Some authors estimate that about one in three children and adolescents with newly diag‐ nosed diabetes has double diabetes. Pozzilli et al reported a prevalence of 4.96% in their un‐ published Italian cohort [1]. The major difficulty with establishing a prevalence rate for double diabetes is that there are no precise definitions for the different types of diabetes pre‐ senting in youth [1]. This is because clinical phenotypes frequently overlap at onset of the disease [1]. For example, obesity and ketoacidosis can be found in both T1D and T2D [23], and the age of diagnosis is now a poorly differentiating factor [24]. In other cases, the clini‐ cal features of double diabetes are not apparent at diagnosis but evolve over time [18].

## **3. Etiology and pathophysiology**

There are genetic, environmental and behavioral factors that affect the pathophysiological processes of T1D and T2D in such as way to result in double diabetes. Obesity is the central pathophysiological mechanism for double diabetes. Obesity may arise from genetic predis‐ position or from environmental factors such as the anabolic role of insulin injection in pa‐ tients with T1D who fail to make the necessary healthy lifestyle changes that are recommended for maintenance of normal weight.

#### **3.1. Genetic factors**

uals have one or more of the anti-islet cell (including glutamic acid decarboxylase, and insulinoma antigen-2) or anti-insulin antibodies. In type 1B these antibodies are absent, but the clinical and biochemical features are similar to 1A. T2D is characterized by insulin resist‐

A new subset of diabetes, called double diabetes is becoming increasingly prevalent as a re‐ sult of the epidemic of childhood obesity [16-18]. In double diabetes, elements of both T1D and T2D co-exist. In this condition, individuals with T1D have insensitivity to insulin that is most often associated with obesity; and individuals with T2D have antibodies against the pancreatic beta cells [14] (Figure 1). Unlike T1D and T2D, there is no consensus on the thera‐

The incidence of both T1D and T2D is rising in children and adolescents [14]. Data from the EURODIAB study indicate that the overall prevalence of T1D among young people under 15 years is increasing by greater than 3% each year, and by more than 6% a year in children aged up to four years [19].Analysis of the 2002 to 2003 data from SEARCH for Diabetes in Youth, a multicenter study funded by the Centers for Disease Control and Prevention and the National Institutes of Health to examine T1D and T2D among children and adolescents in the United States, showed that annually, about 15,000 youth in the United States are new‐ ly diagnosed with T1D, and about 3,700 youth with T2D. The reported rate of new cases

among youth was 19 per 100,000 each year for T1D, and 5.3 per 100,000 for T2D [20].

The prevalence of double diabetes is unknown [16]. However, reports show that about 25% of children with T1D are either overweight or obese [21]. Other reports show that about 35% of children and adolescents with T2D have at least one diabetes-associated antibody [22]. Some authors estimate that about one in three children and adolescents with newly diag‐ nosed diabetes has double diabetes. Pozzilli et al reported a prevalence of 4.96% in their un‐ published Italian cohort [1]. The major difficulty with establishing a prevalence rate for double diabetes is that there are no precise definitions for the different types of diabetes pre‐ senting in youth [1]. This is because clinical phenotypes frequently overlap at onset of the disease [1]. For example, obesity and ketoacidosis can be found in both T1D and T2D [23], and the age of diagnosis is now a poorly differentiating factor [24]. In other cases, the clini‐ cal features of double diabetes are not apparent at diagnosis but evolve over time [18].

There are genetic, environmental and behavioral factors that affect the pathophysiological processes of T1D and T2D in such as way to result in double diabetes. Obesity is the central pathophysiological mechanism for double diabetes. Obesity may arise from genetic predis‐ position or from environmental factors such as the anabolic role of insulin injection in pa‐

ance and absence of diabetes-associated antibodies in serum.

peutic modalities for double diabetes.

146 Hot Topics in Endocrine and Endocrine-Related Diseases

**3. Etiology and pathophysiology**

**2. Prevalence**

Unlike T1D where the *MHC* region of chromosome 6 accounts for approximately 40% of the genetic risk of the disease in concert with other genes [25], and in T2D where genome-wide association studies have identified approximately 50 genetic loci associated with T2D in lean and obese individuals [26-28], there are no distinct genes that are unique to double diabetes. However, it is believed that the major genes that are independently associated with suscept‐ ibility to either T1D (e.g., the MHC and cytotoxic T lymphocyte-associated antigen-4 (*CTLA-4*) [29] or T2D (e.g., the genes encoding adiponectin (*APM1*) and transcription factor 7-like 2 (*TCF7L2*) [30] can serve as genetic determinants for double diabetes, such that the frequency of the major T1D genetic susceptibility gene (*MHC*) is reduced, whereas the ex‐ pression of the genes associated with T2D is enhanced [31].

Apart from the principal genetic determinants of T1D and T2D, there are a number of genes that could potentially lead to an outcome of double diabetes by influencing the pathogenetic processes operating in both T1D and T2D [32]. One of such genes resulting from a genetic variance in insulin receptor substrate 1 (*IRS-1*) plays an important role in insulin resistance, a key component of T2D, and also in β cell apoptosis which is associated with T1D [33]. High mobility group A1 (HMGA1) protein, a product of the Hmga1 gene has been identi‐ fied as a crucial effector in the control of glucose homeostasis, such that impaired HMGA1 function may contribute to the development of specific forms of diabetes [34]. HMGA1-defi‐ cient indiviuduals have reduced insulin receptor expression, reduced insulin signaling and decreased insulin secretion similar to the phenotype of T2D [34].

#### **3.2. Environmental and behavioral factors**

The epidemic of childhood obesity has led to increased diagnosis of metabolic syndrome and T2D in all children including those with existing T1D [35]. Obese or overweight chil‐ dren have been reported to develop T1D at younger ages than children of normal weight [35]. The SEARCH for Diabetes in Youth Study [36] reported an obesity prevalence rate of 12.6% in US youth with T1D. The study also reported a higher prevalence of overweight sta‐ tus (BMI 85th – 95th percentile) among youth with T1D than in those without diabetes (22.1% vs. 16.1%) (P<0.05). Some children with T1D have either a first- or second-degree rel‐ ative with T2D [1]. Furthermore, weight gain is prevalent in adolescents with T1D after at‐ tainment of adult height, which might further impair insulin sensitivity [37].

Therefore, several enviromental factors could lead to the development of double diabetes by their influence on the disease processes of T1D and T2D. Many of the major genetic factors involved in the etiopathogenesis of T2D appear to promote the development of the disease through their influence on obesity and feeding behavior [38]. There is evidence that rapid growth and obesity in early childhood might increase the risk of T1D [35,39]. The strong en‐ vironmental basis for this obesity pandemic and influence on feeding behavior was recently outlined in a World Health Organization Technical Report [40] which states that 'Changes in the world food economy have contributed to shifting dietary patterns, for example, in‐ creased consumption of energy-dense diets high in fat, particularly saturated fat, and low in unrefined carbohydrates. These patterns are combined with a decline in energy expenditure that is associated with sedentary lifestyle, motorized transport, labor-saving devices at home, the phasing out of physically-demanding manual tasks in the workplace, and leisure time that is preponderantly devoted to physically undemanding pastimes'. However, de‐ spite the established association between obesity and the increasing prevalence of T1D, it is unclear how these environmental processes lead to β cell destruction.

T2D, and states that as the population becomes heavier (fatter), diabetes appears earlier,

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149

The accelerator hypothesis is controversial because studies designed to prove its validity have reached various conclusions [35,60-65]. Reports from the United Kingdom indicated a relationship between younger age at diagnosis of T1D and higher body mass index (BMI) in Middlesbrough [35], and Plymouth [64],but not in Birmingham [61]. Other European studies of large cohorts of German and Austrian children with T1D supported the hypothesis [62,63], although studies from Spain and Australia [66,67] did not. Two studies have been conducted in the United States to examine this hypothesis. Dabelea et al [65] tested the hy‐ pothesis in six centers in the US (Cincinnati, Colorado, Hawaii, Seattle, South Carolina, Southern California) and found a significant relationship between BMI standard deviation score (SDS) and age at diagnosis only among patients with low C-peptide values at diagno‐ sis. Evertsen et al [50] reported a significant inverse relationship between age at diagnosis and BMI SDS in their Wisconsin cohort. Thus, there is no consensus on the validity of the

Traditionally, a patient with the classic symptoms of diabetes which include polyuria, poly‐ dipsia, and polyphagia who also has a family history of T2D, obesity, acanthosis nigricans and lack of both ketosis and diabetes-associated autoantibodies is considered to have T2D [68]. On the other hand, patients with T1D are usually thought to be thin, may present with ketosis, and have diabetes associated autoantibodies. [18] Patients with double diabetes pos‐ sess the features of both T1D and T2D which could present siimultaneously at the time of

*Features of Double Diabetes in Child or Adolescent with Pre-existingType 1 diabetes*: The signs and symptoms typical of T2D can develop gradually in a child or adolescent with pre-existing T1D. The rate of the development of these features of increased metabolic load depends on the individual's genetic makeup and his or her degree of weight gain. These patients are usually overweight or obese and require a high dose of insulin to maintain euglycemia be‐ cause of obesity-related insulin resistance [31,69]. Some of these patients may have hyper‐ tension, dyslipidemia, and poor diabetes control. Female adolescent patients may have

*Features of Double Diabetes in Child or Adolescent with Pre-existing Type 2 diabetes*: The presence of increased 'autoimmune load' as marked by the presence of diabetes-associated autoanti‐ bodies in a child or adolescent with all of the typical clinical features of T2D - excess body weight, acanthosis nigricans, high blood pressure, dyslipidemia, polycystic ovary syn‐ drome, positive family history of T2D, belonging to ethnic/racial minority group – is consis‐

thus suggestive of a true acceleration rather than an incidental risk association [59].

hypothesis among children and adolescents with T1D in the United States.

**4. Clinical features**

polycystic ovarian syndrome.

diagnosis, or develop sequentially over time [18].

tent with a diagnosis of double diabetes [31,69].

Several mechanistic models have been proposed to explain this phenomenon. Some reports have linked high titers of glutamic acid decarboxylase autoantibody to an increase in body mass index (BMI) [41] which suggests that increased BMI might favor the development of an autoimmune response towards β cells. This is in line with other reports indicating that a combination of obesity and insulin resistance speeds up the process of beta cell destruction [35,42]. Other proposed mechanisms for beta cell destruction include the the role of upregu‐ lation of autoimmune response by obesity-associated inflammatory cytokines, and hyper‐ leptinemia-associated T-cell activation [43,44].

Other researchers have proposed the following mechanism for obesity-induced insulin re‐ sistance : (a) the liberation of large amounts of non-esterified fatty acids by visceral fat which stimulate neoglucogenesis in the liver and diminish glucose uptake in the muscles; (b) the association of obesity with increased activity of the sympathetic nervous system, which in combination with direct release of tumor necrosis factor, resistin and other adipo‐ cytokines contribute to insulin resistance; (c) the role of the accummulation of local intra‐ myocellular triglycerides on muscle insulin senstivity [45].

In additon to the above mechanistic models, several hypotheses have been advanced to ex‐ plain the association between obesity and rising prevlaence of T1D. The most prominent of these hypotheses is the accelerator hypothesis which states that T1D and T2D are the same disease state set in different genetic backgrounds [46]. It originally proposed three major fac‐ tors as the basis for the development of diabetes: genetic predisposition, insulin resistance and intrinsic rate of beta cell loss. The accelerators have now been reduced to two without altering the premise of the hypothesis [46]. The first is insulin resistance which is believed to accelerate β-cell apoptosis while rendering them more immunogenic. It posits that insulin resistance is the primary driver for the development of diabetes in a susceptible individual and argues that insulin resistance increases through weight gain as does the rate of onset of diabetes [47]. The second accelerator is the hierachy of responsive genes whose reactivity modulates the gradient of β-cell declining function [46].

The central premise of the accelerator hypothesis is based on studies reporting rising inci‐ dence of obesity [6,48] and T1D in children [49,50]. These findings were strengthened by re‐ ports of an association between weight gain and an increased risk to develop diabetes mellitus [51-53], as well as several reports from Europe indicating that an increasing number of children are being diagnosed with T1D at an earlier age [54-58]. This hypothesis proposes a direct cause and effect relationship between obesity and the development of both T1D and T2D, and states that as the population becomes heavier (fatter), diabetes appears earlier, thus suggestive of a true acceleration rather than an incidental risk association [59].

The accelerator hypothesis is controversial because studies designed to prove its validity have reached various conclusions [35,60-65]. Reports from the United Kingdom indicated a relationship between younger age at diagnosis of T1D and higher body mass index (BMI) in Middlesbrough [35], and Plymouth [64],but not in Birmingham [61]. Other European studies of large cohorts of German and Austrian children with T1D supported the hypothesis [62,63], although studies from Spain and Australia [66,67] did not. Two studies have been conducted in the United States to examine this hypothesis. Dabelea et al [65] tested the hy‐ pothesis in six centers in the US (Cincinnati, Colorado, Hawaii, Seattle, South Carolina, Southern California) and found a significant relationship between BMI standard deviation score (SDS) and age at diagnosis only among patients with low C-peptide values at diagno‐ sis. Evertsen et al [50] reported a significant inverse relationship between age at diagnosis and BMI SDS in their Wisconsin cohort. Thus, there is no consensus on the validity of the hypothesis among children and adolescents with T1D in the United States.

## **4. Clinical features**

outlined in a World Health Organization Technical Report [40] which states that 'Changes in the world food economy have contributed to shifting dietary patterns, for example, in‐ creased consumption of energy-dense diets high in fat, particularly saturated fat, and low in unrefined carbohydrates. These patterns are combined with a decline in energy expenditure that is associated with sedentary lifestyle, motorized transport, labor-saving devices at home, the phasing out of physically-demanding manual tasks in the workplace, and leisure time that is preponderantly devoted to physically undemanding pastimes'. However, de‐ spite the established association between obesity and the increasing prevalence of T1D, it is

Several mechanistic models have been proposed to explain this phenomenon. Some reports have linked high titers of glutamic acid decarboxylase autoantibody to an increase in body mass index (BMI) [41] which suggests that increased BMI might favor the development of an autoimmune response towards β cells. This is in line with other reports indicating that a combination of obesity and insulin resistance speeds up the process of beta cell destruction [35,42]. Other proposed mechanisms for beta cell destruction include the the role of upregu‐ lation of autoimmune response by obesity-associated inflammatory cytokines, and hyper‐

Other researchers have proposed the following mechanism for obesity-induced insulin re‐ sistance : (a) the liberation of large amounts of non-esterified fatty acids by visceral fat which stimulate neoglucogenesis in the liver and diminish glucose uptake in the muscles; (b) the association of obesity with increased activity of the sympathetic nervous system, which in combination with direct release of tumor necrosis factor, resistin and other adipo‐ cytokines contribute to insulin resistance; (c) the role of the accummulation of local intra‐

In additon to the above mechanistic models, several hypotheses have been advanced to ex‐ plain the association between obesity and rising prevlaence of T1D. The most prominent of these hypotheses is the accelerator hypothesis which states that T1D and T2D are the same disease state set in different genetic backgrounds [46]. It originally proposed three major fac‐ tors as the basis for the development of diabetes: genetic predisposition, insulin resistance and intrinsic rate of beta cell loss. The accelerators have now been reduced to two without altering the premise of the hypothesis [46]. The first is insulin resistance which is believed to accelerate β-cell apoptosis while rendering them more immunogenic. It posits that insulin resistance is the primary driver for the development of diabetes in a susceptible individual and argues that insulin resistance increases through weight gain as does the rate of onset of diabetes [47]. The second accelerator is the hierachy of responsive genes whose reactivity

The central premise of the accelerator hypothesis is based on studies reporting rising inci‐ dence of obesity [6,48] and T1D in children [49,50]. These findings were strengthened by re‐ ports of an association between weight gain and an increased risk to develop diabetes mellitus [51-53], as well as several reports from Europe indicating that an increasing number of children are being diagnosed with T1D at an earlier age [54-58]. This hypothesis proposes a direct cause and effect relationship between obesity and the development of both T1D and

unclear how these environmental processes lead to β cell destruction.

leptinemia-associated T-cell activation [43,44].

148 Hot Topics in Endocrine and Endocrine-Related Diseases

myocellular triglycerides on muscle insulin senstivity [45].

modulates the gradient of β-cell declining function [46].

Traditionally, a patient with the classic symptoms of diabetes which include polyuria, poly‐ dipsia, and polyphagia who also has a family history of T2D, obesity, acanthosis nigricans and lack of both ketosis and diabetes-associated autoantibodies is considered to have T2D [68]. On the other hand, patients with T1D are usually thought to be thin, may present with ketosis, and have diabetes associated autoantibodies. [18] Patients with double diabetes pos‐ sess the features of both T1D and T2D which could present siimultaneously at the time of diagnosis, or develop sequentially over time [18].

*Features of Double Diabetes in Child or Adolescent with Pre-existingType 1 diabetes*: The signs and symptoms typical of T2D can develop gradually in a child or adolescent with pre-existing T1D. The rate of the development of these features of increased metabolic load depends on the individual's genetic makeup and his or her degree of weight gain. These patients are usually overweight or obese and require a high dose of insulin to maintain euglycemia be‐ cause of obesity-related insulin resistance [31,69]. Some of these patients may have hyper‐ tension, dyslipidemia, and poor diabetes control. Female adolescent patients may have polycystic ovarian syndrome.

*Features of Double Diabetes in Child or Adolescent with Pre-existing Type 2 diabetes*: The presence of increased 'autoimmune load' as marked by the presence of diabetes-associated autoanti‐ bodies in a child or adolescent with all of the typical clinical features of T2D - excess body weight, acanthosis nigricans, high blood pressure, dyslipidemia, polycystic ovary syn‐ drome, positive family history of T2D, belonging to ethnic/racial minority group – is consis‐ tent with a diagnosis of double diabetes [31,69].

## **5. Diagnosis**

There is the need to formulate universal diagnostic criteria to facilitate the recognition of double diabetes either at the time of onset of hyperglycemia or in the course of the disease process. Pozzilli et al [16,31] recently introduced the concept of 'metabolic load' to describe the features of T2D and 'autoimmune load' to describe the features of T1D. They stated that in an obese child or adolescent with hyperglycemia, an increased 'metabolic load' and a re‐ duced 'autoimmune load' are features of double diabetes (Figure 1). Based on this principle, they advanced the following clinical and biochemical guidelines to facilitate the diagnosis of double diabetes:

**6. Treatment**

ensure adequate glycemic control.

bertal development [37].

**6.2. Alternative therapeutic strategies**

to acute and chronic complications of diabetes.

There is no consensus on the best therapeutic regimen for double diabetes. However, be‐ cause insulin resistance is central to the pathophysiological mechanism of double diabetes, optimal management of this condition necessitates the addition of insulin sensitizers to the patient's therapeutic regimen under appropriate clinical circumstances [18]. Intensification of lifestyle modification strategies should be encouraged to maintain normal weight and at‐ tenuate insulin resistance. Finally, because these patients require increased doses of insulin to maintain euglycemia, it is necessary to develop an insulin titration regimen that would

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151

The availability of insulin analogs and diabetes monitoring devices has improved diabetes care around the world. However, according to recent studies, the prevalence of poorly-con‐ trolled diabetes in youth is still high [70]. This poor glycemic control predisposes the youth

A report by the SEARCH for Diabetes in Youth Study group showed that a high proportion of youth with diabetes had high HbA1c values, with 17% of the youth with TIDM, and 27% of those with T2D showing poor control, defined as HbA1c ≥ 9.5% [70]. The American Dia‐ betes Association target values for HbA1c in relation to age are as follows: 7.5-8.5% at age < 6 years, <8% at age 6-12 years, <7.5% at age 13-18 years, and <7.0% at age 19+ years [68]. Thus only a minority of children and adolescents meet the recommended glycemic targets.

The physiological factors that contribute to poor glycemic control in youth are in part relat‐ ed to the hormonal changes in puberty. Puberty is associated with relative insulin resistance, reflected in a two- to threefold increase in the peak insulin response to oral or intravenous glucose [71]; insulin-mediated glucose disposal is approximately 30% lower in adolescents than in prepubertal children or young adults [72]. This physiologic insulin resistance of pub‐ erty is of minimal consequence in the presence of adequate beta-cell function [73]. The cause of this physiologic resistance is likely the transitory increased activity of the growth hor‐ mone-insulin growth factor axis, as well as sex steroids, which coincides with the physiolog‐ ic insulin resistance of adolescence [74] and act as counter-regulatory hormones. As a result of these physiological changes, insulin dosages are often increased to overcome the resist‐ ance to insulin, but metabolic control still frequently worsens during the later stages of pu‐

The increasing insulin resistance and deterioration of glycemic control in adolescents create a great need for alternative therapeutic strategies in adolescents with T1D. One such strat‐ egy is the addition of a drug that improves insulin sensitivity such as metformin, a bigua‐ nide that acts principally by increasing insulin sensitivity in the liver by inhibiting hepatic gluconeogenesis and thereby reducing hepatic glucose production [75]. Other minor mecha‐

**6.1. The burden of poor glycemic control in children and adolescents**


**Figure 1.** The relationship between T1D, T2D and Double Diabetes, TID = type 1 diabetes, T2D = type 2 diabetes, FBG = fasting blood glucose, 2HPP = 2 hour post prandial glucose level; BMI = body mass index; Abs = antibodies

## **6. Treatment**

**5. Diagnosis**

150 Hot Topics in Endocrine and Endocrine-Related Diseases

double diabetes:

T1D.

autoimmunity.

There is the need to formulate universal diagnostic criteria to facilitate the recognition of double diabetes either at the time of onset of hyperglycemia or in the course of the disease process. Pozzilli et al [16,31] recently introduced the concept of 'metabolic load' to describe the features of T2D and 'autoimmune load' to describe the features of T1D. They stated that in an obese child or adolescent with hyperglycemia, an increased 'metabolic load' and a re‐ duced 'autoimmune load' are features of double diabetes (Figure 1). Based on this principle, they advanced the following clinical and biochemical guidelines to facilitate the diagnosis of

**i.** The presence of clinical features of T2D, hypertension, dyslipidemia, increased

**ii.** The presence of a reduced number of clinical features typical of T1D, such as

**iii.** The presence of autoantibodies to islet cells, although with a reduced number and

**Figure 1.** The relationship between T1D, T2D and Double Diabetes, TID = type 1 diabetes, T2D = type 2 diabetes, FBG

= fasting blood glucose, 2HPP = 2 hour post prandial glucose level; BMI = body mass index; Abs = antibodies

classical T1D. Family history for T2D and T1D might be present.

body mass index with increased cardiovascular risk, compared with children with

weight loss, polyuria and polydipsia, development of ketoacidosis; insulin therapy is not the first line of therapy, by contrast to the situation in subjects with classical

titer compared with T1D, and probably a reduced risk associated with the *MHC* locus compared with subjects with T1D. As compared with T1D, where insulin re‐ sistance and obesity are not common features, double diabetes is always character‐ ized by an obese phenotype, with the additional coexistence of β cell There is no consensus on the best therapeutic regimen for double diabetes. However, be‐ cause insulin resistance is central to the pathophysiological mechanism of double diabetes, optimal management of this condition necessitates the addition of insulin sensitizers to the patient's therapeutic regimen under appropriate clinical circumstances [18]. Intensification of lifestyle modification strategies should be encouraged to maintain normal weight and at‐ tenuate insulin resistance. Finally, because these patients require increased doses of insulin to maintain euglycemia, it is necessary to develop an insulin titration regimen that would ensure adequate glycemic control.

## **6.1. The burden of poor glycemic control in children and adolescents**

The availability of insulin analogs and diabetes monitoring devices has improved diabetes care around the world. However, according to recent studies, the prevalence of poorly-con‐ trolled diabetes in youth is still high [70]. This poor glycemic control predisposes the youth to acute and chronic complications of diabetes.

A report by the SEARCH for Diabetes in Youth Study group showed that a high proportion of youth with diabetes had high HbA1c values, with 17% of the youth with TIDM, and 27% of those with T2D showing poor control, defined as HbA1c ≥ 9.5% [70]. The American Dia‐ betes Association target values for HbA1c in relation to age are as follows: 7.5-8.5% at age < 6 years, <8% at age 6-12 years, <7.5% at age 13-18 years, and <7.0% at age 19+ years [68]. Thus only a minority of children and adolescents meet the recommended glycemic targets.

The physiological factors that contribute to poor glycemic control in youth are in part relat‐ ed to the hormonal changes in puberty. Puberty is associated with relative insulin resistance, reflected in a two- to threefold increase in the peak insulin response to oral or intravenous glucose [71]; insulin-mediated glucose disposal is approximately 30% lower in adolescents than in prepubertal children or young adults [72]. This physiologic insulin resistance of pub‐ erty is of minimal consequence in the presence of adequate beta-cell function [73]. The cause of this physiologic resistance is likely the transitory increased activity of the growth hor‐ mone-insulin growth factor axis, as well as sex steroids, which coincides with the physiolog‐ ic insulin resistance of adolescence [74] and act as counter-regulatory hormones. As a result of these physiological changes, insulin dosages are often increased to overcome the resist‐ ance to insulin, but metabolic control still frequently worsens during the later stages of pu‐ bertal development [37].

#### **6.2. Alternative therapeutic strategies**

The increasing insulin resistance and deterioration of glycemic control in adolescents create a great need for alternative therapeutic strategies in adolescents with T1D. One such strat‐ egy is the addition of a drug that improves insulin sensitivity such as metformin, a bigua‐ nide that acts principally by increasing insulin sensitivity in the liver by inhibiting hepatic gluconeogenesis and thereby reducing hepatic glucose production [75]. Other minor mecha‐ nisms include decreasing fatty acid oxidation and intestinal glucose absorption [76], and in‐ creasing peripheral insulin sensitivity by enhancing glucose uptake in the muscles [77]. Metformin has mainly been used in adult patients with T2D and several studies have shown beneficial effects on body weight, blood lipid levels and metabolic control [78-80]. Random‐ ized controlled trials with metformin in adolescents with T2D reported an improvement in fasting plasma glucose level [81]. However, there have been conflicting reports from studies in adolescents with T1D [75-77,82,83]. The benefit was transient in one study [83] and nega‐ tive in another [82]. The main drawback of these studies was the small sample size and lack of reporting on long term benefit and safety of adjunctive therapy in many of them [84].

delivery during the study. Such a comparison is critical because poor glycemic control con‐ tributes to insulin resistance [87] as there is an inverse relationship between glycemic control (as determined by HbA1c) and insulin sensitivity (estimated by glucose infusion rate during

Double Diabetes: The Search for a Treatment Paradigm in Children and Adolescents

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153

In general, patients with double diabetes are overweight or obese and the resultant insulin resistance increases their insulin requirement [1]. However, in addition to requiring a high insulin dose, evidence suggests that many patients often do not have insulin doses titrated sufficiently to achieve target levels of glucose control [89,90]. These patients remain on sub‐ optimal doses of insulin and fail to reach treatment targets [91]. In a recent study Blonde et al [91] demonstrated the efficacy of algorithm-guided, patient titration of once daily long acting insulin in normalizing HbA1c in adult patients with T2D. They conducted a 20-week, randomized, controlled, open label, multicenter, parallel-group study comparing the safety and efficacy of insulin detemir administered once daily in combination with oral antidiabet‐ ic agents when titrated to two fasting plasma glucose targets ( 3.0-5.0 mmol/L versus 4.4.-6.1 mmol/L) for the treatment of T2D in adults. In that study, fasting plasma glucose level de‐ creased throughout the first 8 weeks of the study and then generally remained flat for each treatment group. The combined treatment groups achieved a mean HbA1c level of 6.9% at the end of the study. There were significant reductions in HbA1c in both titration groups: in the 3.9-5.0 mmol/L fasting plasma glucose target group, HbA1c values decreased from a baseline mean of 8% to 6.8% at 20 weeks. In the 4.4-6.1 mmol/L fasting plasma glucose target group, HbA1c values decreased from a 7.9% at baseline to 7.0% at 20 weeks. Overall rates of hypoglycemia episodes were low and were comparable between treatment groups: 7.73 and 5.27 events/subject/year for the 3.9-5 mmol/L and 4.4-6.1 mmol/L groups, respectively. Mean weight changes from baseline to the end of the study were small and did not differ signifi‐

Our group is conducting a randomized control trial to explore the role of protocol-driven treat-to-target regimen in children and adolescents with double diabetes. Given the rising prevalence of obesity in the general population we speculate that many children with T1D will eventually develop double diabetes. Thus, it is timely to devise an appropriate manage‐ ment protocol to treat this burgeoning sub-population. Our aim is to primarily study this group of patients to determine the role of protocol-driven, treat-to-target regimen alone or in combination with metformin therapy in their care. Metformin is approved by the Food and Drug Administration for use in children with T2D, and recently it has been recommend‐ ed that metformin added to insulin therapy might be used in clinical practice in adolescents with T1D who are poorly controlled and show evidence of insulin resistance (double diabe‐ tes) as noted in T2D [84]. Given the conflicting reports on the efficacy of adjunctive metfor‐ min therapy in adolescents with T1D, this double blind, randomized, placebo controlled trial will demonstrate the effect of meformin on HbA1c reduction under optimized insulin titration regimen. Secondly, we will investigate whether a titrated insulin regimen alone would have a superior-, or similar effect to combined metformin and titrated insulin regi‐

euglycemic-hyperinsulinemic clamp) [88].

cantly between groups.

**6.3. The need for an insulin titration regimen for double diabetes**

Evidence for the coexistence of insulin resistance and insulin deficiency in childhood-on‐ set T1D adults has been demonstrated by the insulin-glucose clamp technique [85,86]. Furthermore, two randomized, placebo-controlled trials have investigated the role of ad‐ junctive metformin therapy in adolescents with T1D. In a randomized placebo controlled trial in children with T1D who were treated for 3 months with adjunctive metformin, Sarnblad et al [77] reported a significant decrease in A1c from 9.6% to 8.7%(p<0.05) in the metformin group, compared to 9.5 to 9.2% (p=NS) in the placebo group. In another study, Hamilton et al [75] reported an HbA1c 0.6% lower in the metformin group than in the placebo group (P<0.035), after 3 months of therapy. Mean HbA1c at the end of the study was decreased by 0.3% in the metformin group, while it increased by 0.3% in the placebo group (p=0.03). Both studies reported no difference in mean body mass index and serum lipids in the metformin versus placebo group after 3 months of therapy. Hamilton et al [75] reported no significant changes in mean insulin sensitivity, measured by frequently sampled glucose after intravenous glucose tolerance test, after 3 months of metformin therapy in the metformin versus placebo group. Sarnblad et al [77], using hy‐ perinsulinemic euglycemic clamp study, demonstrated no significant change in insulin sensitivity after 3 months between the groups, but they did report an increase in insulin sensitivity in the metformin group during the study (P<0.05). Hamilton et al [75] report‐ ed a significant change in the mean daily insulin dose in the metformin group in com‐ parison to the placebo group after 3 months of metformin therapy of -0.14 vs. 0.02, P=0.01. However, Sarnblad [77] did not find a significant difference in the daily insulin dosage between the metformin and placebo groups after 3 months of therapy (1.1 vs. 1.3).

The two randomized, controlled studies by Hamilton and Sarnblad did not categorically re‐ cruit children and adolescents with double diabetes. This is important because this sub-set of diabetic youth is known to be insulin resistant and may require a careful titration of insu‐ lin doses. Adjunctive metformin therapy to achieve glycemic control may also be more effec‐ tive in this subset of diabetes patients.

Furthermore, even though these randomized controlled trials were designed to investigate the effectiveness of adjunctive metformin therapy compared to insulin therapy alone, they were not designed to compare metformin adjunctive therapy to protocol-driven, optimized insulin therapy. Neither study demonstrated a strong head-to-head comparison of adjunc‐ tive metformin to patient-directed, treat to target insulin regimen to ensure optimal insulin delivery during the study. Such a comparison is critical because poor glycemic control con‐ tributes to insulin resistance [87] as there is an inverse relationship between glycemic control (as determined by HbA1c) and insulin sensitivity (estimated by glucose infusion rate during euglycemic-hyperinsulinemic clamp) [88].

#### **6.3. The need for an insulin titration regimen for double diabetes**

nisms include decreasing fatty acid oxidation and intestinal glucose absorption [76], and in‐ creasing peripheral insulin sensitivity by enhancing glucose uptake in the muscles [77]. Metformin has mainly been used in adult patients with T2D and several studies have shown beneficial effects on body weight, blood lipid levels and metabolic control [78-80]. Random‐ ized controlled trials with metformin in adolescents with T2D reported an improvement in fasting plasma glucose level [81]. However, there have been conflicting reports from studies in adolescents with T1D [75-77,82,83]. The benefit was transient in one study [83] and nega‐ tive in another [82]. The main drawback of these studies was the small sample size and lack of reporting on long term benefit and safety of adjunctive therapy in many of them [84].

Evidence for the coexistence of insulin resistance and insulin deficiency in childhood-on‐ set T1D adults has been demonstrated by the insulin-glucose clamp technique [85,86]. Furthermore, two randomized, placebo-controlled trials have investigated the role of ad‐ junctive metformin therapy in adolescents with T1D. In a randomized placebo controlled trial in children with T1D who were treated for 3 months with adjunctive metformin, Sarnblad et al [77] reported a significant decrease in A1c from 9.6% to 8.7%(p<0.05) in the metformin group, compared to 9.5 to 9.2% (p=NS) in the placebo group. In another study, Hamilton et al [75] reported an HbA1c 0.6% lower in the metformin group than in the placebo group (P<0.035), after 3 months of therapy. Mean HbA1c at the end of the study was decreased by 0.3% in the metformin group, while it increased by 0.3% in the placebo group (p=0.03). Both studies reported no difference in mean body mass index and serum lipids in the metformin versus placebo group after 3 months of therapy. Hamilton et al [75] reported no significant changes in mean insulin sensitivity, measured by frequently sampled glucose after intravenous glucose tolerance test, after 3 months of metformin therapy in the metformin versus placebo group. Sarnblad et al [77], using hy‐ perinsulinemic euglycemic clamp study, demonstrated no significant change in insulin sensitivity after 3 months between the groups, but they did report an increase in insulin sensitivity in the metformin group during the study (P<0.05). Hamilton et al [75] report‐ ed a significant change in the mean daily insulin dose in the metformin group in com‐ parison to the placebo group after 3 months of metformin therapy of -0.14 vs. 0.02, P=0.01. However, Sarnblad [77] did not find a significant difference in the daily insulin dosage between the metformin and placebo groups after 3 months of therapy (1.1 vs.

The two randomized, controlled studies by Hamilton and Sarnblad did not categorically re‐ cruit children and adolescents with double diabetes. This is important because this sub-set of diabetic youth is known to be insulin resistant and may require a careful titration of insu‐ lin doses. Adjunctive metformin therapy to achieve glycemic control may also be more effec‐

Furthermore, even though these randomized controlled trials were designed to investigate the effectiveness of adjunctive metformin therapy compared to insulin therapy alone, they were not designed to compare metformin adjunctive therapy to protocol-driven, optimized insulin therapy. Neither study demonstrated a strong head-to-head comparison of adjunc‐ tive metformin to patient-directed, treat to target insulin regimen to ensure optimal insulin

1.3).

tive in this subset of diabetes patients.

152 Hot Topics in Endocrine and Endocrine-Related Diseases

In general, patients with double diabetes are overweight or obese and the resultant insulin resistance increases their insulin requirement [1]. However, in addition to requiring a high insulin dose, evidence suggests that many patients often do not have insulin doses titrated sufficiently to achieve target levels of glucose control [89,90]. These patients remain on sub‐ optimal doses of insulin and fail to reach treatment targets [91]. In a recent study Blonde et al [91] demonstrated the efficacy of algorithm-guided, patient titration of once daily long acting insulin in normalizing HbA1c in adult patients with T2D. They conducted a 20-week, randomized, controlled, open label, multicenter, parallel-group study comparing the safety and efficacy of insulin detemir administered once daily in combination with oral antidiabet‐ ic agents when titrated to two fasting plasma glucose targets ( 3.0-5.0 mmol/L versus 4.4.-6.1 mmol/L) for the treatment of T2D in adults. In that study, fasting plasma glucose level de‐ creased throughout the first 8 weeks of the study and then generally remained flat for each treatment group. The combined treatment groups achieved a mean HbA1c level of 6.9% at the end of the study. There were significant reductions in HbA1c in both titration groups: in the 3.9-5.0 mmol/L fasting plasma glucose target group, HbA1c values decreased from a baseline mean of 8% to 6.8% at 20 weeks. In the 4.4-6.1 mmol/L fasting plasma glucose target group, HbA1c values decreased from a 7.9% at baseline to 7.0% at 20 weeks. Overall rates of hypoglycemia episodes were low and were comparable between treatment groups: 7.73 and 5.27 events/subject/year for the 3.9-5 mmol/L and 4.4-6.1 mmol/L groups, respectively. Mean weight changes from baseline to the end of the study were small and did not differ signifi‐ cantly between groups.

Our group is conducting a randomized control trial to explore the role of protocol-driven treat-to-target regimen in children and adolescents with double diabetes. Given the rising prevalence of obesity in the general population we speculate that many children with T1D will eventually develop double diabetes. Thus, it is timely to devise an appropriate manage‐ ment protocol to treat this burgeoning sub-population. Our aim is to primarily study this group of patients to determine the role of protocol-driven, treat-to-target regimen alone or in combination with metformin therapy in their care. Metformin is approved by the Food and Drug Administration for use in children with T2D, and recently it has been recommend‐ ed that metformin added to insulin therapy might be used in clinical practice in adolescents with T1D who are poorly controlled and show evidence of insulin resistance (double diabe‐ tes) as noted in T2D [84]. Given the conflicting reports on the efficacy of adjunctive metfor‐ min therapy in adolescents with T1D, this double blind, randomized, placebo controlled trial will demonstrate the effect of meformin on HbA1c reduction under optimized insulin titration regimen. Secondly, we will investigate whether a titrated insulin regimen alone would have a superior-, or similar effect to combined metformin and titrated insulin regi‐ men in children and adolescents with double diabetes and how this modality of treatment compares to standard insulin therapy.

**Author details**

Benjamin U. Nwosu

**References**

ma 2004;291:2847-2850.

1994;59:307-316.

1995;149:1085-1091.

2005;111:1999-2012.

University of Massachusetts Medical School, Worcester, Massachusetts, USA

diabetes in youth. Diabetes Care;34 Suppl 2:S166-170.

vention. Am J Clin Nutr 2002;75:761-766.

Metab Disord 2004;28:1189-1196.

J Obes Relat Metab Disord 1999;23 Suppl 2:S2-11.

[1] Pozzilli P, Guglielmi C, Caprio S, Buzzetti R: Obesity, autoimmunity, and double

Double Diabetes: The Search for a Treatment Paradigm in Children and Adolescents

http://dx.doi.org/10.5772/53314

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[2] Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, Flegal KM: Prevalence of overweight and obesity among US children, adolescents, and adults, 1999-2002. Ja‐

[3] Flegal KM, Wei R, Ogden C: Weight-for-stature compared with body mass index-forage growth charts for the United States from the Centers for Disease Control and Pre‐

[4] Himes JH, Dietz WH: Guidelines for overweight in adolescent preventive services: recommendations from an expert committee. The Expert Committee on Clinical Guidelines for Overweight in Adolescent Preventive Services. Am J Clin Nutr

[5] Flodmark CE, Lissau I, Moreno LA, Pietrobelli A, Widhalm K: New insights into the field of children and adolescents' obesity: the European perspective. Int J Obes Relat

[6] Ogden CL, Flegal KM, Carroll MD, Johnson CL: Prevalence and trends in overweight

[7] Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM: Prevalence of overweight and obesity in the United States, 1999-2004. Jama 2006;295:1549-1555.

[8] Troiano RP, Flegal KM, Kuczmarski RJ, Campbell SM, Johnson CL: Overweight prevalence and trends for children and adolescents. The National Health and Nutri‐ tion Examination Surveys, 1963 to 1991. Arch Pediatr Adolesc Med

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among US children and adolescents, 1999-2000. Jama 2002;288:1728-1732.

Blonde et al [91] demonstrated that self-titration regimens facilitate empowerment of pa‐ tients, allowing them to become more involved in their treatment, which can result in im‐ proved glycemic control. Patient-directed insulin titration is increasingly important as health care practitioners often do not have the resources to advise patients with the frequen‐ cy needed to effectively titrate their insulin doses to maintain euglycemia. Optimal patient empowerment through self-titration regimens is critical for the motivation to reach treat‐ ment targets.

## **7. Prognosis**

The coexistence of both T1D and T2D in an individual should in principle denote an in‐ creased risk for the complications of both diseases [32]. Therefore, it is possible that these individuals are at higher risk for the microvascular and metabolic complications of T1D and the macrovascular complications of T2D [18]*.* This is supported by investigations by Or‐ chard et al [85,92], in the Epidemiology of Diabetes Complications Study, who reported that patients with T1D who have a positive family history of T2D were at greater risk for cardio‐ vascular disease than those who did not. Furthermore, data from the Diabetes Control and Complications Trial (DCCT) show that weight gain and central obesity are associated with insulin resistance, hypertension, and dyslipidemia in T1D [93], and data from Epidemiology of Diabetes Interventions and Complications (EDIC) Study show that central obesity is an independent risk factor for incident microalbuminuria in individuals with T1D [94]. Howev‐ er, both DCCT and EDIC follow up studies show that intensive diabetes therapy results in a uniform, major reduction in (and significant protection from) microvascular disease [95], even in overweight or obese T1D patients [92]. Thus, there is the need to devise a consensus treatment regimen that would ensure the best glycemic and metabolic outcome for patients with double diabetes.

## **8. Conclusions**

The global pandemic of obesity in children and adolescents has resulted in a new expression of diabetes mellitus known as double diabetes. The entity encompasses the autoimmune load of T1D and the metabolic load of T2D. There is no consensus on the best therapeutic modality for this new expression of diabetes mellitus. However, optimal therapeutic options must address the coexistence of both metabolic and autoimmune components of diabetes mellitus in the patient. There have also been calls to revise the current classification of diabe‐ tes mellitus to take into account the surging prevalence of double diabetes in children and adolescents.

## **Author details**

men in children and adolescents with double diabetes and how this modality of treatment

Blonde et al [91] demonstrated that self-titration regimens facilitate empowerment of pa‐ tients, allowing them to become more involved in their treatment, which can result in im‐ proved glycemic control. Patient-directed insulin titration is increasingly important as health care practitioners often do not have the resources to advise patients with the frequen‐ cy needed to effectively titrate their insulin doses to maintain euglycemia. Optimal patient empowerment through self-titration regimens is critical for the motivation to reach treat‐

The coexistence of both T1D and T2D in an individual should in principle denote an in‐ creased risk for the complications of both diseases [32]. Therefore, it is possible that these individuals are at higher risk for the microvascular and metabolic complications of T1D and the macrovascular complications of T2D [18]*.* This is supported by investigations by Or‐ chard et al [85,92], in the Epidemiology of Diabetes Complications Study, who reported that patients with T1D who have a positive family history of T2D were at greater risk for cardio‐ vascular disease than those who did not. Furthermore, data from the Diabetes Control and Complications Trial (DCCT) show that weight gain and central obesity are associated with insulin resistance, hypertension, and dyslipidemia in T1D [93], and data from Epidemiology of Diabetes Interventions and Complications (EDIC) Study show that central obesity is an independent risk factor for incident microalbuminuria in individuals with T1D [94]. Howev‐ er, both DCCT and EDIC follow up studies show that intensive diabetes therapy results in a uniform, major reduction in (and significant protection from) microvascular disease [95], even in overweight or obese T1D patients [92]. Thus, there is the need to devise a consensus treatment regimen that would ensure the best glycemic and metabolic outcome for patients

The global pandemic of obesity in children and adolescents has resulted in a new expression of diabetes mellitus known as double diabetes. The entity encompasses the autoimmune load of T1D and the metabolic load of T2D. There is no consensus on the best therapeutic modality for this new expression of diabetes mellitus. However, optimal therapeutic options must address the coexistence of both metabolic and autoimmune components of diabetes mellitus in the patient. There have also been calls to revise the current classification of diabe‐ tes mellitus to take into account the surging prevalence of double diabetes in children and

compares to standard insulin therapy.

154 Hot Topics in Endocrine and Endocrine-Related Diseases

ment targets.

**7. Prognosis**

with double diabetes.

**8. Conclusions**

adolescents.

Benjamin U. Nwosu

University of Massachusetts Medical School, Worcester, Massachusetts, USA

## **References**


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**Chapter 7**

**Glucagon-Like Peptide-1 and Its Implications in Obesity**

Glucagon-like peptide (GLP-1) is derived from the processing of the proglucagon gene. This peptide has diverse biological activities affecting peripheral tissues and the central nervous system. Thus, for example, GLP-1 stimulates pancreas insulin secretion in a glucose-depend‐ ent manner after eating, hence its denomination as an "incretin". GLP-1 has also been con‐ sidered an anorexigenic peptide, while also reducing cerebral glucose metabolism in the human hypothalamus and brain stem. These GLP-1 actions in the pancreas and central nerv‐ ous system are achieved through GLP-1 receptors (GLP-1R) that share the same gene se‐ quence in both tissues. In short, GLP-1 is an antidiabetogenic agent due to its action in the pancreas while acting in hypothalamic areas, helping to generate a state of satiety. Interest‐ ingly, GLP-1/exendin-4 administration in obese Zucker rats, which also develop insulin re‐ sistance, hyperinsulinemia and hyperlipidemia, reduces food intake and induced weight

The mid 20th century recorded the first indications that the hypothalamus plays a major role in feeding behaviour and energy homeostasis, whereby the electrical stimulation of the ven‐ tromedial hypothalamus (VMH) suppresses food intake, and the bilateral lesions of these structures induce hyperphagia and obesity. The VMH was therefore called the satiety cen‐ tre. In contrast, alterations in the lateral hypothalamic area (LH) induced the opposite set of responses, and the LH was hence called the hunger centre. At least two kinds of glucose sen‐ sor neurons have been described in the brain: glucose-excited neurons are located mainly in the VMH and are excited by increased glucose levels in the extracellular space, while glu‐ cose-inhibited neurons (mainly present in the LH) are excited by decreases in glucose con‐ centrations. A direct relationship has also been established between the regulation of food

and reproduction in any medium, provided the original work is properly cited.

© 2013 Hurtado et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

intake and energy homeostasis and hypothalamic metabolic sensor activities.

Veronica Hurtado, Isabel Roncero,

http://dx.doi.org/10.5772/54221

loss, which applies to lean rats, too.

**1. Introduction**

Enrique Blazquez, Elvira Alvarez and Carmen Sanz

Additional information is available at the end of the chapter

[95] Effect of intensive diabetes management on macrovascular events and risk factors in the Diabetes Control and Complications Trial. Am J Cardiol 1995;75:894-903.

## **Glucagon-Like Peptide-1 and Its Implications in Obesity**

Veronica Hurtado, Isabel Roncero, Enrique Blazquez, Elvira Alvarez and Carmen Sanz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54221

## **1. Introduction**

creatinine clearance in the epidemiology of diabetes interventions and complications

[95] Effect of intensive diabetes management on macrovascular events and risk factors in the Diabetes Control and Complications Trial. Am J Cardiol 1995;75:894-903.

study. J Am Soc Nephrol 2007;18:235-243.

164 Hot Topics in Endocrine and Endocrine-Related Diseases

Glucagon-like peptide (GLP-1) is derived from the processing of the proglucagon gene. This peptide has diverse biological activities affecting peripheral tissues and the central nervous system. Thus, for example, GLP-1 stimulates pancreas insulin secretion in a glucose-depend‐ ent manner after eating, hence its denomination as an "incretin". GLP-1 has also been con‐ sidered an anorexigenic peptide, while also reducing cerebral glucose metabolism in the human hypothalamus and brain stem. These GLP-1 actions in the pancreas and central nerv‐ ous system are achieved through GLP-1 receptors (GLP-1R) that share the same gene se‐ quence in both tissues. In short, GLP-1 is an antidiabetogenic agent due to its action in the pancreas while acting in hypothalamic areas, helping to generate a state of satiety. Interest‐ ingly, GLP-1/exendin-4 administration in obese Zucker rats, which also develop insulin re‐ sistance, hyperinsulinemia and hyperlipidemia, reduces food intake and induced weight loss, which applies to lean rats, too.

The mid 20th century recorded the first indications that the hypothalamus plays a major role in feeding behaviour and energy homeostasis, whereby the electrical stimulation of the ven‐ tromedial hypothalamus (VMH) suppresses food intake, and the bilateral lesions of these structures induce hyperphagia and obesity. The VMH was therefore called the satiety cen‐ tre. In contrast, alterations in the lateral hypothalamic area (LH) induced the opposite set of responses, and the LH was hence called the hunger centre. At least two kinds of glucose sen‐ sor neurons have been described in the brain: glucose-excited neurons are located mainly in the VMH and are excited by increased glucose levels in the extracellular space, while glu‐ cose-inhibited neurons (mainly present in the LH) are excited by decreases in glucose con‐ centrations. A direct relationship has also been established between the regulation of food intake and energy homeostasis and hypothalamic metabolic sensor activities.

© 2013 Hurtado et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Both AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) and its downstream target p70 ribosomal protein S6 Kinase 1 (S6K1) contribute to detecting cellular energy and integrate nutrient and hormonal signals in order to maintain energy homeostasis in the organism. Thus, the Ser/Thr kinase AMPK is activated during en‐ ergy depletion, when the AMP/ATP ratio increases and triggers a large number of down‐ stream effectors by stimulating ATP-generating catabolic pathways and inhibiting anabolic pathways in order to restore the energy balance. Specifically, it has been reported that fast‐ ing increases, and re-feeding decreases, AMPK activity in several hypothalamic areas. Like‐ wise, the hypothalamic mTOR/S6K1 pathway has also been involved in the control of feeding and in the regulation of energy balances. Thus, mTOR is activated by glucose and amino acids and, therefore, hypothalamic AMPK and mTOR/S6K1 respond to changes in glucose and other nutrients in the opposite way, and their effects on the regulation of food intake may overlap. Our recent results indicate that AMPK and S6K1 are functionally ex‐ pressed in the VMH and LH areas, with differential activation in response to glucose fluctu‐ ations, in both in vitro models of hypothalamic organotypic slice cultures and animals in response to fasting and re-feeding, as well as in Zucker obese rats with a lower activation degree of hypothalamic AMPK in response to fasting.

distinguishable in their ability to produce biological effects through GLP-1 receptors located in pancreatic cells [3], gastric glands [4] and in adipocytes [5], lung [6] and brain [7-10].

Glucagon-Like Peptide-1 and Its Implications in Obesity

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167

**Figure 1.** Posttranslational processing of preproglucagon. GLP: Glucagon-like peptide; GRPP: Glicentin-related pancre‐

In addition, GLP-1 and its own receptors are synthesized in the same brain regions, strongly supporting the actions of this peptide on the CNS. Thus, the perfusion of several brain nu‐ clei with GLP-1 produces a selective release of neurotransmitters [11, 12], and the central and peripheral administration of this peptide inhibits food and drink intake [13-15]. The coexpression of GLP-1R, glucokinase, and glucose transporter protein 2 (GLUT-2) in the neu‐ rons involved in the control of food intake suggests that these cells may play a role in glucose sensing in the brain [14, 16-19]. Furthermore, GLP-1 has beneficial cardiovascular ef‐ fects in humans by lowering blood pressure and improving myocardial function [20, 21], al‐ though in rats this peptide significantly increases arterial blood pressure and heart rate [22, 23]. Interestingly, GLP-1 has proliferative and antiapoptotic actions on pancreatic β-cells [24, 25], and has neurotrophic and neuroprotective features [21]. Considering the functions of

Within the multiple functions of GLP-1, we have selected two important ones, namely, an

atic peptide; IP: Intermediate peptide; MPGF: the major proglucagon fragment; PS: Signal peptide.

GLP-1, its exendin-4 analogue is used in the treatment of type 2 diabetes [26].

incretin and an anorexigenic peptide.

In addition, we have reported that GLP-1/exendin-4 treatment inhibits the activities of AMPK and S6K1 when the activation of these protein kinases peak in both the VMH and LH areas. In pathophysiological situations, as occurs in Zucker obese rats, exendin-4 seems to act as a compensator for the variations in AMPK activity produced either by oscillations in glucose levels or by pathologies such as obesity or episodes of hyperinsulinemia.

In conclusion, it seems that GLP-1/exendin-4 acts in the VMH and LH, modulating the acti‐ vation status of AMPK and S6K1 in response to glucose fluctuations, helping to improve pathophysiological states such as obesity and insulin resistance. The effects of these peptides in the hypothalamus are mediated through the activation of PKA, PKC and PI3K, as well as the phosphatase PP2.

## **2. Glucagon-like peptide-1: Dual role as an incretin and anorexigenic peptide**

Glucagon and related peptides constitute a family derived from the proglucagon molecule, which is identical in sequence in the pancreas, intestine and brain [1], although post-transla‐ tional processing of the precursor yields different products in these organs [2]. (Figure 1)

In gut L-cells, the C–terminal portion of proglucagon is predominantly processed to gluca‐ gon-like peptide-1 (GLP-1) and GLP-2. Further processing of GLP-1 in these cells produces the amidated and truncated forms of the peptide: GLP-1 [7-36] amide, GLP-1 [7-37] and GLP-1 [1-36] amide, with the first two being the biologically active forms, which are cited in the rest of the test as GLP-1. Although the truncated forms of GLP-1 are reported to have strong incretin activity, it is currently known that they are also important in the functioning of other peripheral tissues and the central nervous system. Both forms of the peptide are in‐ distinguishable in their ability to produce biological effects through GLP-1 receptors located in pancreatic cells [3], gastric glands [4] and in adipocytes [5], lung [6] and brain [7-10].

Both AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) and its downstream target p70 ribosomal protein S6 Kinase 1 (S6K1) contribute to detecting cellular energy and integrate nutrient and hormonal signals in order to maintain energy homeostasis in the organism. Thus, the Ser/Thr kinase AMPK is activated during en‐ ergy depletion, when the AMP/ATP ratio increases and triggers a large number of down‐ stream effectors by stimulating ATP-generating catabolic pathways and inhibiting anabolic pathways in order to restore the energy balance. Specifically, it has been reported that fast‐ ing increases, and re-feeding decreases, AMPK activity in several hypothalamic areas. Like‐ wise, the hypothalamic mTOR/S6K1 pathway has also been involved in the control of feeding and in the regulation of energy balances. Thus, mTOR is activated by glucose and amino acids and, therefore, hypothalamic AMPK and mTOR/S6K1 respond to changes in glucose and other nutrients in the opposite way, and their effects on the regulation of food intake may overlap. Our recent results indicate that AMPK and S6K1 are functionally ex‐ pressed in the VMH and LH areas, with differential activation in response to glucose fluctu‐ ations, in both in vitro models of hypothalamic organotypic slice cultures and animals in response to fasting and re-feeding, as well as in Zucker obese rats with a lower activation

In addition, we have reported that GLP-1/exendin-4 treatment inhibits the activities of AMPK and S6K1 when the activation of these protein kinases peak in both the VMH and LH areas. In pathophysiological situations, as occurs in Zucker obese rats, exendin-4 seems to act as a compensator for the variations in AMPK activity produced either by oscillations in

In conclusion, it seems that GLP-1/exendin-4 acts in the VMH and LH, modulating the acti‐ vation status of AMPK and S6K1 in response to glucose fluctuations, helping to improve pathophysiological states such as obesity and insulin resistance. The effects of these peptides in the hypothalamus are mediated through the activation of PKA, PKC and PI3K, as well as

**2. Glucagon-like peptide-1: Dual role as an incretin and anorexigenic**

Glucagon and related peptides constitute a family derived from the proglucagon molecule, which is identical in sequence in the pancreas, intestine and brain [1], although post-transla‐ tional processing of the precursor yields different products in these organs [2]. (Figure 1)

In gut L-cells, the C–terminal portion of proglucagon is predominantly processed to gluca‐ gon-like peptide-1 (GLP-1) and GLP-2. Further processing of GLP-1 in these cells produces the amidated and truncated forms of the peptide: GLP-1 [7-36] amide, GLP-1 [7-37] and GLP-1 [1-36] amide, with the first two being the biologically active forms, which are cited in the rest of the test as GLP-1. Although the truncated forms of GLP-1 are reported to have strong incretin activity, it is currently known that they are also important in the functioning of other peripheral tissues and the central nervous system. Both forms of the peptide are in‐

glucose levels or by pathologies such as obesity or episodes of hyperinsulinemia.

degree of hypothalamic AMPK in response to fasting.

166 Hot Topics in Endocrine and Endocrine-Related Diseases

the phosphatase PP2.

**peptide**

**Figure 1.** Posttranslational processing of preproglucagon. GLP: Glucagon-like peptide; GRPP: Glicentin-related pancre‐ atic peptide; IP: Intermediate peptide; MPGF: the major proglucagon fragment; PS: Signal peptide.

In addition, GLP-1 and its own receptors are synthesized in the same brain regions, strongly supporting the actions of this peptide on the CNS. Thus, the perfusion of several brain nu‐ clei with GLP-1 produces a selective release of neurotransmitters [11, 12], and the central and peripheral administration of this peptide inhibits food and drink intake [13-15]. The coexpression of GLP-1R, glucokinase, and glucose transporter protein 2 (GLUT-2) in the neu‐ rons involved in the control of food intake suggests that these cells may play a role in glucose sensing in the brain [14, 16-19]. Furthermore, GLP-1 has beneficial cardiovascular ef‐ fects in humans by lowering blood pressure and improving myocardial function [20, 21], al‐ though in rats this peptide significantly increases arterial blood pressure and heart rate [22, 23]. Interestingly, GLP-1 has proliferative and antiapoptotic actions on pancreatic β-cells [24, 25], and has neurotrophic and neuroprotective features [21]. Considering the functions of GLP-1, its exendin-4 analogue is used in the treatment of type 2 diabetes [26].

Within the multiple functions of GLP-1, we have selected two important ones, namely, an incretin and an anorexigenic peptide.

#### **2.1. GLP-1 actions as incretin hormone**

The proposals made in 1906 by Moore et al. [27] on the antidiabetogenic effect of intestinal factors, and in 1929 by Zung & La Barre [28] on the release of a substance from intestinal mucosa with properties to decrease glycaemia, signalled the start of the development of the incretin concept and the study of the relationships between the gut and the endocrine pan‐ creas. However, for many years these suggestions were ignored, until the development of radioimmunoassays, when Elrich et al. [29] demonstrated that the insulin secretion response to an oral glucose overload was greater to that obtained after intravenous perfusion with the same amount of glucose. This lends support to the belief that substances from the intestine were involved in the postprandial control of insulin secretion, which was referred to accord‐ ingly as the incretin effect. It is accepted that 20% to 60% of the increase in postprandial in‐ sulin secretion is due to this effect; with the broad oscillation being explained by the amount and composition of food intake.

In addition, many biological effects of GLP-1, other than incretin actions, have been reported in recent decades, representing a good tool for several therapeutic treatments. These GLP-1 effects include properties such as an anorexic peptide, beneficial cardiovascular actions in humans, increased pulmonary surfactant formation in human and experimental animals, pancreatic islet neogenesis and proliferative and antiapoptotic actions. GLP-1 receptors are also widely expressed in the brain [9, 10], where their agonists produce a selective release of neurotransmitters [11, 12] and increase GLP-1 receptor expression in glia after a mechanical lesion of the rat brain has been reported [38]. Accordingly pre-clinical data suggest a neuro‐ protective/neurotrophic function of GLP-1, and some authors have proposed that this pep‐ tide may have a positive potential role for reversing neurodegenerative disorders [21].

Glucagon-Like Peptide-1 and Its Implications in Obesity

http://dx.doi.org/10.5772/54221

169

A number of peptide hormones, previously thought to be specific to the gastroenteropancre‐ atic system and later found also in the mammalian brain, have been shown to modulate ap‐ petite, energy homeostasis and body weight. They have these physiological effects together with other neuropeptides, such as neuropeptide Y (NPY), opioid peptides, galanin, vaso‐ pressin, and GHRH. Peptide Y (Y3-36) is also released from the gastrointestinal tract post‐ prandially, and acts on the NPY Y2 receptor in the arcuate nucleus to inhibit feeding, with a long–term effect [39]. Conversely, other satiety signals induced by gut-brain peptides such as GLP-1 [13-15], GLP-2 [40] and cholecystokinin produced a short-term effect, while insulin and leptin [41] inhibit the appetite by increasing the formation of pro-opiomelanocortin (POMC) and reducing NPY action. In addition, ghrelin, a peptide released by the stomach,

GLP-1 and GLP-2 significantly modify feeding behaviour. The intracerebroventricular (icv) or subcutaneous administration (sc) of GLP-1 produced a marked reduction in food intake and water ingestion [13-15]. Exendin-4 proved also to be a potent agonist of GLP-1 by de‐ creasing both food and water intake in a dose-dependent manner. Pre-treatment with exen‐ din [9-39], an inhibitor of the GLP-1 receptor, reversed the inhibitory effects of GLP-1 and exendin-4. These findings suggest that GLP-1 may modulate both food and water intake through either a central or peripheral mechanism. Similar results have been found in hu‐ mans when the peptide was administered in the periphery [42]. After the subcutaneous ad‐ ministration of GLP-1, it could enter the brain by binding to blood–brain–barrier-free organs such as the subfornical organ and the area postrema [43], or through the choroid plexus,

Several observations suggest a possible action of GLP-1 on thirst-regulatory mechanisms, since GLP-1R mRNA has been located in brain areas related to the control of thirst, such as the preoptic area, glial cells lining the third ventricle and, especially, the neurons of the PVN, which is a key station for water balance regulation through the antidiuretic effects of vasopressin released by its projection to the neurohypophysis [44]. In addition, the icv ad‐ ministration of GLP-1 significantly increases the circulating levels of vasopressin, and the colocalization of the mRNA of the GLP-1 receptor, and vasopressin has been found in the

**2.2. GLP-1 actions in the control of food intake**

is stimulated before meals to facilitate NPY action.

which has a high density of GLP-1 receptors [17].

neurons of the PVN [45].

The functional relationships between the intestine and the pancreatic islet were named by Unger and Eisentraut in 1969 [30] as the enteroinsular axis, while the criteria formulated by Creutzfeldt [31] considered a molecule to be incretin when it is secreted in response to nu‐ trients, and that physiological concentrations increased the secretion of insulin in the pres‐ ence of high glucose concentrations.

The first peptide described with incretin activity was the gastric inhibitory polypeptide (GIP) that went on to be referred to also as the glucose-dependent insulinotropic polypep‐ tide GIP). Thereafter, the observation of incretin activity after the inactivation of GIP sug‐ gested the existence of other molecules with an incretin effect. Thus, in experimental models where GIP was blocked by its own antibodies 50-80% of incretin activity was still observed. We now know that GLP-1 has a greater incretin effect than GIP, being considered the most powerful incretin molecule of all those known. In other words, a molecule with incretin ac‐ tivity may be defined as a hormone of intestinal origin that potentiates the secretion of insu‐ lin after the oral ingestion of nutrients. Knowledge of incretins has been very useful for a better understanding of certain pathophysiological entities [32]. In 1986, Nauck et al. [33] first documented a reduced incretin effect in patients, with type 2 diabetes. It is important to note that Nauck et al. described this reduced effect with GIP and not with GLP-1, because at that time GIP was the only incretin known. However, a year later [34], GLP-1 was identified as an incretin hormone and shown to be more effective than GIP to stimulate insulin secre‐ tion on a molar basis and at an equivalent level of glucose concentration [35]. Both in nondiabetic and type 2 diabetic subjects, GLP-1 was more effective than GIP at enhancing insulin secretion and lowering glucagon concentrations [36].

The recognition that native GLP-1 is quickly degraded by the protease dipeptidyl peptidase IV (DPP-4) led to the development of GLP-1 agonists that are resistant to this enzyme [37]. The degradation by DPP-4 of exenatide and liraglutide and DPP-4 inhibitors (sitagliptin, saxagliptin, vildagliptin and linagliptin) currently represents an effective therapeutic option for patients with type 2 diabetes. Furthermore, several agents have been developed in recent years, including longer acting DPP-4 resistant GLP-1 agonists.

In addition, many biological effects of GLP-1, other than incretin actions, have been reported in recent decades, representing a good tool for several therapeutic treatments. These GLP-1 effects include properties such as an anorexic peptide, beneficial cardiovascular actions in humans, increased pulmonary surfactant formation in human and experimental animals, pancreatic islet neogenesis and proliferative and antiapoptotic actions. GLP-1 receptors are also widely expressed in the brain [9, 10], where their agonists produce a selective release of neurotransmitters [11, 12] and increase GLP-1 receptor expression in glia after a mechanical lesion of the rat brain has been reported [38]. Accordingly pre-clinical data suggest a neuro‐ protective/neurotrophic function of GLP-1, and some authors have proposed that this pep‐ tide may have a positive potential role for reversing neurodegenerative disorders [21].

#### **2.2. GLP-1 actions in the control of food intake**

**2.1. GLP-1 actions as incretin hormone**

168 Hot Topics in Endocrine and Endocrine-Related Diseases

and composition of food intake.

ence of high glucose concentrations.

insulin secretion and lowering glucagon concentrations [36].

years, including longer acting DPP-4 resistant GLP-1 agonists.

The proposals made in 1906 by Moore et al. [27] on the antidiabetogenic effect of intestinal factors, and in 1929 by Zung & La Barre [28] on the release of a substance from intestinal mucosa with properties to decrease glycaemia, signalled the start of the development of the incretin concept and the study of the relationships between the gut and the endocrine pan‐ creas. However, for many years these suggestions were ignored, until the development of radioimmunoassays, when Elrich et al. [29] demonstrated that the insulin secretion response to an oral glucose overload was greater to that obtained after intravenous perfusion with the same amount of glucose. This lends support to the belief that substances from the intestine were involved in the postprandial control of insulin secretion, which was referred to accord‐ ingly as the incretin effect. It is accepted that 20% to 60% of the increase in postprandial in‐ sulin secretion is due to this effect; with the broad oscillation being explained by the amount

The functional relationships between the intestine and the pancreatic islet were named by Unger and Eisentraut in 1969 [30] as the enteroinsular axis, while the criteria formulated by Creutzfeldt [31] considered a molecule to be incretin when it is secreted in response to nu‐ trients, and that physiological concentrations increased the secretion of insulin in the pres‐

The first peptide described with incretin activity was the gastric inhibitory polypeptide (GIP) that went on to be referred to also as the glucose-dependent insulinotropic polypep‐ tide GIP). Thereafter, the observation of incretin activity after the inactivation of GIP sug‐ gested the existence of other molecules with an incretin effect. Thus, in experimental models where GIP was blocked by its own antibodies 50-80% of incretin activity was still observed. We now know that GLP-1 has a greater incretin effect than GIP, being considered the most powerful incretin molecule of all those known. In other words, a molecule with incretin ac‐ tivity may be defined as a hormone of intestinal origin that potentiates the secretion of insu‐ lin after the oral ingestion of nutrients. Knowledge of incretins has been very useful for a better understanding of certain pathophysiological entities [32]. In 1986, Nauck et al. [33] first documented a reduced incretin effect in patients, with type 2 diabetes. It is important to note that Nauck et al. described this reduced effect with GIP and not with GLP-1, because at that time GIP was the only incretin known. However, a year later [34], GLP-1 was identified as an incretin hormone and shown to be more effective than GIP to stimulate insulin secre‐ tion on a molar basis and at an equivalent level of glucose concentration [35]. Both in nondiabetic and type 2 diabetic subjects, GLP-1 was more effective than GIP at enhancing

The recognition that native GLP-1 is quickly degraded by the protease dipeptidyl peptidase IV (DPP-4) led to the development of GLP-1 agonists that are resistant to this enzyme [37]. The degradation by DPP-4 of exenatide and liraglutide and DPP-4 inhibitors (sitagliptin, saxagliptin, vildagliptin and linagliptin) currently represents an effective therapeutic option for patients with type 2 diabetes. Furthermore, several agents have been developed in recent A number of peptide hormones, previously thought to be specific to the gastroenteropancre‐ atic system and later found also in the mammalian brain, have been shown to modulate ap‐ petite, energy homeostasis and body weight. They have these physiological effects together with other neuropeptides, such as neuropeptide Y (NPY), opioid peptides, galanin, vaso‐ pressin, and GHRH. Peptide Y (Y3-36) is also released from the gastrointestinal tract post‐ prandially, and acts on the NPY Y2 receptor in the arcuate nucleus to inhibit feeding, with a long–term effect [39]. Conversely, other satiety signals induced by gut-brain peptides such as GLP-1 [13-15], GLP-2 [40] and cholecystokinin produced a short-term effect, while insulin and leptin [41] inhibit the appetite by increasing the formation of pro-opiomelanocortin (POMC) and reducing NPY action. In addition, ghrelin, a peptide released by the stomach, is stimulated before meals to facilitate NPY action.

GLP-1 and GLP-2 significantly modify feeding behaviour. The intracerebroventricular (icv) or subcutaneous administration (sc) of GLP-1 produced a marked reduction in food intake and water ingestion [13-15]. Exendin-4 proved also to be a potent agonist of GLP-1 by de‐ creasing both food and water intake in a dose-dependent manner. Pre-treatment with exen‐ din [9-39], an inhibitor of the GLP-1 receptor, reversed the inhibitory effects of GLP-1 and exendin-4. These findings suggest that GLP-1 may modulate both food and water intake through either a central or peripheral mechanism. Similar results have been found in hu‐ mans when the peptide was administered in the periphery [42]. After the subcutaneous ad‐ ministration of GLP-1, it could enter the brain by binding to blood–brain–barrier-free organs such as the subfornical organ and the area postrema [43], or through the choroid plexus, which has a high density of GLP-1 receptors [17].

Several observations suggest a possible action of GLP-1 on thirst-regulatory mechanisms, since GLP-1R mRNA has been located in brain areas related to the control of thirst, such as the preoptic area, glial cells lining the third ventricle and, especially, the neurons of the PVN, which is a key station for water balance regulation through the antidiuretic effects of vasopressin released by its projection to the neurohypophysis [44]. In addition, the icv ad‐ ministration of GLP-1 significantly increases the circulating levels of vasopressin, and the colocalization of the mRNA of the GLP-1 receptor, and vasopressin has been found in the neurons of the PVN [45].

The control of feeding behaviour by GLP-1 and exendin-4 has been explored in Zucker obese rats, resulting in a reduction in food intake, with exendin-4 being much more potent than GLP-1. The long-term sc administration of exendin-4 decreased daily food intake and practically blocked weight gain in obese rats. These observations highlight the potential use‐ fulness of exendin-4 as a tool for treating obesity and/or diabetes. Both GLP-1 and exendin-4 control blood glucose through the stimulation of glucose-dependent insulin secretion, the inhibition of glucagon secretion, and delayed gastric emptying [34, 46, 47], which facilitate the decrease in blood glucose in type 1 and type 2 diabetic patients [48]. In the light of these results, different N-terminal substituted GLP-1 analogues resistant to DPP-IV have recently been developed. These resistant analogues have a prolonged metabolic stability in vivo and improved biological activity, which is of great interest in the treatment of type 2 diabetes and/or obesity.

The dual control theory of feeding is based on the homeostatic view of hunger and satiety, together with the consideration of glucose not only as a metabolic fuel, but also as a signal‐ ling molecule, and the existence of specialized neurons containing glucose sensors that acti‐ vate or inhibit feeding when blood glucose levels change. A fall in glucose would activate the LH and, consequently, give rise to hunger. Hunger leads to the consumption of food and thus to an elevation of glucose levels that would activate the VMH, leading to a feeling of

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Over the last 25 years, there has been a dramatic increase in studies on the hypothalamus. Knowledge of the hypothalamus has only recently evolved from anatomical concepts (nu‐ clei, 'areas' and fibre tracts) to neurochemicals (characterizing the distributions of neuropep‐ tides and transmitters and their receptors), focusing on the modulation of feeding behaviour and energy expenditure. We are now beginning to reach the stage of functionally under‐ standing the molecular mechanisms, defining exactly which neuronal populations respond to specific nutritional and related signals, and how they pass on that information. At least 25 transmitters have been suggested to play key roles in feeding behaviour [54]. Accordingly, the dorsomedial and paraventricular regions are important within the hypothalamus, along with the well-established VMH and LH [55]. The VMH, which consists of the ventromedial and arcuate nuclei, respectively, is a key region for integrating the peripheral signals of nu‐ trient status and adiposity. The arcuate nucleus contains neurons with NPY/AgRP and POMC, which have opposing effects on energy homeostasis. Thus, NPY increases food in‐ take and activates energy sparing mechanisms, while melanocortins decrease food intake and increase energy expenditure [56]. On the other hand, the LH, the classical "feeding cen‐ tre", is a heterogeneous area that receives a multitude of neuronal inputs from many areas known to be important in the regulation of energy homeostasis: LH encompassing neurons and terminals containing orexigenic peptides. Basically, the LH contains two distinct neuro‐ nal cell populations that regulate feeding behaviour: containing hypocretin/orexin [57, 58]

In addition to its response to circulating peptides and hormones that reflect energy status, the brain, and specifically the hypothalamus, also senses and responds to changes in blood glucose levels [14, 16, 18, 19, 60]. There are several areas in the brain acting as a glucose sen‐ sor. Examples of these are the hypothalamus [60, 61], nucleus solitarius [62] and amygdala [63]. The glucose sensing neurons located in these areas monitor energy status and initiate the responses to maintain glucose and energy homeostasis. Besides the brain glucose sen‐ sors, there are also glucose sensors located in peripheral tissues including the intestine [64], the carotid body [65] and mesenteric veins [66]. In fact, glucose sensors in the hypothalamus were first discovered in the VMH and LH [57, 58]. Moreover, interstitial glucose levels in the VMH and LH vary with blood glucose concentration [67], and these changes in glucose have been postulated to trigger meal initiation. Since glucose is the brain's primary fuel, it should respond to a severe glucose deficiency. In this way, VMH glucose sensors may play a role in detecting and countering severe glucose deficiency [68]. However, Levin et al. recently showed there was no correlation between VMH glucose levels and spontaneous feeding

satiety that will stop the feeding, and eventually glucose levels would fall again.

and a melanin-concentrating hormone (MCH) [59] (Figure 2).

On the other hand, the icv administration of GLP-2 to mice and rats produced a marked de‐ crease in food intake but not in water ingestion [43]. Surprisingly, this effect was avoided by the administration of exendin [9-39], an antagonist of GLP-1.

## **3. Importance of the VMH and LH in the control of food intake**

In recent years, researchers have been focusing on the relationship between gut hormones and the brain areas controlling appetite, ingestion, food reward and body weight [49, 50].

Both gut and brain are considered the main organs responsible for controlling body weight. The hypothalamus is the focus of many of the peripheral signals and neural pathways that control energy homeostasis and body weight. However, new evidence has been forthcoming in recent years to suggest that human food intake is also controlled by other areas in the cen‐ tral nervous system, such as subcortical and cortical areas.

The hypothalamus regulates body weight by precisely balancing the intake of food, energy expenditure and body fat tissue. The role of the hypothalamus in regulating food intake and body weight was established in 1940 [51] through the classical experiments by Hetherington and Ranson. They placed bilateral electrolytic lesions in a vast region of the hypothalamus, occupied by the dorsomedial and ventromedial areas, the arcuate nucleus, the fornix and a portion of the lateral hypothalamic area (without disturbing the pituitary gland). The results were a marked adiposity characterized by a doubling of body weight and a huge increase in body lipids. A few years later, Anand and Brobeck [52] continued these experiments in greater detail, demonstrating that lesions of the lateral hypothalamus at the level adjacent to the ventromedial nucleus caused loss of appetite, inanition, and even death by starvation. Thus, the lateral hypothalamic area acted as a "feeding centre" and the ventromedial nu‐ cleus as a "satiety centre". Since then, it has been established that the "dual centre model" regulates feeding [53], that the proposed lesioning of the VMH increases appetite, while stimulating the VMH decreases it. By contrast, lesioning or stimulating the LH decreases or induces appetite, respectively.

The dual control theory of feeding is based on the homeostatic view of hunger and satiety, together with the consideration of glucose not only as a metabolic fuel, but also as a signal‐ ling molecule, and the existence of specialized neurons containing glucose sensors that acti‐ vate or inhibit feeding when blood glucose levels change. A fall in glucose would activate the LH and, consequently, give rise to hunger. Hunger leads to the consumption of food and thus to an elevation of glucose levels that would activate the VMH, leading to a feeling of satiety that will stop the feeding, and eventually glucose levels would fall again.

The control of feeding behaviour by GLP-1 and exendin-4 has been explored in Zucker obese rats, resulting in a reduction in food intake, with exendin-4 being much more potent than GLP-1. The long-term sc administration of exendin-4 decreased daily food intake and practically blocked weight gain in obese rats. These observations highlight the potential use‐ fulness of exendin-4 as a tool for treating obesity and/or diabetes. Both GLP-1 and exendin-4 control blood glucose through the stimulation of glucose-dependent insulin secretion, the inhibition of glucagon secretion, and delayed gastric emptying [34, 46, 47], which facilitate the decrease in blood glucose in type 1 and type 2 diabetic patients [48]. In the light of these results, different N-terminal substituted GLP-1 analogues resistant to DPP-IV have recently been developed. These resistant analogues have a prolonged metabolic stability in vivo and improved biological activity, which is of great interest in the treatment of type 2 diabetes

On the other hand, the icv administration of GLP-2 to mice and rats produced a marked de‐ crease in food intake but not in water ingestion [43]. Surprisingly, this effect was avoided by

In recent years, researchers have been focusing on the relationship between gut hormones and the brain areas controlling appetite, ingestion, food reward and body weight [49, 50].

Both gut and brain are considered the main organs responsible for controlling body weight. The hypothalamus is the focus of many of the peripheral signals and neural pathways that control energy homeostasis and body weight. However, new evidence has been forthcoming in recent years to suggest that human food intake is also controlled by other areas in the cen‐

The hypothalamus regulates body weight by precisely balancing the intake of food, energy expenditure and body fat tissue. The role of the hypothalamus in regulating food intake and body weight was established in 1940 [51] through the classical experiments by Hetherington and Ranson. They placed bilateral electrolytic lesions in a vast region of the hypothalamus, occupied by the dorsomedial and ventromedial areas, the arcuate nucleus, the fornix and a portion of the lateral hypothalamic area (without disturbing the pituitary gland). The results were a marked adiposity characterized by a doubling of body weight and a huge increase in body lipids. A few years later, Anand and Brobeck [52] continued these experiments in greater detail, demonstrating that lesions of the lateral hypothalamus at the level adjacent to the ventromedial nucleus caused loss of appetite, inanition, and even death by starvation. Thus, the lateral hypothalamic area acted as a "feeding centre" and the ventromedial nu‐ cleus as a "satiety centre". Since then, it has been established that the "dual centre model" regulates feeding [53], that the proposed lesioning of the VMH increases appetite, while stimulating the VMH decreases it. By contrast, lesioning or stimulating the LH decreases or

**3. Importance of the VMH and LH in the control of food intake**

the administration of exendin [9-39], an antagonist of GLP-1.

tral nervous system, such as subcortical and cortical areas.

induces appetite, respectively.

and/or obesity.

170 Hot Topics in Endocrine and Endocrine-Related Diseases

Over the last 25 years, there has been a dramatic increase in studies on the hypothalamus. Knowledge of the hypothalamus has only recently evolved from anatomical concepts (nu‐ clei, 'areas' and fibre tracts) to neurochemicals (characterizing the distributions of neuropep‐ tides and transmitters and their receptors), focusing on the modulation of feeding behaviour and energy expenditure. We are now beginning to reach the stage of functionally under‐ standing the molecular mechanisms, defining exactly which neuronal populations respond to specific nutritional and related signals, and how they pass on that information. At least 25 transmitters have been suggested to play key roles in feeding behaviour [54]. Accordingly, the dorsomedial and paraventricular regions are important within the hypothalamus, along with the well-established VMH and LH [55]. The VMH, which consists of the ventromedial and arcuate nuclei, respectively, is a key region for integrating the peripheral signals of nu‐ trient status and adiposity. The arcuate nucleus contains neurons with NPY/AgRP and POMC, which have opposing effects on energy homeostasis. Thus, NPY increases food in‐ take and activates energy sparing mechanisms, while melanocortins decrease food intake and increase energy expenditure [56]. On the other hand, the LH, the classical "feeding cen‐ tre", is a heterogeneous area that receives a multitude of neuronal inputs from many areas known to be important in the regulation of energy homeostasis: LH encompassing neurons and terminals containing orexigenic peptides. Basically, the LH contains two distinct neuro‐ nal cell populations that regulate feeding behaviour: containing hypocretin/orexin [57, 58] and a melanin-concentrating hormone (MCH) [59] (Figure 2).

In addition to its response to circulating peptides and hormones that reflect energy status, the brain, and specifically the hypothalamus, also senses and responds to changes in blood glucose levels [14, 16, 18, 19, 60]. There are several areas in the brain acting as a glucose sen‐ sor. Examples of these are the hypothalamus [60, 61], nucleus solitarius [62] and amygdala [63]. The glucose sensing neurons located in these areas monitor energy status and initiate the responses to maintain glucose and energy homeostasis. Besides the brain glucose sen‐ sors, there are also glucose sensors located in peripheral tissues including the intestine [64], the carotid body [65] and mesenteric veins [66]. In fact, glucose sensors in the hypothalamus were first discovered in the VMH and LH [57, 58]. Moreover, interstitial glucose levels in the VMH and LH vary with blood glucose concentration [67], and these changes in glucose have been postulated to trigger meal initiation. Since glucose is the brain's primary fuel, it should respond to a severe glucose deficiency. In this way, VMH glucose sensors may play a role in detecting and countering severe glucose deficiency [68]. However, Levin et al. recently showed there was no correlation between VMH glucose levels and spontaneous feeding [69]. It is therefore unlikely that VMH glucose sensors regulate meal-to-meal food intake, al‐ though it does not rule out a role for glucose sensors in the LH or other brain regions.

The components responsible for glucose sensing in GE neurons seem to be shared with those present in pancreatic beta-cells: GLUT2, as well as glucokinase. Furthermore, GE uses the ATP-sensitive potassium (KATP) channel to sense glucose, as occurs in beta-cells. How‐ ever, while KATP channels are expressed in all GE neurons, only approximately half of

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Multiple subtypes of GE neurons may exist that use alternate glucose sensing strategies. Thus, Claret et al. have shown that transgenic mice lacking the α2 subunit of AMPK, an im‐ portant cellular fuel gauge, also lack GE neurons in ARC [79]. However, other authors [80] have shown that the acute pharmacological activation or inhibition of AMPK had no effect

GI neurons have similar components to GE and, therefore, to beta-cells in glucose sensing, such as glucokinase [78, 81], as well as GLUT2 and GLUT4. However, the signal transduc‐ tion pathway, whereby changes in intracellular ATP alter the activity of GI neurons, is com‐ pletely different. In this case, the activation of α2AMPK by glucose mediates the activation of VMH GI neurons, as described by Murphy et al. [82]. Hypothalamic α2AMPK is a key kinase involved in the energy balance and is a target for a number of hormones and a trans‐ mitter that regulates the energy balance [83-88]. The pharmacological activation of hypo‐ thalamic AMPK increases food intake [89]. It is not therefore surprising that decreased

**4. AMPK, together with mTOR and its downstream target S6K1, integrate**

AMPK is a nutrient and energy sensor. AMPK senses cellular energy availability by detect‐ ing the AMP/ATP ratio. AMPK is activated in low energy states and promotes ATP-generat‐

AMPK is a heterotrimeric complex that contains a catalytic α-subunit (α1 or α2) and two regulatory subunits, β (β1, β2) and γ (γ1, γ2, γ3). The α-subunit contains a kinase domain. The β-subunit contains the regions that permit interaction with other α and γ subunits and a carbohydrate-binding domain that facilitates binding to glycogen. The γ subunit contains four tandem repeats, which are four binding sites for adenosine derivates denominated as

Different isoforms and the alternative splicing of some mRNAs encoding these subunits give rise to a wide range of heterotrimeric combinations. The expression of the catalytic subunits (α1 and α2) is also different. The α2 subunit expression has been found in pan‐ creatic beta-cells, neurons, skeletal muscle and the heart. The liver has 50% of each AMPKα isoform (α1 and α2), while adipose tissue expresses higher levels of the

**nutrient and hormonal signals to maintain energy homeostasis**

ing catabolic pathways and inhibits anabolic reactions [90-92].

CBS motifs (cystathionine β-synthase) [92, 93].

AMPKα1 isoform [93].

VMH GE neurons express glucokinase, and approximately 30% express GLUT2 [78].

on glucose sensing in VMH GE neurons.

glucose activates AMPK in GI neurons.

**Figure 2.** Schematic representation of a hypothalamic slice. Localization of the VMH and LH are indicated. GE and GI neurons are activated or inactivated by a rise in glucose, respectively. The putative components responsible for glu‐ cose sensing in GE and GI are shown.

Glucose sensing neurons are those that alter their frequency of potential actions in response to changes in interstitial glucose levels [60, 70]. There are mainly two neurons whose activity is regulated by alterations in glucose levels [60]: Glucose-excited (GE) neurons that increase their potential frequency in response to increases in interstitial glucose from 0.1 to 2.5 mM glucose. The other kind of neurons (GI) are those that decrease their frequency of potential actions when glucose rises. More recently [71], other neurons have been described that re‐ spond to an increase of more than 5 mM glucose. Thus, high GE (HGE) and high GI (HGI) neurons increase or decrease their frequency of potential actions, respectively, in response to increases in interstitial glucose from 5 to 20 mM, although these neurons are still not thor‐ oughly characterized, and there are doubts about their physiological significance. However, it is important to consider that the interstitial brain glucose concentration is approximately 30% of the concentrations found in the blood. Thus, when the peripheral plasma glucose concentration is 7.6 mM, the interstitial VMH glucose is only 2.5 mM [67]. Decreasing plas‐ ma glucose to 2–3 mM or increasing to 15 mM resulted in brain glucose levels of 0.16 mM and 4.5 mM, respectively [67, 72]. Therefore, glucose concentrations found within the major‐ ity of the brain in vivo under physiological and pathophysiological conditions are within the 0.2 to 5 mM range [73-76], and it seems that the GE and GI neurons are mainly responsible for glucose sensing, since it is unclear whether brain glucose levels ever exceed 5 mM in the presence of an intact blood brain barrier. However, it should be noted that hyperglycemia impairs the integrity of the blood brain barrier [77], raising the question of whether HGE and HGI neurons could have a physiological significance in hyperglycemia-associated path‐ ology. Nevertheless, it seems that glucose sensing neurons could be functioning to protect the brain against a severe energy deficit.

The components responsible for glucose sensing in GE neurons seem to be shared with those present in pancreatic beta-cells: GLUT2, as well as glucokinase. Furthermore, GE uses the ATP-sensitive potassium (KATP) channel to sense glucose, as occurs in beta-cells. How‐ ever, while KATP channels are expressed in all GE neurons, only approximately half of VMH GE neurons express glucokinase, and approximately 30% express GLUT2 [78].

[69]. It is therefore unlikely that VMH glucose sensors regulate meal-to-meal food intake, al‐

**Figure 2.** Schematic representation of a hypothalamic slice. Localization of the VMH and LH are indicated. GE and GI neurons are activated or inactivated by a rise in glucose, respectively. The putative components responsible for glu‐

Glucose sensing neurons are those that alter their frequency of potential actions in response to changes in interstitial glucose levels [60, 70]. There are mainly two neurons whose activity is regulated by alterations in glucose levels [60]: Glucose-excited (GE) neurons that increase their potential frequency in response to increases in interstitial glucose from 0.1 to 2.5 mM glucose. The other kind of neurons (GI) are those that decrease their frequency of potential actions when glucose rises. More recently [71], other neurons have been described that re‐ spond to an increase of more than 5 mM glucose. Thus, high GE (HGE) and high GI (HGI) neurons increase or decrease their frequency of potential actions, respectively, in response to increases in interstitial glucose from 5 to 20 mM, although these neurons are still not thor‐ oughly characterized, and there are doubts about their physiological significance. However, it is important to consider that the interstitial brain glucose concentration is approximately 30% of the concentrations found in the blood. Thus, when the peripheral plasma glucose concentration is 7.6 mM, the interstitial VMH glucose is only 2.5 mM [67]. Decreasing plas‐ ma glucose to 2–3 mM or increasing to 15 mM resulted in brain glucose levels of 0.16 mM and 4.5 mM, respectively [67, 72]. Therefore, glucose concentrations found within the major‐ ity of the brain in vivo under physiological and pathophysiological conditions are within the 0.2 to 5 mM range [73-76], and it seems that the GE and GI neurons are mainly responsible for glucose sensing, since it is unclear whether brain glucose levels ever exceed 5 mM in the presence of an intact blood brain barrier. However, it should be noted that hyperglycemia impairs the integrity of the blood brain barrier [77], raising the question of whether HGE and HGI neurons could have a physiological significance in hyperglycemia-associated path‐ ology. Nevertheless, it seems that glucose sensing neurons could be functioning to protect

cose sensing in GE and GI are shown.

172 Hot Topics in Endocrine and Endocrine-Related Diseases

the brain against a severe energy deficit.

though it does not rule out a role for glucose sensors in the LH or other brain regions.

Multiple subtypes of GE neurons may exist that use alternate glucose sensing strategies. Thus, Claret et al. have shown that transgenic mice lacking the α2 subunit of AMPK, an im‐ portant cellular fuel gauge, also lack GE neurons in ARC [79]. However, other authors [80] have shown that the acute pharmacological activation or inhibition of AMPK had no effect on glucose sensing in VMH GE neurons.

GI neurons have similar components to GE and, therefore, to beta-cells in glucose sensing, such as glucokinase [78, 81], as well as GLUT2 and GLUT4. However, the signal transduc‐ tion pathway, whereby changes in intracellular ATP alter the activity of GI neurons, is com‐ pletely different. In this case, the activation of α2AMPK by glucose mediates the activation of VMH GI neurons, as described by Murphy et al. [82]. Hypothalamic α2AMPK is a key kinase involved in the energy balance and is a target for a number of hormones and a trans‐ mitter that regulates the energy balance [83-88]. The pharmacological activation of hypo‐ thalamic AMPK increases food intake [89]. It is not therefore surprising that decreased glucose activates AMPK in GI neurons.

## **4. AMPK, together with mTOR and its downstream target S6K1, integrate nutrient and hormonal signals to maintain energy homeostasis**

AMPK is a nutrient and energy sensor. AMPK senses cellular energy availability by detect‐ ing the AMP/ATP ratio. AMPK is activated in low energy states and promotes ATP-generat‐ ing catabolic pathways and inhibits anabolic reactions [90-92].

AMPK is a heterotrimeric complex that contains a catalytic α-subunit (α1 or α2) and two regulatory subunits, β (β1, β2) and γ (γ1, γ2, γ3). The α-subunit contains a kinase domain. The β-subunit contains the regions that permit interaction with other α and γ subunits and a carbohydrate-binding domain that facilitates binding to glycogen. The γ subunit contains four tandem repeats, which are four binding sites for adenosine derivates denominated as CBS motifs (cystathionine β-synthase) [92, 93].

Different isoforms and the alternative splicing of some mRNAs encoding these subunits give rise to a wide range of heterotrimeric combinations. The expression of the catalytic subunits (α1 and α2) is also different. The α2 subunit expression has been found in pan‐ creatic beta-cells, neurons, skeletal muscle and the heart. The liver has 50% of each AMPKα isoform (α1 and α2), while adipose tissue expresses higher levels of the AMPKα1 isoform [93].

cells [96], and ATM (ataxia telangiectasia mutated) may also regulate the phosphorylation of Thr172 [97]. The level of Thr172 phosphorylation depends also on the activity of protein phosphatases [98, 99]. The effect of an increase in AMP inhibits phosphatase activity, and considering that LKB1 is constitutively active [100], the response after a rise in AMP increas‐

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AMPK can detect changes in cellular energy state that occur in response to nutrient varia‐ tions. Any cellular or metabolic stress that reduces ATP production (e.g., heat shock, hypo‐ xia, ischemia, glucose deprivation) or accelerates ATP consumption (e.g., contraction of skeletal muscle) will increase the ADP/ATP ratio, which will be amplified by the action of adenylate kinase, resulting in increased AMP/ATP with the consequent activation of AMPK (Figure 3). Once activated, AMPK first directly affects the activity of key enzymes of glucose metabolism and fatty acids, and second, proceeds to the long-term regulation of the tran‐ scriptional control of the main elements involved in these metabolic pathways. The net re‐ sult of the activation of AMPK will restore the energy balance, inhibiting the anabolic pathways responsible for the synthesis of macromolecules, such as proteins and glycogen, and also of the following lipids: fatty acids, triglycerides and cholesterol, while activating the catabolic pathways, such as the oxidation of fatty acids, glucose uptake and glycolysis

The mTOR is a serine/threonine kinase that responds to nutrients and hormonal signals [102-104]. mTOR forms two distinct complexes with different sensitivities to rapamycin: mTORC1 and mTORC2. Both complexes contain mTOR, GβL (G-protein β-protein subu‐ nit-like), mLST8 (mammalian lethal with SEC13 protein) and deptor (DEP domain con‐ taining mTOR interacting protein) [103, 105, 106]. This complex, along with raptor (rapamycin-sensitive adaptor protein of mTOR) and PRAS40 (proline-rich Akt substrate of 40 kDa) forms mTORC1, which is rapamycin and nutrient sensitive. However, mTORC2 comprises mTOR, GβL/mLST8 and deptor together with rictor (rapamycin-in‐ sensitive companion of mTOR), mSin 1 (mammalian stress-activated MAPK-interacting protein 1) and protor 1/2 (protein observed with rictor ½), which is insensitive to acute

mTORC1 regulates metabolism and cell growth in response to several environmental sig‐ nals. The presence of amino acids, growth factors and mitogens stimulates mTORC1, which promotes anabolic processes. The mTORC1 activity phosphorylates multiple substrates, with S6K1 and the initiation factor 4E binding proteins (4E-BPs) being the best characterized [106-108]. The activation of mTORC1 induces the dissociation of 4E-BP from the eukaryotic translation initiation factor 4E (eIF4E), facilitating mRNA translation [109], and the activa‐ tion of S6K1 promotes protein synthesis. Moreover, the mTORC2 function is less well known. It is known that mTORC2 phosphorylates Akt and appears to regulate mainly cell

The effect growth factors have on mTORC1 is mediated through phosphatidylinositol-3,4,5 triphosphate kinase (PI3K) activation, the subsequent activation of phosphoinositide-de‐ pendent kinase (PDK1) and Akt. Once activated, Akt phosphorylates tuberous sclerosis complex 2 (TSC2), suppressing the inhibitory effect of the TSC1-TSC2 complex in mTORC1.

es the phosphorylation of Thr172 and the activation of AMPK.

[101] (Figure 3).

rapamycin (Figure 4)

proliferation and cell survival [110] (Figure 4)

**Figure 3.** Schematic representation of AMPK's subunits and its activation process. AMPK detects changes in the cellu‐ lar energy state that occur in response to nutrient variations or metabolic stress caused by changes in the AMP/ATP ratio. The activation of AMPK triggers key enzymes of glucose metabolism and fatty acids. The long-term effect of AMPK activation is the transcriptional control of the main elements involved in these metabolic pathways.

AMPK serine/threonine kinase activity is stimulated by the phosphorylation of the α-subu‐ nit on the Thr residue (Thr172). This activation process is regulated by several upstream kin‐ ases. The two main kinases in mammals are liver kinase B1 (LKB1), identified as a tumoursuppressor, and the Ca2+/calmodulin-dependent protein kinase (mainly CaMKKβ) [92, 93]. AMPK activity is also allosterically regulated by AMP binding to the γ subunit (Figure 3). Recent studies have found that AMP or ADP binding to the γ regulatory subunit protect the activated phosphorylated form of AMPK [94, 95]. AMP, ADP and ATP bind the γ subunit with similar affinity [94]. AMPK can be activated by increases in AMP and ADP according to changes in the cellular levels of adenosine derivatives. LKB1 phosphorylates AMPK in al‐ most all tissues, while CaMKKβ plays an important role in neurons and T lymphocytes. Other studies have suggested that a member of the MAPKKK family, TAK1 (transforming growth factor β-activated kinase) could be an important AMPK upstream kinase in cardiac cells [96], and ATM (ataxia telangiectasia mutated) may also regulate the phosphorylation of Thr172 [97]. The level of Thr172 phosphorylation depends also on the activity of protein phosphatases [98, 99]. The effect of an increase in AMP inhibits phosphatase activity, and considering that LKB1 is constitutively active [100], the response after a rise in AMP increas‐ es the phosphorylation of Thr172 and the activation of AMPK.

AMPK can detect changes in cellular energy state that occur in response to nutrient varia‐ tions. Any cellular or metabolic stress that reduces ATP production (e.g., heat shock, hypo‐ xia, ischemia, glucose deprivation) or accelerates ATP consumption (e.g., contraction of skeletal muscle) will increase the ADP/ATP ratio, which will be amplified by the action of adenylate kinase, resulting in increased AMP/ATP with the consequent activation of AMPK (Figure 3). Once activated, AMPK first directly affects the activity of key enzymes of glucose metabolism and fatty acids, and second, proceeds to the long-term regulation of the tran‐ scriptional control of the main elements involved in these metabolic pathways. The net re‐ sult of the activation of AMPK will restore the energy balance, inhibiting the anabolic pathways responsible for the synthesis of macromolecules, such as proteins and glycogen, and also of the following lipids: fatty acids, triglycerides and cholesterol, while activating the catabolic pathways, such as the oxidation of fatty acids, glucose uptake and glycolysis [101] (Figure 3).

The mTOR is a serine/threonine kinase that responds to nutrients and hormonal signals [102-104]. mTOR forms two distinct complexes with different sensitivities to rapamycin: mTORC1 and mTORC2. Both complexes contain mTOR, GβL (G-protein β-protein subu‐ nit-like), mLST8 (mammalian lethal with SEC13 protein) and deptor (DEP domain con‐ taining mTOR interacting protein) [103, 105, 106]. This complex, along with raptor (rapamycin-sensitive adaptor protein of mTOR) and PRAS40 (proline-rich Akt substrate of 40 kDa) forms mTORC1, which is rapamycin and nutrient sensitive. However, mTORC2 comprises mTOR, GβL/mLST8 and deptor together with rictor (rapamycin-in‐ sensitive companion of mTOR), mSin 1 (mammalian stress-activated MAPK-interacting protein 1) and protor 1/2 (protein observed with rictor ½), which is insensitive to acute rapamycin (Figure 4)

**Figure 3.** Schematic representation of AMPK's subunits and its activation process. AMPK detects changes in the cellu‐ lar energy state that occur in response to nutrient variations or metabolic stress caused by changes in the AMP/ATP ratio. The activation of AMPK triggers key enzymes of glucose metabolism and fatty acids. The long-term effect of

AMPK serine/threonine kinase activity is stimulated by the phosphorylation of the α-subu‐ nit on the Thr residue (Thr172). This activation process is regulated by several upstream kin‐ ases. The two main kinases in mammals are liver kinase B1 (LKB1), identified as a tumoursuppressor, and the Ca2+/calmodulin-dependent protein kinase (mainly CaMKKβ) [92, 93]. AMPK activity is also allosterically regulated by AMP binding to the γ subunit (Figure 3). Recent studies have found that AMP or ADP binding to the γ regulatory subunit protect the activated phosphorylated form of AMPK [94, 95]. AMP, ADP and ATP bind the γ subunit with similar affinity [94]. AMPK can be activated by increases in AMP and ADP according to changes in the cellular levels of adenosine derivatives. LKB1 phosphorylates AMPK in al‐ most all tissues, while CaMKKβ plays an important role in neurons and T lymphocytes. Other studies have suggested that a member of the MAPKKK family, TAK1 (transforming growth factor β-activated kinase) could be an important AMPK upstream kinase in cardiac

AMPK activation is the transcriptional control of the main elements involved in these metabolic pathways.

174 Hot Topics in Endocrine and Endocrine-Related Diseases

mTORC1 regulates metabolism and cell growth in response to several environmental sig‐ nals. The presence of amino acids, growth factors and mitogens stimulates mTORC1, which promotes anabolic processes. The mTORC1 activity phosphorylates multiple substrates, with S6K1 and the initiation factor 4E binding proteins (4E-BPs) being the best characterized [106-108]. The activation of mTORC1 induces the dissociation of 4E-BP from the eukaryotic translation initiation factor 4E (eIF4E), facilitating mRNA translation [109], and the activa‐ tion of S6K1 promotes protein synthesis. Moreover, the mTORC2 function is less well known. It is known that mTORC2 phosphorylates Akt and appears to regulate mainly cell proliferation and cell survival [110] (Figure 4)

The effect growth factors have on mTORC1 is mediated through phosphatidylinositol-3,4,5 triphosphate kinase (PI3K) activation, the subsequent activation of phosphoinositide-de‐ pendent kinase (PDK1) and Akt. Once activated, Akt phosphorylates tuberous sclerosis complex 2 (TSC2), suppressing the inhibitory effect of the TSC1-TSC2 complex in mTORC1. TSC2 functions as a GTPase-activating protein of Ras homolog enriched in brain (Rheb), which is an mTORC1 activator. Mitogens activating the Ras/MAPK cascade also activate mTORC1. ERK phosphorylates TSC2, inhibiting the TSC1/TSC2 complex and inducing mTORC1 activity [111]. Raptor is additionally phosphorylated by ERK [112] (Figure 4)

mTORC1 activity is inhibited in conditions of energy depletion coordinated with AMPK ac‐ tivity. An increase in AMP/ATP ratio activates AMPK, which phosphorylates the TSC2, and this modification induces the concomitant inhibition of mTORC1 mediated by the TSC1- TSC2 complex [114]. Furthermore, AMPK phosphorylates raptor in mTORC1, which down-

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In addition, energy depletion inhibits mTORC1 by a mechanism that is independent of AMPK activation. This effect is mediated by eliminating the GTP loading of Rheb [116].

Studies in recent years have established a direct relationship between metabolic sensor ac‐ tivity in the hypothalamus and the regulation of food intake, body weight and energy ho‐

AMPK is broadly expressed throughout the brain. The α2 catalytic subunit is present with a high distribution in neurons and activated astrocytes [117]. Hypothalamic AMPK has been assumed to play a role in the central regulation of food intake and energy balance, whereby fasting increases and re-feeding decreases AMPK activity in various hypothalamic nuclei. Alterations of hypothalamic AMPK activity specifically affected α2AMPK and did not

The hypothalamic AMPK role has been studied in vivo by the expression of AMPK mutants: dominant negative AMPK (DN-AMPK) or constitutively active AMPK (CA-AMPK). The ex‐ pression of CA-AMPK in the medial hypothalamus by adenoviruses increased food intake and body weight, whereas the expression of DN-AMPK inhibited them [88]. These altera‐ tions change the hypothalamic neuropeptide expression: the expression of CA-AMPK en‐ hances the effect of fasting, increasing the expression of NPY and AgRP in the ARC and the melanin-concentrating hormone in the lateral hypothalamus, whereas the hypothalamic ex‐ pression of DN-AMPK decreases the expression of orexigenic neuropeptides NPY and

mTORC1 is another metabolic sensor that plays an important role in the regulation of feed‐ ing behaviour and body weight in the hypothalamus [119, 120]. mTOR and its downstream target S6K1 are widely distributed in the rat brain. The activated forms of mTOR and S6K1 are localized mainly in the paraventricular and arcuate nuclei [119], being co-localized in a high percentage of orexigenic neurons that express AgRP/NPY and also in around half the

mTORC1 activation in the rat hypothalamus decreases food intake and body weight. Similar results were found by introducing constitutively active S6K1 mediated by adenovirus into the mediobasal hypothalamus of the rat brain. By contrast, the injection of dominant-nega‐

Food intake leads to periods of fasting and feeding that are associated with substantial changes in the level of available nutrients (e.g., glucose and amino acids) and accompanied

anorexigenic neurons that express POMC/CART in the arcuate nuclei [119].

tive S6K1 leads to an increase in food intake and body weight [121].

**4.1. Expression and regulation of these sensors in the VMH and LH**

regulates this complex [115].

meostasis.

change α1AMPK [88].

AgRP in ARC [88, 118].

*4.1.1. Nutrient regulation*

**Figure 4.** Network of proteins involved in the AMPK/mTOR/S6K1 signalling pathway

mTORC1 is also stimulated by amino acids, especially leucine. This activation pathway is independent of PI3K. The amino-acid regulation of mTOR needs rag GTPases and Rheb. The detailed mechanism of activation is unknown. It has been suggested that rag GTPas‐ es may control the localization of mTOR to specific vesicular membranes containing Rheb-GTP [113].

mTORC1 activity is inhibited in conditions of energy depletion coordinated with AMPK ac‐ tivity. An increase in AMP/ATP ratio activates AMPK, which phosphorylates the TSC2, and this modification induces the concomitant inhibition of mTORC1 mediated by the TSC1- TSC2 complex [114]. Furthermore, AMPK phosphorylates raptor in mTORC1, which downregulates this complex [115].

In addition, energy depletion inhibits mTORC1 by a mechanism that is independent of AMPK activation. This effect is mediated by eliminating the GTP loading of Rheb [116].

### **4.1. Expression and regulation of these sensors in the VMH and LH**

Studies in recent years have established a direct relationship between metabolic sensor ac‐ tivity in the hypothalamus and the regulation of food intake, body weight and energy ho‐ meostasis.

AMPK is broadly expressed throughout the brain. The α2 catalytic subunit is present with a high distribution in neurons and activated astrocytes [117]. Hypothalamic AMPK has been assumed to play a role in the central regulation of food intake and energy balance, whereby fasting increases and re-feeding decreases AMPK activity in various hypothalamic nuclei. Alterations of hypothalamic AMPK activity specifically affected α2AMPK and did not change α1AMPK [88].

The hypothalamic AMPK role has been studied in vivo by the expression of AMPK mutants: dominant negative AMPK (DN-AMPK) or constitutively active AMPK (CA-AMPK). The ex‐ pression of CA-AMPK in the medial hypothalamus by adenoviruses increased food intake and body weight, whereas the expression of DN-AMPK inhibited them [88]. These altera‐ tions change the hypothalamic neuropeptide expression: the expression of CA-AMPK en‐ hances the effect of fasting, increasing the expression of NPY and AgRP in the ARC and the melanin-concentrating hormone in the lateral hypothalamus, whereas the hypothalamic ex‐ pression of DN-AMPK decreases the expression of orexigenic neuropeptides NPY and AgRP in ARC [88, 118].

mTORC1 is another metabolic sensor that plays an important role in the regulation of feed‐ ing behaviour and body weight in the hypothalamus [119, 120]. mTOR and its downstream target S6K1 are widely distributed in the rat brain. The activated forms of mTOR and S6K1 are localized mainly in the paraventricular and arcuate nuclei [119], being co-localized in a high percentage of orexigenic neurons that express AgRP/NPY and also in around half the anorexigenic neurons that express POMC/CART in the arcuate nuclei [119].

mTORC1 activation in the rat hypothalamus decreases food intake and body weight. Similar results were found by introducing constitutively active S6K1 mediated by adenovirus into the mediobasal hypothalamus of the rat brain. By contrast, the injection of dominant-nega‐ tive S6K1 leads to an increase in food intake and body weight [121].

#### *4.1.1. Nutrient regulation*

TSC2 functions as a GTPase-activating protein of Ras homolog enriched in brain (Rheb), which is an mTORC1 activator. Mitogens activating the Ras/MAPK cascade also activate mTORC1. ERK phosphorylates TSC2, inhibiting the TSC1/TSC2 complex and inducing mTORC1 activity [111]. Raptor is additionally phosphorylated by ERK [112] (Figure 4)

176 Hot Topics in Endocrine and Endocrine-Related Diseases

**Figure 4.** Network of proteins involved in the AMPK/mTOR/S6K1 signalling pathway

Rheb-GTP [113].

mTORC1 is also stimulated by amino acids, especially leucine. This activation pathway is independent of PI3K. The amino-acid regulation of mTOR needs rag GTPases and Rheb. The detailed mechanism of activation is unknown. It has been suggested that rag GTPas‐ es may control the localization of mTOR to specific vesicular membranes containing

Food intake leads to periods of fasting and feeding that are associated with substantial changes in the level of available nutrients (e.g., glucose and amino acids) and accompanied by hormonal changes. Several studies describe the effect of glucose on both metabolic sen‐ sors, AMPK and mTOR, in hypothalamic areas. During fasting, the decrease in glucose con‐ centration activates AMPK. This period is also characterized by low levels of glucose and amino acids, and the mTOR complex is kept inactive. The increase in glucose levels after food intake decreases the activity of AMPK and, conversely, the activity of the mTOR/S6K1 pathway is stimulated by higher levels of glucose and amino acids.

the intestinal peptide with orexigenic properties, activates AMPK and stimulates food intake [87, 89]. By contrast, anorexigenic peptides such as leptin decrease AMPK activity in the ARC and PVN [88, 89, 129]. However, leptin treatment increased mTOR and S6K1 hypo‐

Glucagon-Like Peptide-1 and Its Implications in Obesity

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179

It has recently been suggested that ghrelin activates AMPK in presynaptic neurons, induc‐ ing an increase in activity in NPY/AgRP neurons promoting sustained food intake, and this signal stops after leptin is released by adipose tissue, signalling to stimulate POMC neurons inhibiting feeding and also to inhibit the AMPK in the presynaptic neurons, inactivating the

Inoki et al. previously reported that the activation of AMPK induces the inhibition of mTOR activity [114]. It has recently been posited that S6K phosphorylates α2 AMPK. This process

These findings indicate that hypothalamic AMPK and mTOR respond to changes in glu‐ cose and other nutrients in opposite ways, and their effects on the regulation of food in‐

As indicated before, GLP-1 is able to induce several effects contributing to the control of feeding behaviour. It inhibited gastric acid secretion and emptying, stimulated postprandial insulin secretion and inhibited glucagon release. GLP-1 treatment to type 2 diabetic subjects normalized the fasting levels of blood glucose and decreased postprandial glucose levels. We have also reported that GLP-1 reduces glucose metabolism in the human hypothalamus

In general, the brain activity of AMPK is activated by fasting and is inhibited by re-feeding [88, 122, 123], but the effect of glucose on AMPK also regulates *Ampk* expression in VMH [134]. Thus, fasting increased *Ampk* mRNA expression in the hypothalamus of rats, and the

The glucose effect on AMPK might be region-specific in hypothalamic areas that have oppo‐ site effects over the control of feeding behaviour. We have also reported, using rat hypo‐ thalamic slices, that high glucose levels decrease the expression of *Ampk-α2* mRNA, specifically in the LH, but not in the VMH [83]. The decrease in *AMPKα2* expression in re‐ sponse to high glucose levels was reversed by the presence of GLP-1 [83]. Sanz et al. have also reported a different response to glucose in the VMH and LH [136, 137]. The distinctive response in the LH compared to the VMH may be explained by the different role these two

Results obtained from in vivo studies conducted on lean and obese Zucker rats showed that the effects fasting and re-feeding have on the activity of AMPK and S6K in the areas in‐

volved in the control of feeding are modulated by exendin-4 treatment [83].

is necessary for the leptin effects on hypothalamic AMPK activity [132].

**5. Modulation of AMPK and S6K by GLP-1/exendin-4 in these**

thalamic activity [130].

release of NPY/AgRP [131].

take may overlap.

**hypothalamic areas**

and brain stem [133].

ICV administration of GLP-1 reduced that effect [135].

areas have in the control of food intake.

Thus, Kim et al. reported that decreasing intracellular glucose through the supply of 2-deox‐ yglucose increases hypothalamic AMPK activity and food intake. By contrast, hyperglycae‐ mia decreases hypothalamic AMPK activity [122]. It was also observed that AMPK activity is inhibited in arcuate, ventromedial, dorsomedial, paraventricular nuclei and the LH by high glucose and re-feeding. [88]. Increases in α2-AMPK activities in arcuate-ventromedial and paraventricular nuclei are also detected during insulin-induced hypoglycaemic in rats [123]. However, fasted rats recorded a decrease in the number of hypothalamic cells express‐ ing mTOR and S6K1 activated forms specifically in the arcuate nucleus, with these changes responding to the availability of nutrients [119]. Similar findings were subsequently con‐ firmed, also showing that the constitutive activation of S6K in the mediobasal hypothalamic area protects against the harmful effects of a high-fat diet [121].

It has also been established that AMPK and mTOR are involved in the anorexigenic effect induced by high protein diets. Thus, a high protein diet and the intracerebroventricular ad‐ ministration of leucine decreased AMPK phosphorylation in the rat hypothalamus [120]. The activation of hypothalamic mTORC1 is additionally produced by a high protein diet and the intracerebroventricular administration of amino acids or leucine [119, 124].

#### *4.1.2. Gut hormone regulation and signals from energy stores*

The effects of glucose, amino acids and other nutrients are reinforced by the effects of intes‐ tinal peptides, as stated above. They are able to regulate food intake, energetic homeostasis and body weight. The gastrointestinal tract responds to gut contents by secreting hormones, which can serve to inform the CNS of nutrient status. Thus, circulating ghrelin, the only in‐ testinal peptide with orexigenic properties, is high in the period before a meal, and the level declines an hour after eating [125]. Ghrelin stimulates food intake in lean and obese humans [126]. In contrast, anorexigenic intestinal peptides as peptide YY (PYY), pancreatic polypep‐ tide (PP), GLP-1, oxyntomodulin and cholecystokinin are low during the fasting period, and their level increases after a meal, and some of them are released proportionally to the amount of calories ingested (Reviewed in [127]).

Other signals that inform the state of energy stores, such as leptin and insulin levels, are im‐ portant modulators of feeding behaviour. Insulin regulates the storage of nutrients and also informs the brain about the energy balance [128]. Leptin is produced by adipose tissue and informs the brain about the energy storage status [128].

We now know that the function of at least some of these peptides may be mediated by the modulation of hypothalamic metabolic sensors. Thus, it has been reported that hypothala‐ mic AMPK activity is also regulated by several orexigenic and anorexigenic signals. Ghrelin, the intestinal peptide with orexigenic properties, activates AMPK and stimulates food intake [87, 89]. By contrast, anorexigenic peptides such as leptin decrease AMPK activity in the ARC and PVN [88, 89, 129]. However, leptin treatment increased mTOR and S6K1 hypo‐ thalamic activity [130].

by hormonal changes. Several studies describe the effect of glucose on both metabolic sen‐ sors, AMPK and mTOR, in hypothalamic areas. During fasting, the decrease in glucose con‐ centration activates AMPK. This period is also characterized by low levels of glucose and amino acids, and the mTOR complex is kept inactive. The increase in glucose levels after food intake decreases the activity of AMPK and, conversely, the activity of the mTOR/S6K1

Thus, Kim et al. reported that decreasing intracellular glucose through the supply of 2-deox‐ yglucose increases hypothalamic AMPK activity and food intake. By contrast, hyperglycae‐ mia decreases hypothalamic AMPK activity [122]. It was also observed that AMPK activity is inhibited in arcuate, ventromedial, dorsomedial, paraventricular nuclei and the LH by high glucose and re-feeding. [88]. Increases in α2-AMPK activities in arcuate-ventromedial and paraventricular nuclei are also detected during insulin-induced hypoglycaemic in rats [123]. However, fasted rats recorded a decrease in the number of hypothalamic cells express‐ ing mTOR and S6K1 activated forms specifically in the arcuate nucleus, with these changes responding to the availability of nutrients [119]. Similar findings were subsequently con‐ firmed, also showing that the constitutive activation of S6K in the mediobasal hypothalamic

It has also been established that AMPK and mTOR are involved in the anorexigenic effect induced by high protein diets. Thus, a high protein diet and the intracerebroventricular ad‐ ministration of leucine decreased AMPK phosphorylation in the rat hypothalamus [120]. The activation of hypothalamic mTORC1 is additionally produced by a high protein diet

The effects of glucose, amino acids and other nutrients are reinforced by the effects of intes‐ tinal peptides, as stated above. They are able to regulate food intake, energetic homeostasis and body weight. The gastrointestinal tract responds to gut contents by secreting hormones, which can serve to inform the CNS of nutrient status. Thus, circulating ghrelin, the only in‐ testinal peptide with orexigenic properties, is high in the period before a meal, and the level declines an hour after eating [125]. Ghrelin stimulates food intake in lean and obese humans [126]. In contrast, anorexigenic intestinal peptides as peptide YY (PYY), pancreatic polypep‐ tide (PP), GLP-1, oxyntomodulin and cholecystokinin are low during the fasting period, and their level increases after a meal, and some of them are released proportionally to the

Other signals that inform the state of energy stores, such as leptin and insulin levels, are im‐ portant modulators of feeding behaviour. Insulin regulates the storage of nutrients and also informs the brain about the energy balance [128]. Leptin is produced by adipose tissue and

We now know that the function of at least some of these peptides may be mediated by the modulation of hypothalamic metabolic sensors. Thus, it has been reported that hypothala‐ mic AMPK activity is also regulated by several orexigenic and anorexigenic signals. Ghrelin,

and the intracerebroventricular administration of amino acids or leucine [119, 124].

pathway is stimulated by higher levels of glucose and amino acids.

178 Hot Topics in Endocrine and Endocrine-Related Diseases

area protects against the harmful effects of a high-fat diet [121].

*4.1.2. Gut hormone regulation and signals from energy stores*

amount of calories ingested (Reviewed in [127]).

informs the brain about the energy storage status [128].

It has recently been suggested that ghrelin activates AMPK in presynaptic neurons, induc‐ ing an increase in activity in NPY/AgRP neurons promoting sustained food intake, and this signal stops after leptin is released by adipose tissue, signalling to stimulate POMC neurons inhibiting feeding and also to inhibit the AMPK in the presynaptic neurons, inactivating the release of NPY/AgRP [131].

Inoki et al. previously reported that the activation of AMPK induces the inhibition of mTOR activity [114]. It has recently been posited that S6K phosphorylates α2 AMPK. This process is necessary for the leptin effects on hypothalamic AMPK activity [132].

These findings indicate that hypothalamic AMPK and mTOR respond to changes in glu‐ cose and other nutrients in opposite ways, and their effects on the regulation of food in‐ take may overlap.

## **5. Modulation of AMPK and S6K by GLP-1/exendin-4 in these hypothalamic areas**

As indicated before, GLP-1 is able to induce several effects contributing to the control of feeding behaviour. It inhibited gastric acid secretion and emptying, stimulated postprandial insulin secretion and inhibited glucagon release. GLP-1 treatment to type 2 diabetic subjects normalized the fasting levels of blood glucose and decreased postprandial glucose levels. We have also reported that GLP-1 reduces glucose metabolism in the human hypothalamus and brain stem [133].

In general, the brain activity of AMPK is activated by fasting and is inhibited by re-feeding [88, 122, 123], but the effect of glucose on AMPK also regulates *Ampk* expression in VMH [134]. Thus, fasting increased *Ampk* mRNA expression in the hypothalamus of rats, and the ICV administration of GLP-1 reduced that effect [135].

The glucose effect on AMPK might be region-specific in hypothalamic areas that have oppo‐ site effects over the control of feeding behaviour. We have also reported, using rat hypo‐ thalamic slices, that high glucose levels decrease the expression of *Ampk-α2* mRNA, specifically in the LH, but not in the VMH [83]. The decrease in *AMPKα2* expression in re‐ sponse to high glucose levels was reversed by the presence of GLP-1 [83]. Sanz et al. have also reported a different response to glucose in the VMH and LH [136, 137]. The distinctive response in the LH compared to the VMH may be explained by the different role these two areas have in the control of food intake.

Results obtained from in vivo studies conducted on lean and obese Zucker rats showed that the effects fasting and re-feeding have on the activity of AMPK and S6K in the areas in‐ volved in the control of feeding are modulated by exendin-4 treatment [83].

It has been previously reported that the anorexigenic effects produced by the intraperitoneal administration of exendin-4 led to a reduction in food intake and increased the period be‐ tween meals [138]. Additionally, the peripheral administration of exendin-4 and liraglutide regulates food intake by activating the GLP-1 receptors expressed on both vagal afferents and CNS [139]. Recent studies conducted within our group [83] have focused on clarifying the coordinated effects of fasting, re-feeding and exendin-4 administration on the activity of AMPK and S6K in the VMH and LH. The results of these studies show that fasting increases hypothalamic AMPK activity in both areas in lean Zucker rats. However, the subcutaneous administration of exendin-4 over the last hour reversed this effect, whereas exendin-4 acti‐ vated AMPK in animals re-fed for two hours when AMPK activity was markedly inhibited. The activation degree of AMPK after four hours of re-feeding differed in both areas. Thus, the activation level of AMPK in the VMH was similar to fasted rats. However, AMPK activi‐ ty in the LH was still low, and exendin-4 treatment decreased AMPK activity in the VMH, whereas no significant effect was detected in the LH (Figure 5).

Anorexic peptides also regulate the mTOR/S6K1 pathway in hypothalamic areas. Insulin and leptin increases the activated forms of S6K [119]. The administration of exendin-4 also regulates S6K activity and the effect is dependent on the activation status of S6K, as occur‐ red with AMPK. We thus found that S6K activation peaked in animals re-fed for four hours. However, the administration of exendin-4 strongly stimulated S6K activity in animals re-fed for two hours. In contrast, exendin-4 decreased S6K activity in the VMH of lean rats re-fed for four hours [83] (Figure 5).

The use of rat organotypic hypothalamic slices confirmed that AMPK activity at low glucose concentrations was stimulated, and S6K activity was maintained with minimal activation [83]. GLP-1 treatment reversed the effect of glucose on AMPK and did not modify S6K activ‐ ity in the VMH and LH. High levels of glucose stimulated S6K activity in both nuclei, and the presence of GLP-1 reversed such activation. Similar results were found using hypothala‐ mic GT1-7 and neuroblastoma N2A cell lines [83]. The metabolic sensors in these cells re‐ spond to glucose as described above, and GLP-1 treatment reversed the glucose effects [83].

**Figure 5.** Effects of exendin-4 administration in fasted or re-fed lean rats on the activity of AMPK and S6K. Lean Zucker rats were fasted or re-fed for two or four hours. In some cases, the GLP-1 analogue exendin-4 (100 nM) was adminis‐ trated. The activation states of AMPK and S6K were determined by quantifying phospho-specific forms in VMH and LH

Glucagon-Like Peptide-1 and Its Implications in Obesity

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**6. GLP-1/exendin-4 as a compensator of the disturbances in AMPK and**

In obesity, the elevated levels of nutrients and hormonal modifications alter the activity of hypothalamic metabolic sensors. Thus, diet-induced obesity reduced hypothalamic AMPK activity [145]. The GLP-1 receptor agonist exendin-4 is one of the agents used in the treat‐ ment of type 2 diabetes [146] and is a long-acting receptor agonist of GLP-1 that also produ‐

The obese Zucker (fa/fa) rat provides a well-established animal model of insulin resistance and genetic obesity and, in comparison with lean Zucker rats, manifests hyperinsulinemia and hyperlipidemia. We have previously noted that the peripheral long-term subcutaneous administration of exendin-4 decreased food intake and induced weight loss in both obese

areas.

**S6K activities occurring in obesity**

ces weight loss [147-149].

and lean control Zucker rats [15].

The effect of GLP-1 on AMPK activity was also reported in other brain areas. Thus, GLP-1R activation in hindbrain suppressed food intake, and that effect is accompanied by the sup‐ pression of AMPK activity [140].

The complexities of the regulation of hypothalamic AMPK activity have previously been de‐ scribed for some hormones. Thus, the cocaine-and amphetamine-regulated transcript (CART) has been reported to have an anorexic effect after intracerebroventricular adminis‐ tration [141], while CART injected directly into the paraventricular or arcuate nucleus of fasted rats increases food intake [142]. Likewise, differences in the effect of regulatory pepti‐ des on AMPK as a function of nutritional status have been previously described. Ghrelin or cannabinoids have ad libitum effects [143], whereas leptin [88] and adiponectin [144] only have an effect after variable periods of fasting or re-feeding.

#### Glucagon-Like Peptide-1 and Its Implications in Obesity http://dx.doi.org/10.5772/54221 181

It has been previously reported that the anorexigenic effects produced by the intraperitoneal administration of exendin-4 led to a reduction in food intake and increased the period be‐ tween meals [138]. Additionally, the peripheral administration of exendin-4 and liraglutide regulates food intake by activating the GLP-1 receptors expressed on both vagal afferents and CNS [139]. Recent studies conducted within our group [83] have focused on clarifying the coordinated effects of fasting, re-feeding and exendin-4 administration on the activity of AMPK and S6K in the VMH and LH. The results of these studies show that fasting increases hypothalamic AMPK activity in both areas in lean Zucker rats. However, the subcutaneous administration of exendin-4 over the last hour reversed this effect, whereas exendin-4 acti‐ vated AMPK in animals re-fed for two hours when AMPK activity was markedly inhibited. The activation degree of AMPK after four hours of re-feeding differed in both areas. Thus, the activation level of AMPK in the VMH was similar to fasted rats. However, AMPK activi‐ ty in the LH was still low, and exendin-4 treatment decreased AMPK activity in the VMH,

Anorexic peptides also regulate the mTOR/S6K1 pathway in hypothalamic areas. Insulin and leptin increases the activated forms of S6K [119]. The administration of exendin-4 also regulates S6K activity and the effect is dependent on the activation status of S6K, as occur‐ red with AMPK. We thus found that S6K activation peaked in animals re-fed for four hours. However, the administration of exendin-4 strongly stimulated S6K activity in animals re-fed for two hours. In contrast, exendin-4 decreased S6K activity in the VMH of lean rats re-fed

The use of rat organotypic hypothalamic slices confirmed that AMPK activity at low glucose concentrations was stimulated, and S6K activity was maintained with minimal activation [83]. GLP-1 treatment reversed the effect of glucose on AMPK and did not modify S6K activ‐ ity in the VMH and LH. High levels of glucose stimulated S6K activity in both nuclei, and the presence of GLP-1 reversed such activation. Similar results were found using hypothala‐ mic GT1-7 and neuroblastoma N2A cell lines [83]. The metabolic sensors in these cells re‐ spond to glucose as described above, and GLP-1 treatment reversed the glucose effects [83].

The effect of GLP-1 on AMPK activity was also reported in other brain areas. Thus, GLP-1R activation in hindbrain suppressed food intake, and that effect is accompanied by the sup‐

The complexities of the regulation of hypothalamic AMPK activity have previously been de‐ scribed for some hormones. Thus, the cocaine-and amphetamine-regulated transcript (CART) has been reported to have an anorexic effect after intracerebroventricular adminis‐ tration [141], while CART injected directly into the paraventricular or arcuate nucleus of fasted rats increases food intake [142]. Likewise, differences in the effect of regulatory pepti‐ des on AMPK as a function of nutritional status have been previously described. Ghrelin or cannabinoids have ad libitum effects [143], whereas leptin [88] and adiponectin [144] only

whereas no significant effect was detected in the LH (Figure 5).

for four hours [83] (Figure 5).

180 Hot Topics in Endocrine and Endocrine-Related Diseases

pression of AMPK activity [140].

have an effect after variable periods of fasting or re-feeding.

**Figure 5.** Effects of exendin-4 administration in fasted or re-fed lean rats on the activity of AMPK and S6K. Lean Zucker rats were fasted or re-fed for two or four hours. In some cases, the GLP-1 analogue exendin-4 (100 nM) was adminis‐ trated. The activation states of AMPK and S6K were determined by quantifying phospho-specific forms in VMH and LH areas.

## **6. GLP-1/exendin-4 as a compensator of the disturbances in AMPK and S6K activities occurring in obesity**

In obesity, the elevated levels of nutrients and hormonal modifications alter the activity of hypothalamic metabolic sensors. Thus, diet-induced obesity reduced hypothalamic AMPK activity [145]. The GLP-1 receptor agonist exendin-4 is one of the agents used in the treat‐ ment of type 2 diabetes [146] and is a long-acting receptor agonist of GLP-1 that also produ‐ ces weight loss [147-149].

The obese Zucker (fa/fa) rat provides a well-established animal model of insulin resistance and genetic obesity and, in comparison with lean Zucker rats, manifests hyperinsulinemia and hyperlipidemia. We have previously noted that the peripheral long-term subcutaneous administration of exendin-4 decreased food intake and induced weight loss in both obese and lean control Zucker rats [15].

Zucker rats have been used to analyze the exendin-4 effect on the activity of AMPK and S6K in the VMH and LH areas [83]. The results obtained showed that AMPK activity was lower in the obese than in the lean Zucker rats in both areas. Interestingly, the effect of exendin-4 administration on fasted obese Zucker rats was different compared to the lean rats. The ab‐ sence of exendin-4 effect in obese rats maintains AMPK activity at a level of activation simi‐ lar to the lean animals after the administration of exendin-4 [83] (Figure 5).

LH of lean and obese rats after four hours of re-feeding, whereas exendin-4 reduced S6K ac‐

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The prolonged activation of hypothalamic S6K inhibits insulin signalling and contributes to hepatic insulin resistance [150], suggesting that hypothalamic S6K activation would be in‐ volved in the pathogenesis of diet-induced hepatic insulin resistance. Our data indicate that S6K activity in the presence of exendin-4 could be decreased when this protein is maximally activated. This suggests that exendin-4 treatment in diabetic subjects could also improve

We have reported here some of the many actions of GLP-1, such as, its role as an incretin hormone and controlling food intake. Accordingly, we have reviewed the importance of hy‐ pothalamic areas in the control of food intake, such as, for example, the ventromedial and lateral hypothalamus. In parallel, the function of AMPK and the mTOR/S6K pathway has been studied in those areas. Likewise, we have explored the coordinated response of hypo‐ thalamic AMPK and S6K to alterations in nutritional status and energy storage. Our results have revealed both the activation of AMPK and S6K in the VMH and LH in response to changes in glucose concentration or nutritional state, and that GLP-1/exendin-4 acts by counteracting the activation/inactivation of these kinases and contributing to the balance of proper AMPK and S6K activation. It therefore seems that GLP-1/exendin-4 might be acting in the VMH and LH, interacting with the AMPK/S6K signalling pathways, and modulating the activation status of AMPK and S6K in response to nutrient fluctuations. Likewise, GLP-1/exendin-4 would contribute to the normalization of the altered levels of these kinases

Veronica Hurtado1,2,3, Isabel Roncero1,2,3, Enrique Blazquez1,2,3, Elvira Alvarez1,2,3 and

2 Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Spain

1 Department of Biochemistry and Molecular Biology. Faculty of Medicine. University Com‐

3 The Center for Biomedical Research in Diabetes and Associated Metabolic Disorders (CI‐

4 Department of Cellular Biology. Faculty of Medicine. University Complutense of Madrid,

tivity in the VMH of lean Zucker rats but not in their obese counterparts [83].

in pathophysiological states such as obesity, for example.

hepatic insulin resistance.

**7. Conclusions**

**Author details**

Carmen Sanz1,2,3,4

BERDEM), Spain

Spain

plutense of Madrid, Spain

These results suggest that GLP-1/exendin-4 might compensate for the alterations in AMPK, activity produced either by oscillations in glucose levels or by pathologies such as obesity or episodes of hyperinsulinemia (Figure 5, 6).

**Figure 6.** Effects of exendin-4 administration in fasted or re-fed obese rats on the activity of AMPK and S6K. Lean Zucker rats were fasted or re-fed for two or four hours. In some cases, the GLP-1 analogue exendin-4 (100 nM) was administrated. The activation states of AMPK and S6K were determined by quantifying phospho-specific forms in the VMH and LH areas.

Another difference in the level of activation of AMPK between obese and lean Zucker rats was observed after re-feeding for four hours. The AMPK activity in the LH was higher in obese compared to lean animals (Figure 5, 6).

After two hours of re-feeding, exendin-4 treatment increased S6K activity in the VMH and LH in obese rats. Nevertheless, the effect of exendin-4 on S6K activity in the VMH differed between obese and lean rats. Exendin-4 administration did not modify S6K activity in the LH of lean and obese rats after four hours of re-feeding, whereas exendin-4 reduced S6K ac‐ tivity in the VMH of lean Zucker rats but not in their obese counterparts [83].

The prolonged activation of hypothalamic S6K inhibits insulin signalling and contributes to hepatic insulin resistance [150], suggesting that hypothalamic S6K activation would be in‐ volved in the pathogenesis of diet-induced hepatic insulin resistance. Our data indicate that S6K activity in the presence of exendin-4 could be decreased when this protein is maximally activated. This suggests that exendin-4 treatment in diabetic subjects could also improve hepatic insulin resistance.

## **7. Conclusions**

Zucker rats have been used to analyze the exendin-4 effect on the activity of AMPK and S6K in the VMH and LH areas [83]. The results obtained showed that AMPK activity was lower in the obese than in the lean Zucker rats in both areas. Interestingly, the effect of exendin-4 administration on fasted obese Zucker rats was different compared to the lean rats. The ab‐ sence of exendin-4 effect in obese rats maintains AMPK activity at a level of activation simi‐

These results suggest that GLP-1/exendin-4 might compensate for the alterations in AMPK, activity produced either by oscillations in glucose levels or by pathologies such as obesity or

**Figure 6.** Effects of exendin-4 administration in fasted or re-fed obese rats on the activity of AMPK and S6K. Lean Zucker rats were fasted or re-fed for two or four hours. In some cases, the GLP-1 analogue exendin-4 (100 nM) was administrated. The activation states of AMPK and S6K were determined by quantifying phospho-specific forms in the

Another difference in the level of activation of AMPK between obese and lean Zucker rats was observed after re-feeding for four hours. The AMPK activity in the LH was higher in

After two hours of re-feeding, exendin-4 treatment increased S6K activity in the VMH and LH in obese rats. Nevertheless, the effect of exendin-4 on S6K activity in the VMH differed between obese and lean rats. Exendin-4 administration did not modify S6K activity in the

lar to the lean animals after the administration of exendin-4 [83] (Figure 5).

episodes of hyperinsulinemia (Figure 5, 6).

182 Hot Topics in Endocrine and Endocrine-Related Diseases

VMH and LH areas.

obese compared to lean animals (Figure 5, 6).

We have reported here some of the many actions of GLP-1, such as, its role as an incretin hormone and controlling food intake. Accordingly, we have reviewed the importance of hy‐ pothalamic areas in the control of food intake, such as, for example, the ventromedial and lateral hypothalamus. In parallel, the function of AMPK and the mTOR/S6K pathway has been studied in those areas. Likewise, we have explored the coordinated response of hypo‐ thalamic AMPK and S6K to alterations in nutritional status and energy storage. Our results have revealed both the activation of AMPK and S6K in the VMH and LH in response to changes in glucose concentration or nutritional state, and that GLP-1/exendin-4 acts by counteracting the activation/inactivation of these kinases and contributing to the balance of proper AMPK and S6K activation. It therefore seems that GLP-1/exendin-4 might be acting in the VMH and LH, interacting with the AMPK/S6K signalling pathways, and modulating the activation status of AMPK and S6K in response to nutrient fluctuations. Likewise, GLP-1/exendin-4 would contribute to the normalization of the altered levels of these kinases in pathophysiological states such as obesity, for example.

## **Author details**

Veronica Hurtado1,2,3, Isabel Roncero1,2,3, Enrique Blazquez1,2,3, Elvira Alvarez1,2,3 and Carmen Sanz1,2,3,4

1 Department of Biochemistry and Molecular Biology. Faculty of Medicine. University Com‐ plutense of Madrid, Spain

2 Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Spain

3 The Center for Biomedical Research in Diabetes and Associated Metabolic Disorders (CI‐ BERDEM), Spain

4 Department of Cellular Biology. Faculty of Medicine. University Complutense of Madrid, Spain

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**Chapter 8**

**The Insulin-Like Growth**

Emrah Yerlikaya and Fulya Akin

http://dx.doi.org/10.5772/55213

**1. Introduction**

**1.1. Physiology**

Additional information is available at the end of the chapter

**Factor System in the Human Pathology**

Insulin-like growth factors are single chain polypeptides. There are two principle IGFs referred to as IGF-I and IGF-II. IGF-1 is a polypeptide hormone with a molecular weight of 7.6-kDa structurally similar to insulin. In 1957, it is identified by Salmon and Daughaday. Because of the its ability to stimulate the sulfation of the cartilage proteoglycans, it was regarded as a sulphation factor [1]. The IGF-1 gene is on the long arm of chromosome 12q23– 23. IGF-1 gene contains 6 exons [2, 3]. The alternate extension peptide at carboxy terminal, encoded by exons 5 and 6 determines the subforms of IGF-1: IGF-1B and IGF-1A. The most abundant isoform of the IGF-1 (153 aminoacid) is IGF-1A [4, 5]. IGF1B peptide (195 amino acids) is a less abundant IGF1 isoform. IGF-2 is also a peptide with 67 amino acids and molecular weight of 7.4-kDa. IGF-2 is encoded by a gene on the short arm of chromosome 11 at position 15.5. This gene consists of nine exons [6]. In the plasma, 99% of IGFs are bound to a family of binding cysteine-rich proteins. There are six binding proteins (IGFBP-1 to IGFBP-6) [7]. They act as carriers for IGFs in the circulation, regulate the bioavailability of IGFs to spesific tissues and modulates the biological activities of IGF proteins. Six IGFbinding proteins (IGFBPs) can inhibit or enhance the actions of IGFs [8]. Potentiation of IGF activity by some of the IGFBPs, described for IGFBP-1 and IGFBP-3, is also documented for IGFBP-5. Each of IGFBPs is the product of a seperate gene. These genes share a common structural organization in which four conserved exons are located within genes ranging from 5 kb (IGFBP-1) to more than 30 kb (IGFBP-2 and IGFBP-5) [9]. IGFBPs contain N terminal and C terminal domains which are similar in aminoacid sequence. Post-translational modifications of IGFBP, including glycosylation, phosphorylation and proteolysis modify the affinities of the binding proteins to IGF. IGFs mediate their action on target cells by three

> © 2013 Yerlikaya and Akin; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

## **Chapter 8**
