**4. Pathogenesis of type 1 diabetes mellitus**

#### **4.1 Genetic component**

Genetics has an important role in the etiology of type 1 diabetes. However, extra-genetic components influence the penetrance of diabetes susceptibility genes. If data are obtained at a single point in time, the risk of type 1 diabetes mellitus between monozygotic twins can be as low as 30%, but if the monozygotic twins are followed long-term, the cumulative incidence of diabetes reaches 65% (Redondo et al., 2008). In the same cohort of monozygotic twins, the rate of persistent autoantibody positivity, type 1 diabetes mellitus, or both, reached 78% (Redondo et al., 2008).

To better understand the genetic susceptibility to diabetes, candidate gene studies were conducted in order to identify genes that are associated with autoimmune type 1 diabetes.

Human leukocyte antigen (HLA) associations have been long recognized in many autoimmune diseases. In type 1 diabetes mellitus, the HLA on chromosome 6p21 is well described and is considered to play an important role in more than 50% of the familial cases in Caucasians (Noble et al., 1996). HLA DR4-DQ8 or DR3-DQ2 haplotypes are detected in up to 90% of patients with type 1 diabetes mellitus (Devendra & Eisenbarth, 2003). The combination

Americans of all ages and 11.3% of adults aged 20 years and older, according to the National Diabetes Fact Sheet for 2011. About 27% of those with diabetes (approximately 7 million Americans) do not know they have the disease. 1 in every 400 children and adolescents has

Type 1 diabetes mellitus continues to be highly prevalent in many countries, with an overall annual increase estimated at 3% (International Diabetes Federation [IDF] 2010). Worldwide,

The 4th edition of the IDF Diabetes Atlas, released in 2009 at the 20th World Diabetes Congress, estimated that in 2010, 285 million people would have diabetes (6.4% of world's adult population). The same forum predicts that by 2030, 438 million people will have diabetes world-wide. Type 1 diabetes in children is estimated at 480,000 patients worldwide

The natural history of type 1 diabetes is characterized by an autoimmune destruction of the beta cells in the islands of Langerhans in the pancreas. The autoimmune process has cellular and humoral components, leading to the destruction of the beta cells and a decreased insulin secretion. As beta-cell mass declines, insulin secretion decreases until the available insulin no longer is adequate to maintain normal blood glucose levels. After 70-90% of the

The natural history of type 1 diabetes has 4 stages: genetic susceptibility, autoimmune

The rate of beta cell destruction is variable. In some patients years will go by before the onset of diabetes, while other patients may never develop beta cell insufficiency, perhaps due to the regaining of tolerance. Most patients with type 1 diabetes mellitus have one or more susceptible human leukocyte antigen (HLA) class II, and over 90% have beta cell autoantibodies present. The appearance of circulating islet cell autoantibodies is the first

Genetics has an important role in the etiology of type 1 diabetes. However, extra-genetic components influence the penetrance of diabetes susceptibility genes. If data are obtained at a single point in time, the risk of type 1 diabetes mellitus between monozygotic twins can be as low as 30%, but if the monozygotic twins are followed long-term, the cumulative incidence of diabetes reaches 65% (Redondo et al., 2008). In the same cohort of monozygotic twins, the rate of persistent autoantibody positivity, type 1 diabetes mellitus, or both,

To better understand the genetic susceptibility to diabetes, candidate gene studies were conducted in order to identify genes that are associated with autoimmune type 1 diabetes. Human leukocyte antigen (HLA) associations have been long recognized in many autoimmune diseases. In type 1 diabetes mellitus, the HLA on chromosome 6p21 is well described and is considered to play an important role in more than 50% of the familial cases in Caucasians (Noble et al., 1996). HLA DR4-DQ8 or DR3-DQ2 haplotypes are detected in up to 90% of patients with type 1 diabetes mellitus (Devendra & Eisenbarth, 2003). The combination

in 2010, and the number of newly diagnosed cases per year is 75,800 (IDF 2010).

beta cells are destroyed, hyperglycemia develops and diabetes may be diagnosed.

it is more common in males than in females, with a ratio of 1.5.

type 1 diabetes.

**3.1 Natural history** 

process, pre-diabetes and diabetes.

detectable sign of this immune process.

reached 78% (Redondo et al., 2008).

**4.1 Genetic component** 

**4. Pathogenesis of type 1 diabetes mellitus** 

of these 2 types, DR4-DQ8/DR3-DQ2, carries the highest risk and type 1 diabetes mellitus occurs at a very early age in this population. First-degree relatives of the patients who carry the highest risk haplotype combination also have a higher risk of developing diabetes mellitus as compared to the relatives of diabetes patients who do not have this haplotype and who develop type 1 diabetes mellitus later in life (Gillespie et al., 2002).

Another HLA haplotype (DR15-DQ6) might have protective properties, and is found in a much larger percentage in the general population (20%) as compared to less than 1% in patients with type 1 diabetes mellitus (Eisenbarth & Gottlieb, 2004).

HLA haplotypes appear to have an association with islet autoantibodies. Glutamic acid decarboxylase (GAD) antibodies are more frequent in patients with HLA DR3-DQ2, whereas insulin auto-antibodies (IAA) and protein tyrosine phosphatase-like protein antibodies (IA-2 antibodies) are more frequent in patients with HLA DR4-DQ8. Patients that do not have these haplotypes are less likely to develop islet autoantibodies (Achenbach et al., 2005).

Another key genetic factor is the insulin gene (INS), with different forms of the promoter region conferring either protection or increased susceptibility to autoimmune diabetes mellitus (Bennett et al., 1995). The insulin gene contributes 10% to the genetic susceptibility in developing autoimmune diabetes (Bell et al., 1984). The risk of developing diabetes depends on the expression of the insulin protein in the thymus which can cause a defective central tolerance to the insulin molecule. The degree of immune tolerance may be reflected by the less common presence of insulin autoantibodies (IAA) in patients or relatives who have the protective INS class I/III or III/III genotypes (Vafiadis et al., 1997).

Fig. 1. Antigen Presenting Cell. The activation of the T-cell by various stimuli (antigens), is brought by major histocompatibility complex (MHC-HLA II). This figure shows also, inhibitors of T-cell activation: cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphoid tyrosine phosphatase (LYP).

Role of Vitamin D in the Pathogenesis and Therapy of Type 1 Diabetes Mellitus 407

The Th2 cells are protective for the beta cells. They have an inhibitory effect on the Th1 cells, which are destructive to the pancreatic beta cells. In the NOD mouse, it appears that the immunologic self-tolerance to pancreatic beta cells is lost. The disruption of the equilibrium between Th1 and Th2 cells in the thymus and in the periphery is believed to play a crucial role in the pathogenesis of autoimmune diabetes mellitus (Delovitch & Singh, 1997). Once Th1 cells are produced they will secrete interferon (IFN ) and IL-2, leading to the activation of macrophages and cytotoxic T cells, which are destructive to the pancreatic beta cells (Adorini, 2001). The same Th1 cells will stimulate the IgG2a autoantibodies against the islet beta cells autoantigens (Delovitch & Singh, 1997). Autoimmune diabetes can be transferred from a diabetic NOD mouse to an unaffected mouse via T cells (Bendelac et al., 1987). NOD mice develop a spontaneous loss of T-cell tolerance to glutamic acid decarboxylase antibodies (GAD), leading to autoimmune diabetes (Kaufman et al., 1993). In NOD mice, there is an increased resistance to apoptosis in immunocytes (Leijon et al., 1995,

Immune responses to several beta-cell proteins have been described (auto-antigens). Exposure to glutamic acid decarboxylase (GAD65 and GAD67) led to an increased T cell proliferation as early as 4 weeks of life in NOD mice, coinciding with the onset of insulitis (Tisch 1993). Some of the other beta-cell antigens elicited an increased immune response after a few more weeks, but there were other beta-cell antigens that did not trigger an immune reaction (for example, amylin) (Tisch 1993). The same study showed that intrathymic injections of GAD65 had a protective effect from autoimmune diabetes in NOD

GAD65- reactive T cells were proven to have the ability to transfer diabetes to NOD/SCID (severe combined immunodeficiency) mice (Zekzer et al., 1998). To further support the central role of GAD antigen in autoimmune diabetes, the beta-cell-specific suppression of GAD expression in antisense GAD transgenic NOD mice was demonstrated to prevent the

In humans, the pancreas becomes infiltrated with mononuclear cells. Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GAD) and insulinoma associated-2 antibody (IA-2) are demonstrated years before the clinical symptoms of diabetes. (Kulmala et al., 1998) T cell responses to several islet cells antigens (insulin, GAD, IA-2) have been reported in IDDM (MacCuish et al., 1975). The presence of autoantibodies alone does not explain the development of diabetes, since it is recognized now that children born to type 1 diabetic mother with high antibody titers transferred through the umbilical cord do not develop diabetes more often than expected. An interesting case was published by Martin et al in 2001, describing a case of type 1 diabetes mellitus occurring in a patient that had a

The environment is implicated in the pathogenesis of type 1 diabetes mellitus by many

Environmental factors have an important role in initiating an immune process that ultimately leads to pancreatic beta cell destruction and clinically apparent diabetes mellitus. Many environmental factors have been proposed, including viruses (rubella, mumps or coxsackievirus B4), toxic substances and cytotoxins. Nutritional status and diet have also

mice (delaying the onset of disease and decreasing the frequency) (Tisch etal., 1993)

production of diabetogenic T cells and the onset of diabetes (Yoon et al., 1999)

Penha-Goncalves et al., 1995).

hereditary B-cell defect (Martin et al., 2001).

**4.3 Environmental component** 

studies.

T cells are recognized to be a major part of the immune process in diabetes mellitus, and several genes involved in T cell regulation are associated with type 1 diabetes mellitus. Two genes encoding factors that are suppressive to the T cell activation appear to have a close association with autoimmune diabetes: lymphoid tyrosine phosphatase locus (LYP/PTPN22) (Smyth et al., 2004), and cytotoxic T lymphocyte antigen 4 (CTLA-4) (Ueda et al., 2003) (Figure 1), located on chromosome 2q33.

The CTLA-4, which is a T-Lymphocyte receptor, is expressed after T-cell activation (Greenwald et al., 2005). It turns off T-cell responses by inhibiting the production of interleukine-2. CTLA-4 polymorphism in humans has been associated with an increased risk of autoimmune disease, including type 1 diabetes mellitus (Gough et al., 2005).

Another gene linked to an increased risk for type 1 diabetes is the gene for the intercellular adhesion molecule (ICAM-1) (Nejentsev et al., 2003). A recent genome-wide association study described over 40 loci associated with an increased risk for type 1 diabetes (Barrett et al., 2009).

#### **4.2 Autoimmune process**

One of the best animal models for type 1 diabetes mellitus is the nonobese diabetic mouse (NOD). NOD mouse develops type 1 diabetes mellitus spontaneously, over the course of a few months, allowing the investigators to study this process stage by stage. Many reports describe in detail the genetics, the immune process, the influence of the environment and most importantly, the potential therapies to prevent, delay or reverse the destructive process that leads to type 1 diabetes mellitus in this model. Delovitch and Singh (Delovitch & Singh, 1997) reviewed the use of NOD mouse in the studies of type 1 diabetes mellitus. In NOD mice, the first step is the infiltration of the peri-islet regions of the pancreatic islets by dendritic cells (DC) and macrophages, followed by T cells (CD4+ and CD8+). This stage is known as peri-insulitis, occurring around 3-4 weeks of age. It is followed by a slower, progressive T cell destruction of the beta cells (insulitis), by 4-6 months of age (Delovitch & Singh, 1997). Thus, the T cells and the dendritic cells are key players in the immune process leading to type 1 diabetes mellitus.

The dendritic cells (DC) are antigen-presenting cells which originate from the bone marrow. They become active once they capture and process the antigens. After infiltrating the pancreas and undergoing antigenic maturation, DC secrete IL-12 and present the processed antigen (on their surface and in association with the major histocompatibility complex [MHC] class II) to other cells of the immune system (i.e. T cells) (see Fig 1).

T cells are categorized mainly based on their immune actions, achieved via the different cytokines they secrete. Cytokines are classified into two types: type 1 cytokines, which activate the cellular immunity and suppress the humoral immune response, and type 2 cytokines, which activate the humoral immunity and inhibit the cellular immune process (Rabinovitch, 1998).

Th1 cells are preferentially formed from their T cell precursors (T helper 0) under the direct influence of mature DC and IL-12 (Banchereau & Steinman, 1998).

T helper 1 cells (Th1) are involved in cell-mediated immune responses (inflammation, cytotoxicity, delayed hypersensitivity) and produce type 1 cytokines: tumor necrosis factor (TNF), interferon (IFN), and interleukin 2 (IL-2). T helper 2 cells (Th2) are important in humoral immunity (activate B cells and antibody production, down regulating Th 1 cells) and secrete type 2 cytokines: interleukins 4, 5, 6, 9 and 10 (Rabinovitch, 1998) (Fig. 2).

T cells are recognized to be a major part of the immune process in diabetes mellitus, and several genes involved in T cell regulation are associated with type 1 diabetes mellitus. Two genes encoding factors that are suppressive to the T cell activation appear to have a close association with autoimmune diabetes: lymphoid tyrosine phosphatase locus (LYP/PTPN22) (Smyth et al., 2004), and cytotoxic T lymphocyte antigen 4 (CTLA-4) (Ueda

The CTLA-4, which is a T-Lymphocyte receptor, is expressed after T-cell activation (Greenwald et al., 2005). It turns off T-cell responses by inhibiting the production of interleukine-2. CTLA-4 polymorphism in humans has been associated with an increased risk

Another gene linked to an increased risk for type 1 diabetes is the gene for the intercellular adhesion molecule (ICAM-1) (Nejentsev et al., 2003). A recent genome-wide association study described over 40 loci associated with an increased risk for type 1 diabetes (Barrett et

One of the best animal models for type 1 diabetes mellitus is the nonobese diabetic mouse (NOD). NOD mouse develops type 1 diabetes mellitus spontaneously, over the course of a few months, allowing the investigators to study this process stage by stage. Many reports describe in detail the genetics, the immune process, the influence of the environment and most importantly, the potential therapies to prevent, delay or reverse the destructive process that leads to type 1 diabetes mellitus in this model. Delovitch and Singh (Delovitch & Singh, 1997) reviewed the use of NOD mouse in the studies of type 1 diabetes mellitus. In NOD mice, the first step is the infiltration of the peri-islet regions of the pancreatic islets by dendritic cells (DC) and macrophages, followed by T cells (CD4+ and CD8+). This stage is known as peri-insulitis, occurring around 3-4 weeks of age. It is followed by a slower, progressive T cell destruction of the beta cells (insulitis), by 4-6 months of age (Delovitch & Singh, 1997). Thus, the T cells and the dendritic cells are key players in the immune process

The dendritic cells (DC) are antigen-presenting cells which originate from the bone marrow. They become active once they capture and process the antigens. After infiltrating the pancreas and undergoing antigenic maturation, DC secrete IL-12 and present the processed antigen (on their surface and in association with the major histocompatibility complex

T cells are categorized mainly based on their immune actions, achieved via the different cytokines they secrete. Cytokines are classified into two types: type 1 cytokines, which activate the cellular immunity and suppress the humoral immune response, and type 2 cytokines, which activate the humoral immunity and inhibit the cellular immune process

Th1 cells are preferentially formed from their T cell precursors (T helper 0) under the direct

T helper 1 cells (Th1) are involved in cell-mediated immune responses (inflammation, cytotoxicity, delayed hypersensitivity) and produce type 1 cytokines: tumor necrosis factor (TNF), interferon (IFN), and interleukin 2 (IL-2). T helper 2 cells (Th2) are important in humoral immunity (activate B cells and antibody production, down regulating Th 1 cells)

and secrete type 2 cytokines: interleukins 4, 5, 6, 9 and 10 (Rabinovitch, 1998) (Fig. 2).

[MHC] class II) to other cells of the immune system (i.e. T cells) (see Fig 1).

influence of mature DC and IL-12 (Banchereau & Steinman, 1998).

of autoimmune disease, including type 1 diabetes mellitus (Gough et al., 2005).

et al., 2003) (Figure 1), located on chromosome 2q33.

al., 2009).

**4.2 Autoimmune process** 

leading to type 1 diabetes mellitus.

(Rabinovitch, 1998).

The Th2 cells are protective for the beta cells. They have an inhibitory effect on the Th1 cells, which are destructive to the pancreatic beta cells. In the NOD mouse, it appears that the immunologic self-tolerance to pancreatic beta cells is lost. The disruption of the equilibrium between Th1 and Th2 cells in the thymus and in the periphery is believed to play a crucial role in the pathogenesis of autoimmune diabetes mellitus (Delovitch & Singh, 1997). Once Th1 cells are produced they will secrete interferon (IFN ) and IL-2, leading to the activation of macrophages and cytotoxic T cells, which are destructive to the pancreatic beta cells (Adorini, 2001). The same Th1 cells will stimulate the IgG2a autoantibodies against the islet beta cells autoantigens (Delovitch & Singh, 1997). Autoimmune diabetes can be transferred from a diabetic NOD mouse to an unaffected mouse via T cells (Bendelac et al., 1987). NOD mice develop a spontaneous loss of T-cell tolerance to glutamic acid decarboxylase antibodies (GAD), leading to autoimmune diabetes (Kaufman et al., 1993). In NOD mice, there is an increased resistance to apoptosis in immunocytes (Leijon et al., 1995, Penha-Goncalves et al., 1995).

Immune responses to several beta-cell proteins have been described (auto-antigens). Exposure to glutamic acid decarboxylase (GAD65 and GAD67) led to an increased T cell proliferation as early as 4 weeks of life in NOD mice, coinciding with the onset of insulitis (Tisch 1993). Some of the other beta-cell antigens elicited an increased immune response after a few more weeks, but there were other beta-cell antigens that did not trigger an immune reaction (for example, amylin) (Tisch 1993). The same study showed that intrathymic injections of GAD65 had a protective effect from autoimmune diabetes in NOD mice (delaying the onset of disease and decreasing the frequency) (Tisch etal., 1993)

GAD65- reactive T cells were proven to have the ability to transfer diabetes to NOD/SCID (severe combined immunodeficiency) mice (Zekzer et al., 1998). To further support the central role of GAD antigen in autoimmune diabetes, the beta-cell-specific suppression of GAD expression in antisense GAD transgenic NOD mice was demonstrated to prevent the production of diabetogenic T cells and the onset of diabetes (Yoon et al., 1999)

In humans, the pancreas becomes infiltrated with mononuclear cells. Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GAD) and insulinoma associated-2 antibody (IA-2) are demonstrated years before the clinical symptoms of diabetes. (Kulmala et al., 1998) T cell responses to several islet cells antigens (insulin, GAD, IA-2) have been reported in IDDM (MacCuish et al., 1975). The presence of autoantibodies alone does not explain the development of diabetes, since it is recognized now that children born to type 1 diabetic mother with high antibody titers transferred through the umbilical cord do not develop diabetes more often than expected. An interesting case was published by Martin et al in 2001, describing a case of type 1 diabetes mellitus occurring in a patient that had a hereditary B-cell defect (Martin et al., 2001).

#### **4.3 Environmental component**

The environment is implicated in the pathogenesis of type 1 diabetes mellitus by many studies.

Environmental factors have an important role in initiating an immune process that ultimately leads to pancreatic beta cell destruction and clinically apparent diabetes mellitus. Many environmental factors have been proposed, including viruses (rubella, mumps or coxsackievirus B4), toxic substances and cytotoxins. Nutritional status and diet have also

Role of Vitamin D in the Pathogenesis and Therapy of Type 1 Diabetes Mellitus 409

contains cholecalciferol (vitamin D3), originating from animal sources, and ergocalciferol

Regardless of their source, once they enter into the circulation, forms of inactive vitamin D3 or D2 bind to the vitamin D-binding protein (DBP) and are transported to the liver. The inactive vitamin D is activated through a 2-step hydroxylation process via two hydroxylases that belong to the cytochrome P450- dependent steroid hydroxylases (CYP450). In the liver, vitamin D undergoes the first hydroxylation at C-25 via some of the CYP 450 vitamin D 25 hydroxylases, forming calcidiol (25-hydroxyvitamin D) (Prosser & Jones, 2004). This is the major circulating form of vitamin D. At the level of the proximal renal tubule, 25-OH vitamin D is further hydroxylated to calcitriol (1,25 dihydroxyvitamin D, the active form of

Both calcidiol and calcitriol are inactivated via the 25-hydroxyvitamin D3-24-hydroxylase

1-hydroxylase has been described in many extrarenal tissues: macrophages, monocytes, and placenta, rendering these cells capable of synthesizing 1-,25(OH)2D3from 25(OH)vitamin D (Weisman et al., 1979, Bhalla et al., 1983, Stoffels et al., 2007, Adams et al., 1983). The activity of 1-hydroxylase in the immune cells is not under the regulation of parathyroid hormone and 1-,25(OH)2D3, but rather under immune cytokine regulation. A defect in the up-regulation of 1-hydroxylase after immune stimulation is described in NOD mouse (Overbergh et al., 2000). Extrarenal distribution of 1-hydroxylase becomes

VDR is part of the nuclear receptor super family of ligand-activated transcription factors, which also includes glucocorticoid, thyroid hormone and estrogen receptors. The gene for VDR is located on chromosome 12q12-14, and shows great polymorphism (Haussler et al., 1998). After 1,25 (OH)2D3 binds to VDR, it induces conformational changes that facilitate heterodimerization with the retinoid X receptor and the recruitment of nuclear receptor coactivator proteins, which then act on the chromatin. The specific DNA sequence that is ultimately affected by the vitamin D is known as the vitamin D responsive element (VDRE)

The discovery of the vitamin D receptor (VDR) on the immune cells (Strugnell & DeLuca, 1997), led to the hypothesis that vitamin D could affect the autoimmune processes. However, in VDR deficient mice models, there is no increase in autoimmune diseases

The protective effects of vitamin D in several autoimmune diseases have been described in animal models (experimental autoimmune encephalomyelitis (Lemire, 1995), murine models of human multiple sclerosis and murine models of rheumatoid arthritis (Cantorna et al., 1996). In other autoimmune diseases, like psoriasis, vitamin D analogues are the mainstay of

The extraskeletal effects of 1-,25(OH)2D3 can usually be observed only at very high concentrations (10-10mol/l), higher than physiological levels needed for calcium balance (concentrations that could probably be achieved in specific target tissues via the macrophages' 1-hydroxylase) (Mathieu et al., 2005). Thus a risk of hypercalcemia and other side effects of 1-25(OH)2D3 could occur if it were used for its anti-autoimmune properties. Numerous vitamin D analogs have been developed to exert extraskeletal effects, with less pronounced action on the calcium metabolism. Most of these analogs are used for laboratory

vitamin D) by the 1-hydroxylase (1(OH)ase, CYP27B1) (Prosser & Jones, 2004).

(CYP24), forming the inactive metabolite 24,25- dihydroxyvitamin D (Holick, 1999).

important in understanding the extra-skeletal effects of vitamin D.

(Carlberg & Polly, 1998).

(Mathieu et al., 2001)

treatment today.

(vitamin D2), deriving from plants (Holick, 1999).

been implicated as potential players in type 1 diabetes pathogenesis: vitamin D deficiency, early protein diet exposure or exposure to cow's milk in infancy.

Viruses are among the main culprits studied. Before the eradication of rubella in most countries, congenital rubella was strongly associated with the development of type 1 diabetes mellitus (Menser et al., 1978). A recent meta analysis of observational studies has shown an association between type 1 diabetes and enterovirus infection (Yeung 2011).

While some theories implicate viral infections in the pathogenesis of type 1 diabetes, a recent hypothesis argues that a decreased exposure to microbes may contribute to the current increase in autoimmune disease. This theory is known as "the hygiene hypothesis" (Gale, 2002).

It is a known fact that the incidence of autoimmune diabetes follows a geographical pattern, with many studies reporting an association between type 1 diabetes and vitamin D status. A few large ecological studies describe a pattern of geographical variation, with an increased incidence of type 1 diabetes in the areas located north of the equator. Furthermore, seasons appear to also influence the incidence of type 1 diabetes, with the highest incidence during winter and the lowest during summer. The month of birth during springtime is associated with a higher risk of type 1 diabetes (Kahn et al., 2009), a finding that could be explained by possible low circulating vitamin D levels in both mother and fetus through the winter months of the pregnancy.

In order to develop more information about environmental factors that play a role in the pathogenesis of diabetes, an international initiative (the Environmental Determinants of Diabetes in the Young) will be following thousands of infants with an increased genetic risk from birth until adolescence and will gather data about infectious agents, dietary or other environmental factors.

Typically, the treatment for type 1 diabetes mellitus involves insulin therapy, but in the last few years new therapies have been approved as well (for example, Symlin). For newly diagnosed patients with autoimmune diabetes, combination therapy has been suggested in an attempt to minimize beta cell destruction and prolong pancreatic function. The new therapeutic options include: immunotherapy, vaccines, drugs that influence T cell action, anti-inflammatory drugs (for example, one time use of anti-IL-1R drug), or long-term treatment with B cell components to induce regulatory T cells (oral or nasal insulin, insulin peptide therapy, GAD-Alum or the proinsulin DNA vaccines). Glucagon-like peptide 1 related drugs (GLP-1) could be also considered as a therapeutic option because they promote peritubular pancreatic cell growth (Von Herrath, 2010).

### **5. Vitamin D**

Although initially described as a "vitamin", vitamin D is now recognized to be a hormone, synthesized in the human body and exerting its action on other organs via a nuclear receptor (vitamin D receptor, VDR).

Even though vitamin D can be obtained from the diet in small quantities, the main source of vitamin D is the skin. Under the direct influence of ultra violet B light (UVB light), 7 dehydrocholesterol (DHC) (provitamin D3) is converted into pre-vitamin D3, which is then further converted into cholecalciferol (vitamin D3) via thermal isomerization. Interestingly, if pre-vitamin D3 continues to be exposed to UVB, it will be converted into biologically inactive metabolites (tachysterol and lumisterol), preventing a potential UVB- induced vitamin D intoxication (Holick, 1999) The other source of vitamin D is the diet, which

been implicated as potential players in type 1 diabetes pathogenesis: vitamin D deficiency,

Viruses are among the main culprits studied. Before the eradication of rubella in most countries, congenital rubella was strongly associated with the development of type 1 diabetes mellitus (Menser et al., 1978). A recent meta analysis of observational studies has shown an association between type 1 diabetes and enterovirus infection (Yeung 2011). While some theories implicate viral infections in the pathogenesis of type 1 diabetes, a recent hypothesis argues that a decreased exposure to microbes may contribute to the current increase in autoimmune disease. This theory is known as "the hygiene hypothesis"

It is a known fact that the incidence of autoimmune diabetes follows a geographical pattern, with many studies reporting an association between type 1 diabetes and vitamin D status. A few large ecological studies describe a pattern of geographical variation, with an increased incidence of type 1 diabetes in the areas located north of the equator. Furthermore, seasons appear to also influence the incidence of type 1 diabetes, with the highest incidence during winter and the lowest during summer. The month of birth during springtime is associated with a higher risk of type 1 diabetes (Kahn et al., 2009), a finding that could be explained by possible low circulating vitamin D levels in both mother and fetus through the

In order to develop more information about environmental factors that play a role in the pathogenesis of diabetes, an international initiative (the Environmental Determinants of Diabetes in the Young) will be following thousands of infants with an increased genetic risk from birth until adolescence and will gather data about infectious agents, dietary or other

Typically, the treatment for type 1 diabetes mellitus involves insulin therapy, but in the last few years new therapies have been approved as well (for example, Symlin). For newly diagnosed patients with autoimmune diabetes, combination therapy has been suggested in an attempt to minimize beta cell destruction and prolong pancreatic function. The new therapeutic options include: immunotherapy, vaccines, drugs that influence T cell action, anti-inflammatory drugs (for example, one time use of anti-IL-1R drug), or long-term treatment with B cell components to induce regulatory T cells (oral or nasal insulin, insulin peptide therapy, GAD-Alum or the proinsulin DNA vaccines). Glucagon-like peptide 1 related drugs (GLP-1) could be also considered as a therapeutic option because they

Although initially described as a "vitamin", vitamin D is now recognized to be a hormone, synthesized in the human body and exerting its action on other organs via a nuclear

Even though vitamin D can be obtained from the diet in small quantities, the main source of vitamin D is the skin. Under the direct influence of ultra violet B light (UVB light), 7 dehydrocholesterol (DHC) (provitamin D3) is converted into pre-vitamin D3, which is then further converted into cholecalciferol (vitamin D3) via thermal isomerization. Interestingly, if pre-vitamin D3 continues to be exposed to UVB, it will be converted into biologically inactive metabolites (tachysterol and lumisterol), preventing a potential UVB- induced vitamin D intoxication (Holick, 1999) The other source of vitamin D is the diet, which

early protein diet exposure or exposure to cow's milk in infancy.

promote peritubular pancreatic cell growth (Von Herrath, 2010).

(Gale, 2002).

winter months of the pregnancy.

environmental factors.

**5. Vitamin D** 

receptor (vitamin D receptor, VDR).

contains cholecalciferol (vitamin D3), originating from animal sources, and ergocalciferol (vitamin D2), deriving from plants (Holick, 1999).

Regardless of their source, once they enter into the circulation, forms of inactive vitamin D3 or D2 bind to the vitamin D-binding protein (DBP) and are transported to the liver. The inactive vitamin D is activated through a 2-step hydroxylation process via two hydroxylases that belong to the cytochrome P450- dependent steroid hydroxylases (CYP450). In the liver, vitamin D undergoes the first hydroxylation at C-25 via some of the CYP 450 vitamin D 25 hydroxylases, forming calcidiol (25-hydroxyvitamin D) (Prosser & Jones, 2004). This is the major circulating form of vitamin D. At the level of the proximal renal tubule, 25-OH vitamin D is further hydroxylated to calcitriol (1,25 dihydroxyvitamin D, the active form of vitamin D) by the 1-hydroxylase (1(OH)ase, CYP27B1) (Prosser & Jones, 2004).

Both calcidiol and calcitriol are inactivated via the 25-hydroxyvitamin D3-24-hydroxylase (CYP24), forming the inactive metabolite 24,25- dihydroxyvitamin D (Holick, 1999).

1-hydroxylase has been described in many extrarenal tissues: macrophages, monocytes, and placenta, rendering these cells capable of synthesizing 1-,25(OH)2D3from 25(OH)vitamin D (Weisman et al., 1979, Bhalla et al., 1983, Stoffels et al., 2007, Adams et al., 1983). The activity of 1-hydroxylase in the immune cells is not under the regulation of parathyroid hormone and 1-,25(OH)2D3, but rather under immune cytokine regulation. A defect in the up-regulation of 1-hydroxylase after immune stimulation is described in NOD mouse (Overbergh et al., 2000). Extrarenal distribution of 1-hydroxylase becomes important in understanding the extra-skeletal effects of vitamin D.

VDR is part of the nuclear receptor super family of ligand-activated transcription factors, which also includes glucocorticoid, thyroid hormone and estrogen receptors. The gene for VDR is located on chromosome 12q12-14, and shows great polymorphism (Haussler et al., 1998). After 1,25 (OH)2D3 binds to VDR, it induces conformational changes that facilitate heterodimerization with the retinoid X receptor and the recruitment of nuclear receptor coactivator proteins, which then act on the chromatin. The specific DNA sequence that is ultimately affected by the vitamin D is known as the vitamin D responsive element (VDRE) (Carlberg & Polly, 1998).

The discovery of the vitamin D receptor (VDR) on the immune cells (Strugnell & DeLuca, 1997), led to the hypothesis that vitamin D could affect the autoimmune processes. However, in VDR deficient mice models, there is no increase in autoimmune diseases (Mathieu et al., 2001)

The protective effects of vitamin D in several autoimmune diseases have been described in animal models (experimental autoimmune encephalomyelitis (Lemire, 1995), murine models of human multiple sclerosis and murine models of rheumatoid arthritis (Cantorna et al., 1996). In other autoimmune diseases, like psoriasis, vitamin D analogues are the mainstay of treatment today.

The extraskeletal effects of 1-,25(OH)2D3 can usually be observed only at very high concentrations (10-10mol/l), higher than physiological levels needed for calcium balance (concentrations that could probably be achieved in specific target tissues via the macrophages' 1-hydroxylase) (Mathieu et al., 2005). Thus a risk of hypercalcemia and other side effects of 1-25(OH)2D3 could occur if it were used for its anti-autoimmune properties. Numerous vitamin D analogs have been developed to exert extraskeletal effects, with less pronounced action on the calcium metabolism. Most of these analogs are used for laboratory

Role of Vitamin D in the Pathogenesis and Therapy of Type 1 Diabetes Mellitus 411

tolerogenic DC with special endocytic properties (Ferreira et al., 2009). Adorini et al published a paper describing how 1,25(OH)2 D3 can change the dendritic cells into a tolerogenic phenotype which is thought to induce T regulatory cells and inhibit

**DC**

**Th**

**Ag**

**CD40 MCH II**

**CD40LTCR**

**CD80/86**

**CD28/CTLA4**

**Th1 Th1**

Fig. 2. The immunomodulatory effects of 1,25(OH)2D3. At the level of the antigen-

of MHC class II-complexed antigen and of co-stimulatory molecules, in addition to

induction of regulatory T cells. Both Th2 and Tregs can inhibit Th1 cells through the production of counteracting or inhibitory cytokines. Together, these immunomodulatory effects of 1,25(OH)2D3 can lead to the protection of target tissues, such as β cells, in autoimmune diseases and transplantation. CD40L, CD40 ligand; Mf, macrophage; Tc, cytotoxic T cell; TGF- β, transforming growth factor β; Th1,T helper type 1; TNF-, tumor

necrosis factor ; Treg, regulatory T cell. This figure was published in Trends in

Endocrinology and Metabolism Vol.16 No.6 August 2005. Vitamin D and type 1 diabetes mellitus: state of art. Chantal Mathieu and Klaus Badenhoop. Copyright @ Elsevier 2005.

Descriptions of the VDR on T lymphocytes lead to the subsequent investigation of vitamin D actions on these immune cells. Interestingly, 1-hydroxylase is not expressed in the T cells, and vitamin D activated in the macrophages acts on the T cells, suggesting an

Rigby and his team proved that cytokine production by T cells is influenced by vitamin D analogs: IL-2 and IFN are inhibited (Rigby et al., 1987), while production of some of the

presenting cell (such as dendritic cells; DCs), 1,25(OH)2D3 inhibits the surface expression

production of the cytokine IL-12, thereby indirectly shifting the polarization of T cells from a Th1 towards a Th2 phenotype. In addition, 1,25(OH)2D3 has immunomodulatory effects directly at the level of the T cell, by inhibiting the production of the Th1 cytokines IL-2 and IFN-γ and stimulating the production of Th2 cytokines. Moreover, 1,25(OH)2D3 favors the

**Th1**

**Th1**

**Th2 Th2**

**Th2**

**IL-10 TGF-**

**Treg**

**IL-4 IL-5 IL-10**

**Th1**

autoimmune diseases, like type 1 diabetes (Adorini, 2003) (Fig 2).

**IL-12**

**IFN-**

**1,25(OH)2D3**

**IL-2**

**M**

**IL-1 TNF free radicals**

**Tc**

Used with permission.

autocrine action of 1,25 dihydroxyvitamin D3.

type 2 cytokines (IL-4, 5, and 10) is enhanced (Boonstra et al., 2001)

**cell**

research purposes, but some are part of standard treatment for certain autoimmune diseases (for example, calcipotriol for psoriasis).

There are several theories that attempt to explain the link between Vitamin D and autoimmune diabetes. This relationship appears to be complex, with actions at multiples levels: genetic, autoimmune and also direct action on the pancreatic beta cells.
