**3. Vitamin D in maintaining pancreatic β-cell function and regulating insulin sensitivity**

As demonstrated in the previous section, VD is one of the key players in the control of immune homeostasis, and here, we will examine in more detail the molecular mechanisms, showing how inadequate VD status and inflammation can

**193**

**Figure 2.**

*protein gene.*

*Vitamin D Deficiency and Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.89543*

secretion, and insulin signaling (**Figure 2**).

contribute to pancreatic β-cell dysfunction and the formation of insulin resistance (IR). Comprehensive results of experimental and clinical studies have shown that vitamin D is a potential regulator of pancreatic β-cell survival, Ca2+ levels, insulin

Vitamin D plays an immunomodulatory role in preventing pancreatic β-cell dysfunction and death, via VDR, which is expressed along with Cyp27B1 in APCs, activated T cells, and islet pancreatic β-cells [12]. These effects have been demonstrated in many studies of nonobese diabetic mice using 1,25(OH)2D or analogs. Conversely, 1,25(OH)2D-deficient mice showed a tendency to develop more aggressive form of T1D, if the deficiency is present at an early age. The bioactive form of VD protects against the development of insulitis in the pancreas or reduces their

*Mechanisms of glucose homeostasis deregulation related to vitamin D deficiency status. Cyp27B1, 25-hydroxyvitamin D 1-alpha-hydroxylase gene; Vdr, vitamin D receptor gene; Vdbp, vitamin D-binding* 

### *Vitamin D Deficiency and Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.89543*

*Vitamin D Deficiency*

cytic activity [1, 3].

of the anti-inflammatory cytokines.

not shown any promising result [10, 11].

**insulin sensitivity**

not respond to vitamin D after the IL-10 stimulus, regardless of their high phago-

Vitamin D shows an inhibitory action on the adaptive immune system, the responses of which include the ability of T and B lymphocytes to produce cytokines and immunoglobulins, respectively, to specifically combat antigens presented to them by macrophages and dendritic cells (DCs). Experimental studies have yielded encouraging results on the immunomodulatory effect of calcitriol on T helper (Th) cells. In particular, 1,25(OH)2D was shown to suppress the immune responses mediated by Th1 cells capable of producing such pro-inflammatory cytokines as IL-2, IL-6, interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) [4]. The lack of IFN-γ prevents further antigen presentation to T lymphocytes and their recruitment, while lower IL-2 production impedes T lymphocyte proliferation and differentiation. It has been recently demonstrated that calcitriol also increases formation and activity of CD4+/CD25+ regulatory T cells (Treg) as seen by elevated FoxP3 and IL-10 expression [5]. Increased levels of IL-10 as well as other cytokines with anti-inflammatory properties, induced by calcitriol, block Th1 differentiation, thus shifting the balance from Th1 to Th2 cell phenotype [6]. Many of the effects of VD on Th1 cells, which were previously considered to be implicated in the pathogenesis of several autoimmune diseases, can now be attributable, at least in part, to the inhibitory action of 1,25(OH)2D on the formation and activity of Th17 cells, producing IL-17 [5]. The overall impact of VD on Th cells is related to the suppression of antigen-presenting cells (APCs) of the innate immune system, including the most potent dendritic cells. This modulatory effect of 1,25(OH)2D induces a "tolerogenic state" associated with the differentiation of Treg cells, autoreactive T cell apoptosis, reduced production of inflammatory cytokines, and increased levels

Chromatin immunoprecipitation assay revealed VDR binding to a VDRE in the proximal area of IL-10 promoter in antibody-producing cells of the immune system, or B-cells [7]. 1,25(OH)2D blocked the proliferation of activated B-cells and stimulated their apoptosis. It also inhibited maturation of activated B-cells into plasma cells and memory cells that is consistent with the inhibitory action of VD on the secretion of IgM and IgG [8]. Several observational trials showed an inverse relationship between

Due to the ability of VD to suppress the adaptive immune system, the role of VD deficiency and supplementation in inflammatory and autoimmune diseases acquires more comprehensive support. In a number of animal models, including autoimmune diabetes, inflammatory arthritis, experimental allergic encephalitis, and different mouse models of enterocolitis, calcitriol prevented the initiation and reduced the disease progression. However, despite strong experimental evidence, human studies are less convincing to prove a role for VD in the modulation of adaptive immune system of individuals affected by autoimmune diseases. In this respect, some trials have confirmed beneficial effect of VD on different inflammatory disease progression, inflammatory markers, and T cell subsets, whereas others have

**3. Vitamin D in maintaining pancreatic β-cell function and regulating** 

As demonstrated in the previous section, VD is one of the key players in the control of immune homeostasis, and here, we will examine in more detail the molecular mechanisms, showing how inadequate VD status and inflammation can

serum IgE and 25OHD levels, while others indicated a positive correlation [9].

**192**

contribute to pancreatic β-cell dysfunction and the formation of insulin resistance (IR). Comprehensive results of experimental and clinical studies have shown that vitamin D is a potential regulator of pancreatic β-cell survival, Ca2+ levels, insulin secretion, and insulin signaling (**Figure 2**).

Vitamin D plays an immunomodulatory role in preventing pancreatic β-cell dysfunction and death, via VDR, which is expressed along with Cyp27B1 in APCs, activated T cells, and islet pancreatic β-cells [12]. These effects have been demonstrated in many studies of nonobese diabetic mice using 1,25(OH)2D or analogs. Conversely, 1,25(OH)2D-deficient mice showed a tendency to develop more aggressive form of T1D, if the deficiency is present at an early age. The bioactive form of VD protects against the development of insulitis in the pancreas or reduces their

#### **Figure 2.**

*Mechanisms of glucose homeostasis deregulation related to vitamin D deficiency status. Cyp27B1, 25-hydroxyvitamin D 1-alpha-hydroxylase gene; Vdr, vitamin D receptor gene; Vdbp, vitamin D-binding protein gene.*

severity through a dual mechanism of action on both pancreatic β-cells and immune cells [13].

In pancreatic islets, 1,25(OH)2D decreases, as was shown in *in vitro* and *in vivo* experiments, the expression of pro-inflammatory cytokines (e.g., IL-6), which are involved in the pathogenesis of T1D, making β-cells less chemoattractive and less prone to inflammation [14]. This leads to a decrease in T-cell recruitment and infiltration, an increase in regulatory cells, and a delay in the autoimmune process. In addition, 1,25(OH)2D reduces the expression of MHC class I, leading to a decrease in the vulnerability of islet β-cells to the action of cytotoxic T lymphocytes [13, 14].

At the level of the immune system, 1,25(OH)2D inhibits the differentiation and maturation of DCs and promotes their apoptosis, preventing them from becoming APCs, which is the first step in initiating an immune response. It has also been shown that 1,25(OH)2D restores suppressor cells, reduces cytokine formation by Th1 cells responsible for β-cell death, and shifts the immune response toward Th2 cell activation, leading to more benign inflammatory response in pancreatic islets. 1,25(OH)2D suppresses the formation of IL-6, a direct stimulator of Th17 cells involved in the pathogenesis of various autoimmune diseases, including T1D [15]. On the other hand, 1,25(OH)2D exerts an antiapoptotic effect on cytokine-induced apoptosis of pancreatic β-cells. It induces and maintains high protein levels of the A20 (anti-inflammatory protein; inhibits NF-κB signaling), leading to a decrease in nitric monoxide (NO) levels. In fact, NO is able to directly induce β-cell dysfunction and death, or indirectly may affect β-cell function through the induction of Fas expression. Fas is a transmembrane cell surface receptor and a member of the TNF receptor superfamily. Activation of these receptors occurs under the influence of inflammatory cytokines secreted by mononuclear cells that infiltrate islet cells. Reduction of the NO level leads to inhibition of all the above mechanisms and allows realizing cytoprotective effect on islet β-cells. The ability of 1,25(OH)2D to counteract the cytokine-induced expression of Fas in human pancreatic islets at both mRNA and protein levels, modulating the cell death signal cascades and preventing β-cell apoptosis, was established [16].

Several trials have reported that VD deficiency caused impairment of glucosemediated secretion of insulin in rat pancreatic β-cells, which was restored after VD supplementation. However, the results of clinical studies are not unambiguous as VD adequacy was not always associated with the improvement of insulin secretion. This stimulatory effect of VD is important for the prevention of T2D and may have different explanations. The bioactive form of VD is able to induce insulin secretion through direct binding of VDR-RXR complex to VDRE previously identified in the promoter of insulin gene in pancreatic β-cells [17]. In accordance with this finding, mice with a lack of functional VDR showed impaired insulin secretion after stimulation with glucose [18]. It is noteworthy that VDRE can stimulate not only the transcription of the insulin gene but also many other genes involved in the organization of the cytoskeleton, cell growth, differentiation, and survival of pancreatic β-cells.

In addition to genomic effects, rapid nongenomic mechanism of VD action appears to be involved in depolarization-stimulated insulin exocytosis by regulating intracellular Ca2+. This effect of calcitriol is realized through a membrane VDRmediated increase in the synthesis of inositol trisphosphate and phospholipase C that promotes the release of Ca2+ from endoplasmic reticulum and diacylglycerolmediated PKC activation. In turn, activated PKC phosphorylates the ATP-dependent K+ channels and L-type voltage-dependent Ca2+ channels. Ultimately, these effects lead to depolarization of the cytoplasmic membrane and the opening of Ca2+ L-type and T-type channels that increase intracellular Ca2+ level and, accordingly, insulin secretion [19]. Activation of PKA signaling pathways by calcitriol, apparently, is also involved in the regulation of L-type voltage-dependent Ca2+ channels.

**195**

membrane [26].

*Vitamin D Deficiency and Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.89543*

plasma membrane Ca2+-ATPase, and Na+

of the renin-angiotensin-aldosterone system [22].

tissue, liver, and muscle, thereby reducing glucose uptake [24].

An important endocrine and metabolic organ, playing a crucial role in glucose homeostasis and energy balance, is adipose tissue. This tissue is also the major site of VD storage in an organism that can sequestrate a fat-soluble prehormone and significantly decrease its level in blood circulation. It was found that VD exerts an important effect on the expression of genes implicated in promoting adipogenesis and adipose tissue remodeling. VDR is expressed in adipocytes in early stages of adipogenesis and mediates inhibitory effect of calcitriol on adipocyte differentiation through Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling pathways [25]. Suppressive action of 1,25(OH)2D on transcription factors, such as PPARγ and CAAT/enhancer-binding protein α (C/EBPα), alters the expression of numerous genes involved in lipolysis, lipogenesis, secretion of adipokines, insulin sensitivity (via GLUT4 expression), and transfer of fatty acids across the

The association between obesity, IR, and VD deficiency is a subject of intense research. A characteristic feature of hypertrophic enlargement of adipose tissue is elevated release of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, MCP1, and resistin) by adipose-resident macrophages and activated T lymphocytes, whereas secretion of adiponectin, an anti-inflammatory and insulin-sensitizing bioregulatory molecule, by adipocytes is reduced [27]. Thus, one of the harmful consequences of obesity is impaired secretion of adipokines and systemic inflammation, which coexists with IR and favors the development of T2D as a key contributing factor. VD is known to protect against IR associated with inflammation by modulating the function of immune cells and secretion of adipokines (adiponectin and

It has been suggested that increased intracellular Ca2+ induced by VD may enhance the expression of cAMP-responsive element-binding protein (CREB), responsible for maintaining efficient transcription of insulin gene and insulin exocytosis, as well as for the glucose sensing and pancreatic β-cell survival [20]. Increased expression of proteins involved in providing low resting Ca2+ level, such as calcium-binding proteins (parvalbumin, calbindin-D28k, and calbindin-D9k),

nism by which vitamin D affects insulin secretion [21]. Furthermore, preclinical studies have shown that VD improves β-cell function by reducing the excess activity

Optimal intracellular levels of Ca2+ are essential not only for the proper function of pancreatic β-cells but also for insulin-responsive tissues, including liver, adipose tissue, and skeletal muscles. Impaired regulation of extracellular and intracellular Ca2+ concentrations due to abnormal transduction of insulin signaling in target tissues may evoke dephosphorylation and decreased activity of glucose transporter-4 (GLUT-4), leading to a phenomenon known as peripheral insulin resistance. The results of several studies confirmed that VD deficiency is involved in the onset of IR. Moreover, an adequate VD level was shown to improve insulin resistance associated with T2D [23]. In addition to the effect of calcitriol on insulin sensitivity related to regulation of extracellular Ca2+ concentration and its influx into cells through cell membranes, the active metabolite of VD seems to be an inducer of insulin receptor expression, which in turn improves insulin sensitivity [23]. Another mechanism underlying the beneficial effects of calcitriol on insulin sensitivity is related to activation of the peroxisome proliferator-activated receptor delta (PPARδ) [22]. Activated PPARδ, as a transcription factor, reduces fatty acids-evoked IR in adipose tissue and skeletal muscles. A secondary elevation of parathyroid hormone (PTH) in response to VD deficiency can also increase the concentration of intracellular Ca2+ in insulin-sensitive tissues and exacerbate IR by decreasing the number of GLUT1 and GLUT4 in the cell membranes of adipose

/Ca2+-exchanger, can be another mecha-

#### *Vitamin D Deficiency and Diabetes Mellitus DOI: http://dx.doi.org/10.5772/intechopen.89543*

*Vitamin D Deficiency*

cells [13].

severity through a dual mechanism of action on both pancreatic β-cells and immune

In pancreatic islets, 1,25(OH)2D decreases, as was shown in *in vitro* and *in vivo* experiments, the expression of pro-inflammatory cytokines (e.g., IL-6), which are involved in the pathogenesis of T1D, making β-cells less chemoattractive and less prone to inflammation [14]. This leads to a decrease in T-cell recruitment and infiltration, an increase in regulatory cells, and a delay in the autoimmune process. In addition, 1,25(OH)2D reduces the expression of MHC class I, leading to a decrease in the vulnerability of islet β-cells to the action of cytotoxic T lymphocytes [13, 14]. At the level of the immune system, 1,25(OH)2D inhibits the differentiation and maturation of DCs and promotes their apoptosis, preventing them from becoming APCs, which is the first step in initiating an immune response. It has also been shown that 1,25(OH)2D restores suppressor cells, reduces cytokine formation by Th1 cells responsible for β-cell death, and shifts the immune response toward Th2 cell activation, leading to more benign inflammatory response in pancreatic islets. 1,25(OH)2D suppresses the formation of IL-6, a direct stimulator of Th17 cells involved in the pathogenesis of various autoimmune diseases, including T1D [15]. On the other hand, 1,25(OH)2D exerts an antiapoptotic effect on cytokine-induced apoptosis of pancreatic β-cells. It induces and maintains high protein levels of the A20 (anti-inflammatory protein; inhibits NF-κB signaling), leading to a decrease in nitric monoxide (NO) levels. In fact, NO is able to directly induce β-cell dysfunction and death, or indirectly may affect β-cell function through the induction of Fas expression. Fas is a transmembrane cell surface receptor and a member of the TNF receptor superfamily. Activation of these receptors occurs under the influence of inflammatory cytokines secreted by mononuclear cells that infiltrate islet cells. Reduction of the NO level leads to inhibition of all the above mechanisms and allows realizing cytoprotective effect on islet β-cells. The ability of 1,25(OH)2D to counteract the cytokine-induced expression of Fas in human pancreatic islets at both mRNA and protein levels, modulating the cell death signal cascades and

Several trials have reported that VD deficiency caused impairment of glucosemediated secretion of insulin in rat pancreatic β-cells, which was restored after VD supplementation. However, the results of clinical studies are not unambiguous as VD adequacy was not always associated with the improvement of insulin secretion. This stimulatory effect of VD is important for the prevention of T2D and may have different explanations. The bioactive form of VD is able to induce insulin secretion through direct binding of VDR-RXR complex to VDRE previously identified in the promoter of insulin gene in pancreatic β-cells [17]. In accordance with this finding, mice with a lack of functional VDR showed impaired insulin secretion after stimulation with glucose [18]. It is noteworthy that VDRE can stimulate not only the transcription of the insulin gene but also many other genes involved in the organization of the cytoskeleton, cell growth, differentiation, and survival of pancreatic β-cells. In addition to genomic effects, rapid nongenomic mechanism of VD action appears to be involved in depolarization-stimulated insulin exocytosis by regulating intracellular Ca2+. This effect of calcitriol is realized through a membrane VDRmediated increase in the synthesis of inositol trisphosphate and phospholipase C that promotes the release of Ca2+ from endoplasmic reticulum and diacylglycerolmediated PKC activation. In turn, activated PKC phosphorylates the ATP-dependent

 channels and L-type voltage-dependent Ca2+ channels. Ultimately, these effects lead to depolarization of the cytoplasmic membrane and the opening of Ca2+ L-type and T-type channels that increase intracellular Ca2+ level and, accordingly, insulin secretion [19]. Activation of PKA signaling pathways by calcitriol, apparently, is also

involved in the regulation of L-type voltage-dependent Ca2+ channels.

preventing β-cell apoptosis, was established [16].

**194**

K+

It has been suggested that increased intracellular Ca2+ induced by VD may enhance the expression of cAMP-responsive element-binding protein (CREB), responsible for maintaining efficient transcription of insulin gene and insulin exocytosis, as well as for the glucose sensing and pancreatic β-cell survival [20]. Increased expression of proteins involved in providing low resting Ca2+ level, such as calcium-binding proteins (parvalbumin, calbindin-D28k, and calbindin-D9k), plasma membrane Ca2+-ATPase, and Na+ /Ca2+-exchanger, can be another mechanism by which vitamin D affects insulin secretion [21]. Furthermore, preclinical studies have shown that VD improves β-cell function by reducing the excess activity of the renin-angiotensin-aldosterone system [22].

Optimal intracellular levels of Ca2+ are essential not only for the proper function of pancreatic β-cells but also for insulin-responsive tissues, including liver, adipose tissue, and skeletal muscles. Impaired regulation of extracellular and intracellular Ca2+ concentrations due to abnormal transduction of insulin signaling in target tissues may evoke dephosphorylation and decreased activity of glucose transporter-4 (GLUT-4), leading to a phenomenon known as peripheral insulin resistance. The results of several studies confirmed that VD deficiency is involved in the onset of IR. Moreover, an adequate VD level was shown to improve insulin resistance associated with T2D [23]. In addition to the effect of calcitriol on insulin sensitivity related to regulation of extracellular Ca2+ concentration and its influx into cells through cell membranes, the active metabolite of VD seems to be an inducer of insulin receptor expression, which in turn improves insulin sensitivity [23]. Another mechanism underlying the beneficial effects of calcitriol on insulin sensitivity is related to activation of the peroxisome proliferator-activated receptor delta (PPARδ) [22]. Activated PPARδ, as a transcription factor, reduces fatty acids-evoked IR in adipose tissue and skeletal muscles. A secondary elevation of parathyroid hormone (PTH) in response to VD deficiency can also increase the concentration of intracellular Ca2+ in insulin-sensitive tissues and exacerbate IR by decreasing the number of GLUT1 and GLUT4 in the cell membranes of adipose tissue, liver, and muscle, thereby reducing glucose uptake [24].

An important endocrine and metabolic organ, playing a crucial role in glucose homeostasis and energy balance, is adipose tissue. This tissue is also the major site of VD storage in an organism that can sequestrate a fat-soluble prehormone and significantly decrease its level in blood circulation. It was found that VD exerts an important effect on the expression of genes implicated in promoting adipogenesis and adipose tissue remodeling. VDR is expressed in adipocytes in early stages of adipogenesis and mediates inhibitory effect of calcitriol on adipocyte differentiation through Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling pathways [25]. Suppressive action of 1,25(OH)2D on transcription factors, such as PPARγ and CAAT/enhancer-binding protein α (C/EBPα), alters the expression of numerous genes involved in lipolysis, lipogenesis, secretion of adipokines, insulin sensitivity (via GLUT4 expression), and transfer of fatty acids across the membrane [26].

The association between obesity, IR, and VD deficiency is a subject of intense research. A characteristic feature of hypertrophic enlargement of adipose tissue is elevated release of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, MCP1, and resistin) by adipose-resident macrophages and activated T lymphocytes, whereas secretion of adiponectin, an anti-inflammatory and insulin-sensitizing bioregulatory molecule, by adipocytes is reduced [27]. Thus, one of the harmful consequences of obesity is impaired secretion of adipokines and systemic inflammation, which coexists with IR and favors the development of T2D as a key contributing factor. VD is known to protect against IR associated with inflammation by modulating the function of immune cells and secretion of adipokines (adiponectin and

leptin). It has been reported in numerous trials using animal models and in several human observational studies that higher VD levels are accompanied by lower inflammatory markers including TNF-α, IL-6, and C-reactive protein in healthy persons, and in those with inflammation-associated diseases, such as arteriosclerosis, inflammatory polyarthritis, and diabetes [28]. As for adipokines, positive correlation was shown between VD and adiponectin, and inverse correlation between VD and leptin [29]. Finally, VD by targeting mitochondrial respiratory functions through multiple mechanisms also attenuates oxidative stress and exerts key beneficial effects on controlling inflammation, impaired energy metabolism, and cell apoptosis. However, this topic is beyond the scope of this chapter. Vitamin D functions associated with the regulation of β-cell function and insulin sensitivity are summarized in **Figure 2**.
