Abstract
Context: Due to the biochemical role of vitamin D (Vit D) in the endocrine system, especially its potential anti-inflammatory and immune-modulating properties, there is an increased interest in its potential role in the prevention and control of diabetes mellitus.
Objective: This study evaluated the potential therapeutic efficacy of Vit D in averting the detrimental effects of both types of diabetes mellitus.
Materials and methods: A total of 50 male Wistar rats were allotted into five groups: a placebo group; a nongenetic model of type 1 diabetes in rats (T1D), injected with a single dose of streptozotocin (STZ; 65 mg/kg, ip); a nongenetic model of type 2 diabetes in rats (T2D), given a short-term high-fat diet followed by a single low dose of STZ (35 mg/kg, ip); fourth and fifth groups that were gastrogavaged with Vit D (10 IU/kg) three days after the induction of T1D and T2D, respectively, which was continued daily throughout the experiment.
Results: Vit D (10 IU/kg/60 days) significantly (p < 0.05) decreased fasting plasma glucose, ketoacidosis (decreased non-esterified fatty acid and β-hydroxyl butyric acid), pro-inflammatory interleukin-6, HbA1c in T1D and T2D and insulin resistance index by 33% in T2D. Interestingly, Vit D significantly (p < 0.05) increased fasting plasma insulin by 144% in T1D, plasma Ca level, insulin sensitivity index, and β-cell function index in T1D and T2D.
Discussion and conclusion: Vit D ameliorated the deleterious biochemical impact of diabetes mellitus, likely by increasing insulin secretion and sensitivity, ameliorating the β-cell function, and decreasing the number of pro-inflammatory cytokines and insulin resistance.
Introduction
Type 1 (T1D) and type 2 (T2D) diabetes are considered multifactorial diseases in which both genetic predisposition and environmental factors participate in their development. Diabetes is ranked as the fifth leading cause of death in the United States, and the number of people with diabetes in the world are expected to approximately double between 2000 and 2030 (Danescu et al., Citation2009). In light of these data, it is imperative to discover and implement preventive measures to address this growing epidemic. Vitamin D (Vit D, calciferol) is a steroid-derived fat-soluble vitamin obtained from food, but most people achieve their Vit D needs (85–90%) by endogenous synthesis through direct ultraviolet B-mediated synthesis in the skin (Mohr et al., Citation2008). By the action of UVB light (290–315 nm), the B ring of 7-dehydrocholesterol (pro-vitamin D) can be broken to form precholecalciferol (pre-vitamin D), which rapidly isomerizes to Vit D in a thermosensitive process (Bikle, Citation2009). Vit D itself is biologically inert and requires two successive hydroxylation reactions to be activated. The first hydroxylation occurs in the liver and is conducted by 25-hydroxylases, which convert Vit D into 25(OH) D. The second occurs mainly in the kidneys and is conducted by 1α-hydroxylase, which converts 25(OH) D to 1,25(OH) 2D, the active form of Vit D (Mathieu et al., Citation2005). The production of 1,25(OH) 2D in the kidney is strictly regulated by several factors, such as 1,25(OH) 2D itself, PTH, and the serum concentrations of calcium and phosphate (Norman, Citation2008). Many extrarenal tissues also express 1α-hydroxylase, including pancreatic islets, antigen-presenting cells, liver, and skeletal muscles (Brannon et al., Citation2008). These cells are capable of producing local concentrations of 1,25(OH) 2D, which have autocrine and paracrine actions. This extrarenal production plays an important role in the modulation of immune responses and the regulation of cell differentiation, proliferation, and apoptosis (Baeke et al., Citation2008). The 1α-hydroxylase present in extrarenal tissues is identical to the renal 1α-hydroxylase, but the regulation of its expression is different. The extrarenal 1α-hydroxylase is primarily regulated by immune signals from interferon-γ and other cytokines (Mathieu et al., Citation2005). The Vit D-binding protein (DBP) binds and transports Vit D and its metabolites in plasma. Only 1,25(OH) 2D is metabolically active and exerts its effects mainly by activating the Vit D receptor (Vit DR) which is a member of the nuclear receptor superfamily of ligand-activated transcription factors (Bikle, Citation2009). The binding of 1,25(OH) 2D to Vit DR leads to the transcription of regulated genes (over 200 genes). These Vit DRs are not only expressed in classical Vit D responsive target tissues, but are also expressed in a broad range of other tissues (Norman, Citation2008). The discovery that Vit DRs are widely expressed in the immune system, led to the recognition of the central immunomodulatory role for 1,-25(OH) 2D (Baeke et al., Citation2008). The objective of this study is to evaluate the potential therapeutic efficacy of Vit D in averting the detrimental effects of both types of diabetes mellitus and to determine the possible mechanisms involved.
Materials and methods
Chemicals
STZ and other reagent kits were purchased from Sigma Chemicals (St Louis, MO). Vitamin D (cholecalciferol) (Vidrop®) was obtained from Medical Union Pharmaceuticals (Abu Sultan, Ismailia, Egypt), the recommended dose is 10 IU/kg (Pittas, Citation2006).
Animals
A total of 50 male Wistar rats (six-weeks-old) obtained from the Department of Animal Science, Faculty of Science, were used in this experiment. They were kept in rat cages in a well-ventilated house, with a temperature range of 27–30 °C, and a 12 h natural light and 12 h darkness cycle, with free access to tap water and dry rat pellets. They were allowed to acclimate for 15 days prior to the experiment. The animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), and the study protocol was approved by the local authorities. Rats were sorted into five equal groups of 10 rats in each group: a placebo group was injected ip with citrate buffer (pH 4.5); a nongenetic model of T1D in rats was injected with a single dose of streptozotocin (STZ; 65 mg/kg, ip); a nongenetic model of T2D in rats was given a short-term HFD feeding followed by a single low dose of STZ (35 mg/kg, ip); a fourth group was gastrogavaged with Vit D as vidrop® (10 IU/kg) three days after the induction of T1D, which was continued daily throughout the experiment; and a fifth group was gastrogavaged with Vit D as vidrop® (10 IU/kg) three days after the induction of T2D, which was continued daily throughout the experiment. The experimental period lasted for 60 days. The induction of T1D and T2D was monitored by the measurement of the plasma glucose and insulin levels before starting the experiment.
Samples
Blood samples (3 ml) were obtained from the orbital venous sinus of overnight-fasted placebo, diabetic-control, and diabetic-treated rats (10–12 h). Ethical protocol of sample collection was achieved as approved by the Ethical Committee. These samples were collected in heparinized tubes and divided into two portions. The first one was centrifuged at 3000 rpm for 10 min to separate the plasma. FPG was estimated immediately, and the residual plasma was stored at −20 °C for other biochemical assays. The second portion of the blood sample was used for the determination of HbA1c. Successive blood samples were then collected from all groups at 30, 60, 90, and 120 min following the administration of a glucose solution (3 g/kg b.wt.) orally by gastric intubation. The blood samples were centrifuged, and the plasma was obtained for the determination of the glucose concentration and insulin level. Plasma glucose was assayed according to Trinder (Citation1969), and nonesterified fatty acid (NEFA) and β-hydroxyl butyric acid (BHBA) were analyzed according to Duncombe (Citation1964). Commercial radioimmunoassay kits were used to measure the concentration of insulin (Wilson & Miles, Citation1977). Interleukin-6 was measured using a commercial immunoassay kit, according to Bazan (Citation1990). HbA1c was assayed according to Jeppsson et al. (Citation2002). The plasma Ca level was measured according to Gindler and King (Citation1972). Insulin resistance was evaluated by a homeostasis model assessment of insulin resistance (HOMA-IR) according to Matthews et al. (Citation1985); this index largely reflects hepatic IR. The ISOGTT index, which is a measure of insulin sensitivity, was assayed according to Matsuda and DeFronzo (Citation1999); this index reflects whole-body insulin sensitivity. β-Cell function was calculated according to Wareham et al. (Citation1995), which is a widely used measure of β-cell function.
Statistical analysis
The results are expressed as the mean ± SE. Data analysis was performed using one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered statistically significant.
Results
Changes in plasma glucose, insulin, insulin sensitivity, insulin resistance, and β-cell function
The induction of T1D significantly (p < 0.05) increased the plasma glucose levels by approximately 280% and significantly (p < 0.05) decreased the plasma insulin level and β-cell function by approximately 60% and 64%, respectively, compared to rats in the control group. The induction of T2D significantly (p < 0.05) increased the plasma glucose levels by 245% and insulin resistance by approximately 200%, and significantly (p < 0.05) decreased insulin sensitivity by 50% compared to the placebo group. In contrast, supplementation with Vit D ameliorated and mitigated the adverse effects associated with DM and significantly (p < 0.05) decreased the plasma glucose concentrations in T1D and T2D and insulin resistance by approximately 33% in T2D, with significant (p < 0.05) increase in the plasma insulin levels by approximately 144% in T1D, insulin sensitivity, and β-cell function of the pancreas in T1D and T2D ().
Table 1. Effect of Vit D on FPG, FPI, insulin sensitivity, insulin resistance, and β-cell function in T1D and T2D.
Table 2. Effect of Vit D on plasma glucose during OGTT in T1D and T2D.
Table 3. Effect of Vit D on plasma insulin levels during OGTT in T1D and T2D.
Changes in nonesterified fatty acids and β-hydroxy butyric acid
shows that the induction of T1D and T2D significantly (p < 0.05) increased plasma NEFA and BHBA. The adverse effects associated with both types of DM were ameliorated when co-treated with Vit D, resulting in significant (p < 0.05) decrease in plasma NEFA and BHBA compared to nontreated diabetic rats.
Table 4. Effect of Vit D on NEFA, BHBA, Ca, IL-6 and HbA1c% in T1D and T2D.
Changes in plasma calcium, interleukin-6, and glycosylated hemoglobin
As shown in , the induction of T1D and T2D significantly (p < 0.05) increased glycosylated hemoglobin, interleukin-6, and significantly (p < 0.05) decreased the plasma Ca level compared to the placebo group. However, co-administration of Vit D significantly (p < 0.05) decreased glycosylated hemoglobin, interleukin-6, and significantly (p < 0.05) increased the plasma Ca level in T1D and T2D.
Discussion
The above findings suggest the role of Vit D in both the occurrence and treatment of DM (Forouhi et al., Citation2008). The results of this study revealed that Vit D supplementation regulates glucose/insulin homeostasis and is able to increase the β-cell function and insulin sensitivity while decreasing insulin resistance, the number of proinflammatory cytokines, and ketoacidosis associated with diabetes mellitus, as shown in . The obtained results are consistent with previous observations, as the observed effects of Vit D supplementation on glycemic control included a deceased glycosylated hemoglobin concentration in Vit D-replete, T1D patients and an improved disposition index in adults at high risk of T2D (Kayaniyil et al., Citation2011; Mitri et al., Citation2011a). Vit D intake >500 IU/day decreased the fasting glucose and the risk of T2D by 13% compared to vitamin D intake <200 IU/day, and individuals with the highest Vit D status (>25 ng/ml) had a 43% lower risk of T2D compared to those in the lowest group (<14 ng/ml); (Danescu et al., Citation2009; Mitri et al., Citation2011b).
A small-scale analysis revealed that daily calcium (500 mg) and Vit D (700 IU) supplementation for three years prevented a further increase in fasting blood glucose in a subgroup with impaired fasting blood glucose (100–125 mg/dl) at baseline (Pittas et al., Citation2006, Citation2007). Additionally, from data obtained in an observational study, the authors suggested that by extrapolation, Vit D replenishment in Vit D-deficient healthy adults may improve plasma insulin levels more compared to either rosiglitazone or metformin treatment (Chiu et al., Citation2004). Moreover, in an experimental animal model of T2D (obese Wistar rats) without Vit D deficiency, Vit D was found to decrease plasma glucose concentrations by as much as 40% (de Souza Santos & Vianna, Citation2005). The maternal serum 25-hydroxy Vit D3 concentrations in both the GDM and IGT groups at 24–28 weeks of gestation were significantly lower than that of the non-GDM controls and inversely related to the fasting glucose and insulin concentrations (Daga et al., Citation2012; Nikooyeh et al., Citation2011).
Palomer et al. (Citation2008) found that Vit D replenishment improves glycemia and insulin secretion in patients with T2D and established hypovitaminosis D. Boucher (Citation2011) reported that the mechanism of action of Vit D in T2D is thought to be mediated not only through the regulation of calcium trafficking in β-islet cells – which regulate insulin synthesis, secretion, sensitivity, and high calcium intake and that have been found to be inversely associated with body weight and fatness – but also through direct action on pancreatic β-cell function mediated by the binding of the active form of 1,25-dihydroxyvitamin D to the vitamin D receptor, which is expressed in β-cells in which the presence of the Vit D response element in the human insulin gene promoter and transcriptional activation of the human insulin gene caused by 1,25-dihydroxyvitamin D further supports a direct effect of Vit D on insulin synthesis and secretion. Alternatively, the activation of Vit D may occur within β-cells through 25(OH) d-1α-hydroxylase (CYP27B1), which is expressed in β-cells.
In peripheral insulin-target cells, active Vit D metabolites may enhance insulin sensitivity in several ways, including through an increase in the expression of insulin receptors (Maestro et al., Citation2002), the activation of transcription factors important in glucose homeostasis (Dunlop et al., Citation2005), or indirectly via the regulation of calcium, which is essential for insulin-mediated intracellular processes. Takiishi et al. (Citation2010) revealed that T1D and T2D patients have a higher incidence of hypovitaminosis D and pancreatic tissue (more specifically, the insulin-producing beta-cells), and they also reported that numerous cell types of the immune system express Vit DR and DBP and that some allelic variations in genes involved in Vit D metabolism and Vit DR are associated with glucose (in) tolerance, insulin secretion, sensitivity, and inflammation. The same authors showed that pharmacologic doses of 1,25-dihydroxyvitamin D (1,25(OH) (2) D) prevent insulitis in mice, possibly through immune modulation as well as through direct effects on β-cell function. Likewise, 1-α,25(OH)(2)D(3) prevents T1D in animal models, modifies T-cell differentiation, modulates dendritic cell action, and induces cytokine secretion, shifting the balance to regulatory T cells (Mathieu et al., Citation2005).
Vit D has recently been associated with several contributing factors known to be linked to the development of T2D, including defects in pancreatic β-cell function, insulin sensitivity, and systemic inflammation (Danescu et al., Citation2009; Jódar-Gimeno & Muñoz-Torres, Citation2012). A study conducted by Bourlon et al. (Citation1999) showed that 1,25(OH) 2D could activate the de novo biosynthesis of insulin in islets from Vit D-deficient rats after stimulating the islets with glucose, which increases the rate of conversion of proinsulin to insulin. Critical analysis of mice with an engineered deletion of the Vit DR revealed an increased sensitivity to autoimmune diseases, such as inflammatory bowel disease or T1D, after exposure to predisposing factors (Bouillon et al., Citation2008). Other epidemiological data have associated Vit D deficiency and an increased prevalence of these autoimmune diseases with, for example, a three-fold increase in T1D when the Vit D deficiency was present in early life (Hypponen et al., Citation2001). Furthermore, it has been suggested that in Vit D deficiency, the 1,25-(OH) 2D-mediated attenuation of pathological Th1 immune responses is impaired, thus explaining the increased risk for Th1-mediated autoimmune diseases (Peterlik & Cross, Citation2005). Similarly, a 1,25-(OH) 2D shift from a Th1 to a Th2 cytokine expression profile locally in the pancreas and in the pancreas-draining lymph nodes has been observed (Overbergh et al., Citation2000). The same authors revealed that when 1,25-(OH) 2D is administered in mice of older age having autoimmune β-cell destruction, it could still block diabetes progression and induce the immune shift in response to autoantigens but not to disease-irrelevant self or foreign antigens.
Other studies have demonstrated a protective effect of 1,25-(OH) 2D (incubation periods ranging between 48 and 72 h) or its analogs on cytokine-induced β-cell dysfunction and death (Isaia et al., Citation2001; Riachy et al., Citation2001). In healthy subjects, the plasma levels of cytokine TNF-α are inversely related to glucose oxidative metabolism and whole glucose disposal (Paolisso et al., Citation1998). The same authors revealed that Vit D metabolites downregulate this cytokine. Vit D deficiency may also impair insulin secretion through its associated increase in PTH levels. It was proposed that Vit D deficiency-associated hyperparathyroidism may actually cause a paradoxical increase in the intracellular calcium level [Ca]I, and this PTH-induced increase in [Ca]i may in turn impair the calcium signal required for glucose-induced insulin secretion (Fujita & Palmieri, Citation2000).
In contrast to the present study, 1,25-dihydroxyvitamin D treatment (1 μg/d for 4 days) had no effect on fasting or stimulated glucose, insulin, C-peptide, or glucagon concentrations in 35 diabetic subjects (Orwoll et al., Citation1994). Taylor and Wise (Citation1998) reported that Vit D supplementation in three cases of British Asians with Vit D deficiency and T2D led to increased insulin resistance and deterioration of glycemic control. Wu et al. (Citation2009) suggested that calcium intake or systemic Vit D status, after adjusting for the intake of dairy products, is associated with decreased insulin secretion in men and women. There is no clinical impact of hypovitaminosis D on metabolic or glycemic control or on markers of systemic inflammation in Chinese T2D, and the correction of Vit D deficiency would not have great clinical utility on glucose tolerance or insulin sensitivity as a diabetes therapeutic agent in established diabetes (Luo et al., Citation2009; Tai et al., Citation2008).
The discrepancy in the results is difficult to account for, but this discrepancy may be explained by different populations studied, a possibility that there may be a different response to Vit D among different ethnic groups and the existence of DNA sequence variations (polymorphisms) for the Vit DR gene that may account for a variability in the endocrine action of Vit D (Palomer et al., Citation2008) or differences in methodological approaches.
Conclusion
Vit D ameliorated and mitigated the deleterious biochemical impacts associated with both types of diabetes mellitus, most likely by increasing insulin secretion and sensitivity, ameliorating the β-cell function, and decreasing the number of pro-inflammatory cytokines and insulin resistance. Remarkably, the increased plasma Ca level, which is essential for insulin-mediated intracellular processes, could attribute partly to the antidiabetic activities of Vit D. All these improvements may decrease the metabolic disruptions associated with DM, including ketoacidosis.
Acknowledgements
We are grateful for the assistance of the Faculty of Veterinary Medicine, Damanhur University. Authors acknowledge the scholars whose articles are cited and included in references of this manuscript.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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