Associations between Vitamin D and Type 2 Diabetes Mellitus: The Role of Vitamin D Receptor and Binding Protein

Background: Type 2 diabetes mellitus (T2DM) is a chronic disease that is characterized by β-cell dysfunction and resistance for insulin. Vitamin D is necessary for insulin secretion so it is a crucial factor in the development of T2DM. This study was done to investigate the association between serum 25-hydroxy Vitamin D [25(OH)3D], VDR (Vitamin D receptor) and VDBP (Vitamin D binding protein) with type 2 diabetic patients compared to control subjects. Subjects and Methods: This study carried out 110 female patients who were previously diagnosed with type 2 diabetes and 110 age, sex and weight matched as controls. All participants were subjected to full history taking, clinical examination and assessment of fasting blood glucose, HbA1c , lipid profile, 25-hydroxy Vitamin D [25(OH)3D], VDR and VDBP. Results: Results showed that the level of 25(OH)3D was significantly lower in diabetic group compared to controls and was significantly negatively correlated with glycated hemoglobin, serum total cholesterol and low density lipoprotein cholesterol in type 2 DM. between D and VDBP levels. D deficiency is in diabetic patients and associated with poor control and outcome. This suggests a role of D in the pathogenesis and control of T2DM. Serum VDBP in diabetes may be independent to the level of 25(OH)3D and further


Introduction
T2DM (Type-2 diabetes mellitus) is a chronic disease characterized by both β-cell dysfunction and increased insulin resistance [1]. The prevalence of T2DM continues to rise not only in developing countries, but also in developed countries now [2]. Several genetic and environmental factors can result in the progressive loss of β-cell function that manifests clinically as hyperglycemia. Once hyperglycemia occurs, patients are at risk of developing chronic complications [3]. Vitamin D is one of fat-soluble vitamin with steroid nucleus; it is described as a hormone and acts through intracellular receptors, which belong to the thyroid-steroid receptor superfamily [4].
Total serum levels of 25(OH)3D are a sensitive indicator for Vit D deficiency [5]. Vitamin D deficiency occurs worldwide due to insufficient sunlight and/or dietary intake common in adults. Vitamin D insufficiency is often detected in diabetic patients and it is believed to be linked to the disease development and severity [6].
The main carrier of Vitamin D in the serum is Vitamin D-binding protein (VDBP). It is a low-molecular weight glycoprotein (58 kDa) which significantly predicts the bioavailability of active levels of (25(OH)3D) in the bloodstream [7].
The VDBP/25(OH)D complex formation, its filtration and reabsorption through receptor-mediated uptake in proximal renal tubular cells are vital for activation of Vitamin D [8].
Vitamin D is involved in skeletal development, thyroidal metabolism, immune response regulation, cardiovascular health and glucose-mediated insulin secretion via regulation of insulin receptor expression [9]. There is emerging evidence that low 25(OH)3D levels may be associated with increased risk of the MetS (metabolic syndrome), which represents a cluster of risk factors for type 2 diabetes [10]. Vitamin D supplementation may increase insulin production and secretion by acting via the regulation of the Vitamin D receptors dependent calcium and phosphorus metabolism cascade in pancreatic beta cells [11] [12].
There are several mechanisms proposed to relate the role of Vitamin D with the development of diabetes mellitus. Some of these include expression of Vitamin D receptors in the beta cells of pancreas, role of Vitamin D in maintenance of normal calcium homeostasis which plays a major role in insulin secretion, presence of Vitamin D receptor in skeletal muscle, improvement of insulin mediated glucose utilization following Vitamin D therapy, role of cytokines like Interleukin 6 and TNF alpha (Tumour Necrosis factor alpha) in causing insulin resistance and down regulation of cytokine production by Vitamin D [10]. Vitamin D may act on pancreatic beta cells in two possible pathways; Vitamin D may act directly to induce beta-cell insulin secretion by increasing the intracellular calcium concentration or it may mediate activation of beta-cell calcium-dependent endopeptidases to produce the cleavage that facilitates the conversion of pro-insulin to insulin [13].
In peripheral insulin-target tissues, Vitamin D might directly enhance insulin action through stimulation of the expression of insulin receptors [14].

Subjects
This study was carried out by cooperation between Medical Biochemistry and

Exclusion Criteria
Included patients currently on oral steroid, endocrine disorders or patients on drugs that could have effect on bone (antiepileptic, corticosteroids, antidepressants, Vitamin D and calcium).

Methods
8 ml of venous blood were withdrawn from every subject after a 12 h fasting and dividing into three tubes: One ml of blood was transferred into sodium fluoride containing tube for enzymatic colorimetric determination of blood glucose. was defined as Vitamin D intoxication [20].
The remaining 1 ml from the blood sample was placed in EDTA tube for quantitative colorimetric determination of glycated hemoglobin as percent of total hemoglobin using kits supplied by Teco diagnostics, USA [21].

Statistical Analysis
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. For qualitative data we used number and percent. The Kolmogorov-Smirnov test used to verify the normality of distribution. For quantitative data we used mean, standard deviation and median. Significance of the obtained results was at the 5% level.

Results
Diabetic cases were significantly associated with higher frequency of smoking, hypertension, higher BMI, wider WC, higher HA1C, TC, LDL, FBG and metabolic syndrome. Lower Vitamin D level was significantly associated with DM cases when compared to control group. VDR and VDBP did not differ significantly between both groups (Table 1).
Decreasing Vitamin D level was significantly associated with increasing BMI, WC, HA1C, TC, LDL, as well as with decreeing VDR. No significant association was found between Vit D and VDBP levels when DM cases were classified according to Vitamin D level (Table 2).
There were no significant differences found in Vitamin D level, VDR and VDBP according to studied data (nationality, work, smoking and hypertension) in DM cases (Table 3).
There was significant negative correlation between Vit D level and BMI, WC, HA1C, TC and LDL. There was significant negative correlation between VDR andHA1C, TC and LDL on the other hand; there was significant positive correlation between VDR and Vit D level (Table 4).
There was significant negative correlation VDBP level and VDR. Otherwise, no significant correlations were found between Vitamin D levels, VDR, VDBP with other studied parameters in DM cases (Table 4).   (Table 5).
Smoking, HTN, higher BMI, WC, TC, LDL and lower Vit D were associated with risk of DM in univariable analysis. However, taking significant covariates into multivariable analysis revealed that higher TC, LDL and lower Vit D were considered as independent predictors of T2DM (Table 6).
E. S. Arafat et al.

Discussion
Type 2 diabetes mellitus (T2DM) is a heterogeneous group of disorders resulting from the combination of genetic, behavioral, nutritional, and environmental risk factors. The pathogenesis of T2DM involves deficiency in insulin secretion and insulin resistance [22].
The nutritional risk factors play an important role in pancreatic β-cell physiology and their effects on insulin secretion [23]. Vitamin D is a critical and essential micronutrient for human health; it has a potential effect on pancreatic insulin secretion and insulin action [22].
Our study's aim is to study the association between serum 25-hydroxy Vitamin D [25(OH)3D], VDR &VDBP and type 2 diabetic patients.
In the present study, analysis of the demographic and clinical data of two groups revealed that, smoking, hypertension and obesity were significantly high in diabetic patients group. These results agree with that reported by Mohammad et al. [24]. Several studies reported that elevations of both BMI and waist circumference are associated with increased incidence of diabetes [24] [25]. This is explained by the fact that obesity links to insulin resistance by increasing production of adipokines/cytokines, including tumor necrosis factor-α, resistin and retinol-binding protein that contribute to insulin resistance [26].
In the present study, diabetic cases were significantly associated with higher This result can be explained by the fact that dyslipidemia association with atherosclerosis and the progression of atherosclerosis in diabetes is mainly due to the associated hyperglycemia, obesity and insulin resistance. Excess free fatty acids (FFA) liberation from adipose tissue occurs in T2DM due to insulin resistance. To a large extent lipoproteins hepatic metabolism is controlled by insulin [28].
The co-occurrence of metabolic risk factors in patient group (abdominal obesity, hyperglycemia, dyslipidemia and hypertension) suggested the existence of a metabolic syndrome.
In our study, the level of Vitamin D is significantly lower in type 2 diabetic patients than controls as shown in Figure 1(a). Many studies observed that Vit D level is significantly lower in type 2 diabetes [1] [29] [30]. This result can be explained by the fact that Vitamin D deficiency plays a role in the pathogenesis of T2DM. T2DM manifests as a result of insulin resistance, increased hepatic glucose production and β-cell failure. Vitamin D deficiency increases insulin resistance and decreases insulin secretion [31].
In our study, VDR and VDBP did not differ significantly between both groups as shown in Figure 1 urine as a part of albuminuria that indicates tubular dysfunction in diabetes [32].
In our study, as shown in Figure 2 Vit D level showed significant negative correlation with BMI, WC, Wortsman et al. [33]      concentration was a strong biomarker of diabetes risk [22].
The levels of Vitamin D, Vitamin D binding protein and Vitamin D receptors (VDR) have effect on the role of Vitamin D in stimulating insulin release [41].
Our study confirms the association between Vitamin D deficiency and type 2 diabetes.