Abstract
Introduction. It was found that vitamin D may have a direct effect on adipocyte differentiation and metabolism and might be involved in the glucose regulation of insulin secretion, as suggested from the discovery of a nuclear localization of 1,25-(OH)2D3 in pancreatic islets. In recent years, several polymorphisms in the VDR gene which are able to alter the activity of VDR protein have been described. The BsmI and FokI polymorphisms were described in relation to obesity and type 2 diabetes.
The aim of the study was to find whether there are associations between BsmI and FokI polymorphisms and anthropometric (BMI, WHR, BP) and biochemical parameters describing metabolic syndrome.
Materials and methods. Studied were 176 randomly selected men aged 25–65 years (mean: 51.99 years) with a mean BMI of 28.06 kg/m2. Two polymorphisms of the VDR gene (FokI and BsmI) were explored using the PCR-RFLP method. Serum glucose, insulin, total cholesterol, LDL, HDL, and TG were measured using commercially available kits.
Results. It was found that BB carriers tend to have higher BMI (29.00 ± 3.74 versus 26.81 ± 3.76, p = 0.024) and waist circumference (101.79 ± 10.59 versus 96.23 ± 10.35, p = 0.014) compared with the bb genotypes. Similarly, FF and Ff carriers had higher fasting insulin levels than the ff genotypes (12.30 ± 10.26 versus 9.76 ± 5.88, p = 0.001 and 9.76 ± 5.88 vs. 6.35 ± 2.64, p = 0.008), and lover cHDL levels in comparison to ff genotypes (52.28 ± 10.02 versus 60.63 ± 16.58, p = 0.015 and 53.70 ± 12.03 versus 60.63 ± 16.58, p = 0.032. Besides these, no significant differences were found.
Conclusions. The BsmI VDR polymorphism seems to influence BMI, while the FokI VDR polymorphism appears to affect insulin sensitivity and serum cHDL level.
Introduction
Among the fundamental biological effects of the action of the active form of vitamin D, i.e. 1,25(OH)(2)D(3), are the control of calcium and bone metabolism and the regulation of the proliferation and differentiation of many cell types, in addition to its immunoregulatory, antiangiogenic, and antioxidant properties Citation[1-4]. It was also found that vitamin D might be involved in glucose homeostasis by influencing the regulation of insulin secretion Citation[2],Citation[4,5].
The pleiotropic effect of vitamin D action is mediated by the binding of the active metabolite 1,25 (OH)(2)D(3) to a specific cytosolic/nuclear vitamin D receptor (VDR) and by non-genomic pathways through a membrane receptor and second messengers Citation[2]. The gene encoding VDR is located in the long arm of chromosome 12 (locus 12q12-q14) Citation[1,2].
In recent years, several fragment polymorphisms in the VDR gene which are potentially able to alter the activity of VDR protein and which occur frequently in the population have been described Citation[1],Citation[6]. They are associated with a variety of physiological and pathological phenotypes in many populations. Among these polymorphisms, the FokI vitamin D receptor gene start codon polymorphism and the BsmI vitamin D receptor gene polymorphism located in intron 8 play important roles Citation[1,2]. Both polymorphism are useful genetic markers in determining BMD and the risk of osteoporosis and have been described in relation to obesity and type 2 diabetes mellitus Citation[1,2],Citation[5],Citation[7,8].
The aim of our study was to find whether there is an association between the BsmI and FokI polymorphisms and anthropometric and biochemical parameters characterizing metabolic syndrome.
Materials and methods
Study group
The study group consisted of 176 unrelated randomly selected men aged 25–65 years (mean age: 51.99 ± 10.73 years) from the population of Wroclaw. A complete physical examination, which also included medical history, was performed by a physician for all subjects. The patients were informed about the aim and method of the study. The study protocol was approved by the Bioethics Committee of the Medical University of Wroclaw, Poland.
At baseline, anthropometric measurements such as age, weight, height, waist and hip circumference, body mass index (BMI), waist-to-hip ratio (WHR), and diastolic and systolic blood pressure (BP) were taken for all subjects. The clinical characteristics of the study population are presented in .
Table I. Characteristic of the study group
Biochemical measurements
Blood samples were obtained in the morning from the antecubital vein for analysis of serum glucose, insulin, total cholesterol, HDL cholesterol (cHDL), LDL cholesterol (cLDL), and triglyceride (TG) concentrations. The biochemical parameters were measured according to standard enzymatic methods using commercially available kits.
Genetic polymorphism analysis
Genomic DNA was obtained from the leukocytes of the peripheral blood by phenol extraction. The blood samples were collected by venepuncture and frozen in the presence of EDTA. The two polymorphisms of the VDR gene (FokI and BsmI) were explored using the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method. Genomic DNA (10 ng) was amplified in a final 20 µl reaction mixture containing: 10 µl RedTaqReadymix PCR solution (20 mM Tris-HCL, 100 mM KCL, 3 mM MgCl2, 0.002% gelatin, 0.4 mMdNTP solution, 0.06 DNA polymerase U Taq/µl) (Sigma), 5.4 µl ddH2O, 2 µl Q-solution (Qiagen), and 0.3 pmol of each starter.
In the case of the FokI polymorphism, the starter pair used was F (forward): 5'-AGCTGGCCCTGGCACTGACTCTGCTCT-3’ and R (reverse): 5'-AT GGAAACACCTTGCTTCTTCTCCCTC-3’, and in the case of BsmI it was F (forward): 5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3’ and R (reverse): 5'-AACCAGCGGGAAGTCAAGGG-3’. The PCR conditions were 30 cycles: denaturation at 94°C for 60 s, binding at 55°C for 1 min, extension at 72°C for 3 min, and 1 cycle final extension at 72°C for 7 min. The products of the PCR reaction were digested using the respective restriction enzymes for FokI (INC Biomedicals Inc) for 3 hours at 37°C and BsmI (Mva 12691, MBI Ferments) overnight at 37°C. Electrophoretic separation of the obtained fragments was carried out in 2% agarose gel.
The following allelic variations were observed:
for the FokI polymorphism, a 265-bp fragment for the uncleaved allele (called “F”) and 196-bp and 69-bp fragments for the cleaved allele (called “f”)
for the BsmI polymorphism, an 870-bp fragment for the uncleaved allele (called “B”) and 640-bp and 230-bp fragments for the cleaved allele (called “b”).
Statistical analysis
The results are given as means and standard deviations in the tables. One-way analysis of variance (ANOVA) was used to examine a variability of variables in genotypes. Differences between groups (genotypes) were assessed using the Tukey's LSD test (post-hoc test). Allelic frequencies were estimated by gene counting and genotype distribution of the polymorphism and were tested against Hardy-Weinberg equilibrium by Chi-square analysis. In all statistical comparisons, p < 0.05 was defined as significant. All statistical calculations were performed using Statistica 6.0 and Microsoft Excel software.
Results
The mean age of examined men was 51.99 ± 10.73 years, mean BMI 28.06 ± 4.34 kg/m2, mean weight 84.81 ± 13,45 kg, mean hip circumference 103.8 ± 8.71 cm, mean waist circumference 99.38 ± 11.25 cm, mean WHR 0.913 ± 0.08, mean systolic blood pressure 132.16 ± 17.17 mmHg, and mean diastolic blood pressure 84.77 ± 12.88 mmHg ().
The prevalence of each genotype in the study population was: BB 11.38% (n = 19), Bb 59.88% (n = 100), bb 28.74% (n = 48) and FF 31.21% (n = 54), Ff 59.54% (n = 103), ff 9.25% (n = 16) ( and ).
Table II. Mean values of the examined parameters according to BsmI polymorphism (analysis of variance)
Table III. Mean values of examined parameters according to FokI polymorphism (analysis of variance)
We found that BB carriers tended to have statistically significant higher BMI than did the bb genotypes (29.00 ± 3.74 versus 26.81 ± 3.76, p = 0.024). Moreover, we observed that BB carriers had greater waist circumference than the Bb and bb genotypes (101.79 ± 10.59 versus 96.23 ± 10.35, p = 0.014). Similarly, FF and Ff carriers had significantly higher fasting insulin levels compared with the ff genotypes (12.30 ± 10.26 versus 9.76 ± 5.88, p = 0.001, and 9.76 ± 5.88 versus 6.35 ± 2.64, p = 0.008). In addition, we found an association of the FokI polymorphism with cHDL levels. Serum cHDL concentrations were significantly lover in patients with FF and Ff genotypes compared with those with the ff genotypes (52.28 ± 10.02 versus 60.63 ± 16.58, p = 0.015 and 53.70 ± 12.03 versus 60.63 ± 16.58, p = 0.032).
Beside these, no significant differences were found. Other anthropometrics and biochemical parameters, such as diastolic and systolic blood pressure, body mass, WHR, and the concentrations of total cholesterol, cLDL, and TG, were similar in the carriers of the different genotypes. In contrast to cHDL levels, a great difference in glucose concentration in the men depending on the FokI polymorphism was not statistically significant (p = 0.082) ().
In the case of both polymorphisms the observed departures from Hardy-Weinberg equilibrium were not significant (for FokI Chi2 = 11.08, p = 0.004, for BsmI Chi2 = 9.07, p = 0.01).
Discussion
The vitamin D endocrine system is without doubt involved in a wide variety of biological processes. Variations in this endocrine system can lead to several common chronic diseases, such as osteoarthrosis, diabetes, cancer, tuberculosis, and cardiovascular disorders Citation[3],Citation[6]. The gene for the vitamin D receptor (VDR) is one of the genes that has been most extensively studied, mainly in relation to BMD, due to the important role of vitamin D in bone metabolism. It is expressed in a large number of different tissues Citation[1],Citation[7],Citation[9]. Among the many types of cells that respond to vitamin D are adipocytes and pancreatic beta-cells Citation[4,5],Citation[8]. Thanks to this, VDR genotype can function as a determinant of body composition and also play an important role in glucose homeostasis and in the mechanism of insulin release Citation[4],Citation[7,8],Citation[10].
In this report we demonstrated relationships between BsmI and FokI vitamin D receptor gene polymorphisms and anthropometric and biochemical parameters describing metabolic syndrome in a male population. These findings indicate that the BsmI VDR polymorphism seems to influence BMI and the FokI polymorphism insulin sensitivity and cHDL concentration. Additionally, we found great differences in glucose levels in men depending on the FokI polymorphism, but they were not statistically significant. In the case of triglyceride, LDL and total cholesterol levels and systolic and diastolic blood pressure values we found no statistically significant correlations with the particular genotypes.
These results allow us to suggest that genetic variations in the VDR gene correlate with fat mass and body weight. Other authors have reached similar conclusions. Grundberg et al., for example, noted that individuals with the BB genotype were characterized by greater body weight and fat mass compared with subjects with the bb genotype Citation[9]. Sweeney et al. observed trends for an association between the VDR FokI ff genotype and greater WHR Citation[11]. It should be stressed that both studies investigated groups of women. For comparison, our results showed that men with the BB genotype had statistically significant greater waist circumference and BMI compared with those having the Bb and bb genotypes. These observations support the influence of VDR gene polymorphism status on the development of the abdominal obesity typical of metabolic syndrome. Unfortunately, there are few publications related to this issue.
Genetic variations in the VDR gene can also play an important role in the pathogenesis of type 2 diabetes mellitus. This assumption is in accordance with previously described findings of other authors. It is widely known that vitamin D participates in the regulation of insulin secretion and can influence insulin sensitivity Citation[4,5],Citation[7,8]. Scragg et al. indicated a possible role of VDR allelic variations in the mechanism of glucose homeostasis Citation[12]. Based on results of an epidemiological study, these authors suggested that a high level of vitamin D is associated with increased insulin sensitivity and with a decreased risk of type 2 diabetes Citation[12]. In a study by Ortlepp et al. analysing the BsmI polymorphism it was observed that VDR genotype is associated with altered fasting glucose levels in young men Citation[13]. Similarly, Oh and Barrett-Connor indicated on the basis of their results that the BsmI polymorphism may be associated with insulin resistance in a non-diabetic Caucasian population Citation[10].
VDR is not a major candidate gene for type 2 diabetes mellitus, but it can be related to the susceptibility to obesity in subjects with early-onset type 2 diabetes Citation[7]. Different data were reported by Malecki et al. Their examination of BsmI, TagI, and FokI VDR polymorphism did not provide evidence for their association with type 2 diabetes mellitus in a Polish population Citation[14]. The pathophysiological mechanism of the connection between allelic variations of the VDR gene and type 2 diabetes mellitus development still remains unclear.
Among many other types of cells, VDR is also expressed on preadipocytes Citation[4],Citation[7],Citation[9]. Thereby, vitamin D can be directly involved in the regulation of adipocyte differentiation and metabolism and indirectly by its modulation of insulin secretion. Lenoir et al. showed in vitro on the Ob 17 cell line that vitamin D inhibits the differentiation of preadipocytes Citation[15]. Kawada et al. had similar results Citation[16]. Moreover, other authors have also described a possible impact of vitamin D on lipoprotein lipase expression Citation[17,18].
Abdominal type of obesity and insulin resistance are the main factors which lead to the development type 2 diabetes. Many studies have indicated the relationship of obesity with a higher risk of vitamin D deficiency Citation[4,5],Citation[7]. It is speculated that an association of VDR polymorphism with obesity in type 2 diabetic subjects can be related to an allelic modulation of insulin secretion Citation[4]. It has been demonstrated that vitamin D deficiency induces type 2 diabetes through impaired insulin secretion involving VDR on pancreatic beta cells Citation[19].
Vitamin D and its receptor, which is expressed on many cell types of the immune system, play an important role in the pathogenesis of type 1 diabetes mellitus Citation[4],Citation[20,21]. This probably results from a reduction in the immunomodulatory action of vitamin D Citation[19]. The role of the VDR gene as a genetic determinant that leads to autoimmune destruction of pancreatic beta cells and, further, to the development of type 1 diabetes mellitus is still debated.
The role of VDR gene polymorphisms in the regulation of blood pressure and volume homeostasis it is not well known. Experimental studies have demonstrated that a lack of vitamin D action leads to hypertension in mice. This observation has been supported by data on humans. Li et al. have suggested that vitamin D is a potent endocrine suppressor of rennin biosynthesis through a VDR-dependent mechanism regulating the rennin-angiotensin system, which plays a central role in the regulation of blood pressure Citation[22]. Some researches have shown that vitamin D can reduce blood pressure in hypertensive patients Citation[23]. The influence of the BsmI vitamin D receptor gene polymorphism and 25-hydroxyvitamin D on arterial pressure in healthy individuals was examined by Muray et al. Citation[24,25].
They observed that healthy men with higher levels of vitamin D had higher levels of systolic and diastolic blood pressure. Moreover, men with the bb genotype had the highest levels of systolic blood pressure, but the blood pressure values in women were not influenced by vitamin D or by BsmI genotype. These data allow us to assume that the pathophysiological interaction between vitamin D and sex hormones plays an important role in the control of blood pressure Citation[24,25]. The contribution of genetic factors to the development of hypertension has also been confirmed in studies carried out by Kulah et al. Citation[26]. They evaluated the relationships of target organ damage and 24-hour ambulatory blood pressure monitoring with the vitamin D receptor gene FokI polymorphism in patients with essential hypertension. Their results indicated a negative correlation between vitamin D level and blood pressure values in the group of patients with the ff/Ff genotype. Additionally, on the basis of retinal examination these authors showed that the degree and presence of retinopathy were significantly higher in Ff/ff carriers compared with FF individuals Citation[26]. In contrast to the cited studies, our findings did not indicate significant dependencies of diastolic and systolic blood pressure on particular genotypes.
Nevertheless, there is clear evidence of relationships between vitamin D deficiency and high values of blood pressure. However, functional, clinical studies are necessary to establish the significance of the vitamin D endocrine system in the pathogenesis of blood pressure. The low-calcemic vitamin D analogues may be used as a new class of anti-hypertensive agents to control rennin production and blood pressure in the future. However, knowing the status of VDR gene polymorphisms will be helpful in preventing target organ damage in hypertensive patients.
In summary, we conclude that vitamin D receptor polymorphisms play important biological roles, notably in calcium metabolism, but also in glucose homeostasis and the regulation of insulin secretion. The results of our study suggest an association between BsmI VDR polymorphism and susceptibility to obesity and between FokI VDR polymorphism and insulin sensitivity. A limitation of this report is the small population of only male patients.
Therefore, more genetic epidemiological studies in larger populations of both men and women are needed for a better understanding of the relationship between VDR variations and various phenotypes for insulin secretion, insulin sensitivity, and anthropometric data and their potential clinical implications.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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