| Background:Recent studies have provided new insights into the function of vitamin D. In addition to the classical effects in maintaining calcium homostasis, vitamin D has been shown to be closely associated with immune system, cell's proliferation and differentiation, and other endocrine glands. Accumulating evidence suggests vitamin D play an important role in decreasing the risk of many chronic illnesses, including common cancers, autoimmune diseases, infectious diseases, and cardiovascular disease. Moreover, vitamin D deficiency was also found to be associated with metabolic syndrome (MS), but it was still a controversial topic. In addition, whether vitamin D deficiency can lead to the development of obesity, the basic component of metabolism syndrome, and whether it have direct effect on lipid metabolism are not clearly defined.Objective:1) To detect the association of vitamin D deficiency and metabolism syndrome in the clinical case control study. To investigate the changes in serum 25(OH)D concentration after the metabolic disorders improved, and to explore if vitamin D deficiency is a risk factor for metabolic syndrome.2) To explore the effects of 1,25(OH)2D3 on the 3T3-L1 cell proliferation and differentiation in vitro study.Methods:1.Clinical study.1) The case control study. One hundred and seventy MS patients and 63 healthy people from Pinggu district Beijing were incruited in this study. The healthy people as control group were of normal serum glucose concentration, blood pressure, and BMI. Biochemical parameters and authropometry were evaluated. And serum 25(OH)D concentrations were compared between the subjects and controls to explore the association of serum 25(OH)D and MS.2) The prospective intervention study. Life style intervention including diet instruction and exercises were conducted in the MS patients for one year, and some of them received medication therapy for hypertension and diabetes. Biochemical parameters and authropometry were reevaluated after one year intervention to explore the association of MS and serum 25(OH)D concentration.2. The in vitro study.1) In order to study the effect of 1,25(OH)2D3 on the proliferation of 3T3-L1 preadipocytes, the cell were cultured with AIMV serum-free medium for seven days. Thereafter, the 3T3-L1 cells proliferation was determined by MTT spectrophotometry.2) In order to investigate the effects of 1,25(OH)2D3 on the differentiation of 3T3-L1 preadipocytes, the cells were cultured with medium containing insulin, dexamethasone and IBMX to induce differentiation. After 12 days, red oil O staining and dye extraction as well as intracellular triglyceride assays were performed to determine the amount of intracellular lipid, which can serve as a marker of adipocyte differentiation.3) the 3T3-L1 were collected after 4 days and 8 days cultured in the differentiation medium and after 7 days cultured in the common medium. Then the total RNA was extracted and reverse transcripted into cDNA. After that, the relative mRNA levels were determined by Real-time PCR for genes listed below:gene for proliferation and apoptosis:Cyclin D1, Bax and BCL-2; transcription factors critical for the maintenance of differentiation:PPARγ,CEBP/a;critical enzymes for lipid metabolism: ACC1,ACS1,G3DPH and HSL; adipokines:adiponectin,visfatin.Results:1.Clinical study. The percent of vitamin D deficiency was higher in the patients with MS than those without MS (90.1% vs 79.4%,OR=2.192, P=0.021). MS was significantly associated with BMI and age[r=1.857(1.549,2.226), P＜0.0005, r=1.126 (1.066,1.188), P＜0.0005]. No association was found between MS and serum 25(OH)D and parathyroid hormone (PTH) concentration. The serum 25(OH)D concentration of obesity was lower than people without obesity (13.97±4.17 vs 15.22±4.80 ng/ml, P=0.041). Multiple line regression demonstrated that serum 25(OH)D concentration was negative associated with high density lipoprotein cholesterol(HDL-C)(B=-0.188, P=0.025), total cholesterol (TC)(B=-0.189, P=0.025). After one year intervention, the weight, BMI and waist circumference(WC) decreased, at the same time, the serum 25(OH)D concentration increased significantly (14.40±4.03 vs 16.11±4.02 ng/ml, P<0.0005). Multiple line regression demonstrated△25(OH)D was prospective associated with△triglyceride (TG) (B=0.195,P=0.014).2)The in vitro study.1,25(OH)2D3 inhibit 3T3-L1 cell proliferation in dose-dependent manner. When 1,25(OH)2D3 concentration was 10-7M,10-8M and 10-9M, the relative OD value was 39.8±4.0%,48.4±6.9% and 64.3±9.7%(P＜0.05). When the concentration fell to less than 10-10M, the proliferation inhibition effect decreased dramatically, with relative OD value of 79.8±9.9%(P＜0.05). Both semi-quantitative red oil O stain method and triglyceride assays showed that 1,25(OH)2D3 inhibited the differentiation of 3T3-L1 preadipocytes in dose-dependent manner. At the 12th day, the intracellular triglyceride content decreased by 13.3% and 9.8% respectivelywhen the 1,25(OH)2D3 concentration was 10-8M and 10-9M (P=0.013,P=0.027) compared with control group, and the effect disappeared when the concentration was less than 10-10M. When 3T3-L1 preadipocytes exposed to 1,25(OH)2D3 at 10-8M, the relative mRNA concentration of Cyclin Dl and transcript factors PPARy and C/EBPa reduced significently, and the critical lipid synthesis enzymes G3PDH, ACC1, and ACS1 increased dramatically, while the critical lipid catabolic enzymes HSL reduced remarkably, and the adipokines Adiponectin and Visfatin reduced significantly. When the 1,25(OH)2D3 concentration was 10-10M, the effects disappeared.Conclusion:1. The percent of vitamin D deficiency was higher in the patients with MS than those without MS. MS was positively associated with BMI and age. No association was found between MS and serum 25(OH)D and parathyroid hormone (PTH) concentration. After one year intervention, the serum 25(OH)D concentration increased following the decrease of FBG, weight,WC, BMI and TG.2.1,25(OH)2D3 can inhibit 3T3-L1 preadipocytes proliferation and differentiation in does-dependent manner, possibly through the inhibition effect it exert on the Cyclin D1 and transcription factors PPARy and C/EBPa. 1,25(OH)2D3 can stimulate the critical lipid synthesis enzymes G3PDH,ACC1,ACS1 expression and inhibit the critical lipid catabolic enzymes HSL and the adipokines Adiponectin and Visfatin expression. |
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