Effect And Mechanism Of Vitamin D₃ On Islet β Cell Function In Prediabetes Population

BackgroundPrediabetes,an intermediate state between normoglycemia and diabetes mellitus（DM）,is a critical period for preventing the development of the type 2 diabetes mellitus（T2DM）.Early intervention in people with prediabetes could reduce the risk of T2DM by 40-70%.Vitamin D is an essential fat-soluble micronutrient with various biological activities.Observational studies suggested that vitamin D deficiency might be a risk factor for impaired insulin secretion and T2DM.However,results of randomized controlled trials（RCT）on the relationship between vitamin D supplementation and glucose metabolism are still equivocal,and well-designed RCTs are needed to validate these findings.Mechanically,chronic hyperglycemia drives an increased rate of proinsulin synthesis,which imposes a synthesis burden on isletβcells,and increases the likelihood of protein misfolding and endoplasmic reticulum stress（ERS）.Previous studies indicated that vitamin D might play a role in inhibiting ERS,and we hypothesized that vitamin D could improveβcell dysfunction by inhibiting pancreatic ERS.Therefore,we performed an RCT in the Chinese population with prediabetes to determine the effect of vitamin D on pancreaticβ-cell function,and established the DM rat model to explore the possible mechanism.Objectives1.To evaluate the effect of vitamin D₃ and calcium supplementation alone or in combination on pancreaticβ-cell function,insulin secretion,and insulin sensitivity in the Chinese population with prediabetes.2.To explore the effects of vitamin D₃ supplementation on bioavailable 25（OH）D（bio-25（OH）D）and free 25（OH）D levels,and the association of bio-25（OH）D and free25（OH）D with the improvement ofβcell function.3.To assess the protective effect of vitamin D₃ on pancreaticβ-cell function,and investigate whether the potential mechanism is related to ERS induced apoptosis.Methods1.Effect of vitamin D₃ and/or calcium supplementation on pancreaticβ-cell function in subjects with prediabetes:a randomized,placebo-controlled trialThis was a 24-week,randomized,placebo-controlled trial conducted in Xuchang City,Henan.All potential subjects underwent the 75g oral glucose tolerance test（OGTT）.Based on the inclusion and exclusion criteria,243 subjects with prediabetes were randomly assigned to receive 1600 IU/daily vitamin D and/or 500 mg/daily calcium for 24 weeks（vitamin D group,n=60;calcium group;n=60;vitamin D+calcium,n=61;placebo group,n=62）.The participants completed the comprehensive questionnaire survey,physical examination,OGTT,and blood sample collection.Levels of glucose,lipid,insulin,PTH,25（OH）D,albumin,and vitamin D binding protein（VDBP）were detected.We selected the disposition index（DI）as the primary endpoint to evaluate isletβcell function,and calculated the levels of bio-25（OH）D and free 25（OH）D according to the published and accepted formulas.The effects of vitamin D₃ and calcium were evaluated by repeated measures of variance analysis and generalized estimated equation.Multiple linear regression was used to analyze the association between DI and the levels of bio-25（OH）D and free 25（OH）D.The study protocol was approved by the Life Science Ethics Committee of Zhengzhou University,and signed informed consent were provided by all participants.2.The effect and mechanism of vitamin D₃ in improving islet function in diabetic rats by inhibiting endoplasmic reticulum stressFour-week-old specific pathogen-free male SD rats（n=72）were randomly divided into normal control group（NC group,n=10）and diabetic model group（n=62）.The rats in NC group were given a normal chow diet for the whole process,while the rats in the model group were fed with a high-fat diet for four weeks.Then,rats in the model group were intraperitoneally injected with 2%STZ at a dose of 35 mg/kg to establish the diabetic rat model.We randomly divided the DM rats into model control group（MC,n=10）,low-dose vitamin D group（LVD,41.67 IU/kg,n=10）,medium-dose vitamin D group（MVD,166.67 IU/kg,n=10）,high-dose vitamin D group（HVD,520.83 IU/kg,n=10）and positive control group（PC,metformin 200 mg/kg,n=10）.Rats in NC and MC groups were given 2 m L/kg corn oil daily.After administration of vitamin D₃ for ten weeks,all rats were anesthetized,and blood samples were collected from abdominal aorta.Pancreatic tissues were immediately removed and stored frozen.We detected the levels of FPG,lipids,insulin,and 25（OH）D in blood samples,and quantified the protein expressions of VDR,GRP78,IRE1,p-IRE1,CHOP,Bax,and Bcl-2 in pancreatic tissues by Western blot.Immunohistochemistry was used to evaluate the content of insulin in islets,and TUNEL assay was employed to score the apoptosis of pancreatic tissue.One-way ANOVA was used to compare the differences of these quantitative indicators among various groups.Results1.Effect of vitamin D and/or calcium supplementation on pancreaticβ-cell function in subjects with prediabetes:a randomized,placebo-controlled trial（1）Two hundred and forty-three subjects with prediabetes（mean age 56 years,65.84%females）were enrolled in the RCT.After 24 weeks of intervention,a total of212 subjects were followed up,with a follow-up rate of 87.2%.At baseline,there was no significant difference among the four groups in both demographic and clinical characteristics（P>0.05）.After the intervention,25（OH）D levels in the vitamin D+calcium group and the vitamin D group were significantly improved（P<0.001）.（2）After 24 weeks of intervention,compared with those who did not receive vitamin D supplement,levels of TC（change=-0.13 mmol/L,95%CI:-0.22,-0.03;P=0.011）and LDL-C（change=-0.09 mmol/L,95%CI:-0.18,0.00;P=0.039）were significantly decreased in participants supplemented with vitamin D.（3）Compared with placebo group,the insulin secretion index（IGI）was significantly improved in vitamin D+calcium（change=0.28,95%CI:0.10,0.47;P=0.003）and calcium group（change=0.20,95%CI:0.02,0.39;P=0.033）;however,there were no significant differences in the changes of insulin sensitivity indexes among four groups（P>0.05）.（4）After 24 weeks of intervention,the DI of vitamin D+calcium group was significantly higher than that at baseline（6.61±0.80 vs.6.40±0.71,P=0.032）.Meanwhile,there was a marginal increase of DI in the vitamin D+calcium group compared with placebo（change=0.21,95%CI:-0.02,0.43;P=0.068）,which was more evident in older and baseline vitamin D insufficient subjects.（5）At baseline,25（OH）D level was highly positively correlated with free25（OH）D（r=0.793,P<0.001）and bio-25（OH）D（r=0.770,P<0.001）.After 24 weeks of intervention,the levels of free 25（OH）D and bio-25（OH）D in the vitamin D+calcium group and vitamin D group were significantly higher than those in the placebo group（P<0.001）.（6）At 24 weeks,the change of DI was positively correlated with the evolution of free 25（OH）D（β=0.013,P=0.044）,but not with the shift of 25（OH）D levels（P>0.05）.2.The effect and mechanism of vitamin D in improving islet function in diabetic rats by inhibiting endoplasmic reticulum stress（1）A stable and effective DM rat model was established by high-fat diet combined with low-dose STZ.Typical characteristics of diabetes（polydipsia,polyuria,more food,weight loss）were also observed in the DM rats.Our study showed that supplementation with vitamin D could alleviate the symptoms of polydipsia and weight loss in DM rats（P<0.05）.（2）The improvement of 25（OH）D levels in DM rats showed a dose-response relationship with the increase of vitamin D dose（P<0.05）.Compared with the MC group,FPG and TG levels in the HVD group were significantly decreased（P<0.05）,while HOMA-βand HDL-C were significantly increased（P<0.05）.（3）The results of HE staining showed that compared with the MC group,the number of islets tended to be increased,and the cells in islets tended to be intact in all the vitamin D intervention groups.The results of insulin immunohistochemistry indicated that insulin levels in islets of DM rats were obviously increased in MVD and HVD groups（P<0.05）.TUNEL staining suggested that vitamin D₃ could significantly improve the apoptosis of pancreatic tissue in DM rats（P<0.05）.（4）Compared with the MC group,SOD levels in serum and pancreas of DM rats were increased in MVD and HVD groups（P<0.05）,while levels of MDA reduced significantly（P<0.05）.In addition,serum TNF-αand IL-1βlevels in MVD and HVD groups were also substantially decreased（P<0.05）.（5）Results of western blot showed that compared with the MC group,vitamin D could significantly promote the expression of VDR and Bcl-2（P<0.05）,and inhibit the expression of GRP78,p-IRE1/IRE1,CHOP,and Bax（P<0.05）.Conclusions1.Vitamin D₃ supplementation for 24 weeks could effectively reverse vitamin D deficiency and regulate lipid metabolism in prediabetic subjects;while combination with calcium,the insulin secretion could improve significantly.Especially,there was a noticeable improvement ofβ-cell function after vitamin D₃ and calcium treatment in participants with vitamin D insufficiency.2.Bio-25（OH）D and free 25（OH）D were better indicators of vitamin D status than serum 25（OH）D levels.The effect of vitamin D₃ intervention onβ-cell function in prediabetic subjects was due to the increased circulating free 25（OH）D levels rather than 25（OH）D levels.3.Vitamin D₃ played a beneficial role in alleviating symptoms of DM rats（polydipsia and weight loss）,reducing blood glucose and lipids,and improving pancreaticβcell function in a dose-dependent manner.4.Vitamin D₃ might improve isletβ-cell function by inhibiting ERS induced apoptosis through the IRE1/CHOP signaling pathway,and play an important role in anti-inflammatory and antioxidant processes.