| Part One GLP-1RA promotes brown adipogenesis of C3H10T1/2 mesenchymal stem cellsObjective: Previous studies have revealed the vital role of glucagon-like peptide-1 receptor agonist(GLP-1RA)in weight loss due to stimulate adipocyte browning.In this study,we investigated the contribution of the differentiation to brown adipocytes from C3H10T1/2 mesenchymal stem cells(MSCs)promoted by one of the GLP-1 receptor(GLP-1R)agonist--liraglutide.The role of PI3K/AKT/mTOR signaling pathway was also studied in this process.Methods: C3H10T1/2 MSCs were induced to differentiate into mature brown adipocytes and treated with liraglutide(10 nM and 100 nM)with or without PI3 K inhibitor 10μmol LY294002,the control group was not intervened by liraglutide and(or)LY294002.The cell apoptosis and proliferation were determined by Annexin V method and Cell Counting kit-8.The Oil red O staining was used for lipid droplet staining.The quantitative realtime PCR and western blot were employed to determine the expression of adipogenic(PRDM16,PPARγ,PGC-1α,CIDE-A,UCP-1)and mitochondrial genes(CytoC,Cox II),mitochondrial DNA(mtDNA).Western blot analyses were used for quantification of protein levels in PI3K/AKT/mTOR signaling pathway.Results: 1.Comparing with control group,the apoptosis of brown adipocytesdecreased and the viability of cells increased gradually with the increase of the concentration of liraglutide.In the 4,6,8 days of cell differentiation,the multilocular lipid droplets intervened by 10 nM liraglutide were more than control group,and the Oil red O stainings were also higher(P < 0.05 for all).In the2,4,6,8 days of cell differentiation,the multilocular lipid droplets intervened by 10 nM liraglutide were more than control group,and the Oil red O stainings were also higher(P < 0.05 for all).2.In the cell differentiation,comparing with control group,the expressions of brown adipocyte specific(PRDM16,PPARγ,PGC-1α,CIDE-A,UCP-1)and mitochondrial genes(CytoC,Cox II),mtDNA were promoted gradually by the inceasing concentration of liraglutide(P < 0.05 for all).3.Liraglutide treatment increased the levels of phosphorylated AKT and mTOR(P < 0.05 for all)during differentiation from C3H10T1/2 MSCs to brown adipocytes comparing with control group,and the phosphorylated AKT and mTOR were more higher in 100 nM liraglutide group than 10 nM liraglutide and control group(P < 0.05 for all).4.LY294002 not only attenuated differentiation of C3H10T1/2 MSCs into brown adipocytes,but also reduced Oil red O staining,the expressions of brown adipocyte specific(PRDM16,PPARγ,PGC-1α,CIDE-A,UCP-1)and mitochondrial genes(CytoC,Cox II),mtDNA,phosphorylated AKT and mTOR levels comparing with control group(P < 0.05 for all).5.However,co-treatment with liraglutide and LY294002,the Oil red O staining,expression of adipogenic(PRDM16,PPARγ,PGC-1α,CIDE-A,UCP-1)and mitochondrial genes(CytoC,Cox II),mtDNA,and phosphorylated AKT and mTOR levels were decreased compared to C3H10T1/2MSCs only treated with liraglutide 100nM(P < 0.05 for all).Conclusion: In the differentiation from C3H10T1/2 MSCs to mature brown adipocytes,GLP-1RA not only inhibited apoptosis of cells,increased proliferation of cells and formation of multilocular lipid droplets during differentiation,but alsopromotes brown adipogenesis of C3H10T1/2 mesenchymal stem cells,and the PI3K/AKT/mTOR signaling pathway is involved in GLP-1RA-mediated promotion of differentiation.Part Two Effects of GLP-1RA on fat distribution and pancreatic β-cell function in type 2 diabetes patients with visceral obesityObjective: Comparing with Western patients of type 2 diabetes,Chinese diabetic patients are not obese.But Chinese nonobese adults(BMI < 25 kg/m~2)have a greater number of visceral obesity.GLP-1RA can control blood glucose and weight,protect pancreatic β-cells,decrease visceral and hepatic fat depots in obese type 2diabetes(T2DM)and prediabetes.The aim of this research was to investigate the effect of fat distribution and pancreatic β-cells function of GLP-1RA(exenatide)on T2 DM patients with visceral obesity.Research Methods: Forty-nine nonobese T2 DM patients with visceral obesity were enrolled,and all subjects were intervened and treated for 24 weeks.Before and after intervention,weight,waist circumference,BMI,systolic blood pressure,diastolic blood pressure,fasting plasma glucose(FPG),2h postprandial blood glucose(2h PBG),glycated hemoglobin(Hb A1c),lipids,liver and kidney function,FGF-21 and 100g steamed bread test were measured.The subcutaneous and visceral adipose tissue(SAT,VAT)were measured by abdominal MRI,liver fat content(LFC)was measured by1H-MRS.The indexes of Matsuda and MBCI were calculated to evaluate insulin sensitivity and pancreatic β cell function.The t-test of paired samples,the non-parametric Wilcoxon test and symbol rank test,the mixed-effect model controlling for age and sex,and the Pearson correlation analysis were used to statistical analyses.Results: 1.The data of 40 completers were analyzed finally.Comparing with basic status,after intervention of 24 weeks,FPG(9.14 ± 2.41 mmol/L vs 8.01 ± 2.03mmol/L),2h PBG(15.95 ± 4.28 mmol/L vs 14.38 ± 3.37 mmol/L),Hb A1c(8.43 ±1.06 % vs 7.05 ± 1.04 %)decreased significantly(P < 0.05 for all).2.After intervention,there was no significant change of Matsuda index,but the MBCI index increased significantly(5.44 ± 3.33 vs 7.27 ± 5.04)(P = 0.045).3.Compaing with the data of pre-treatment,VAT(80.56 ± 34.26 cm~2 vs 66.82 ± 30.07 cm~2),LFC(22.96 ±3.02 % vs 9.83 ± 2.38 %)and SAT(129.85 ± 43.73 cm~2 vs 114.18 ± 44.39 cm~2)all decreased significantly(P < 0.01 for all).5.Compaing with the data of pre-treatment,FGF-21 decreased(372.79 ± 244.56 pg/ml vs 198.64 ± 147.02 pg/ml)(P< 0.01),and positively correlated with LFC(r = 0.248,P = 0.027).Conclusions: Exenatide could not only improve glycaemic metabolism,control weight,enhance pancreatic β-cell function,but also decrease visceral and subcutaneous adiposity,liver fat content in T2 DM patients with visceral adiposity. |
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