小G蛋白二磷酸鸟苷解离刺激因子调节小鼠脂肪细胞肥大及糖代谢紊乱的初步研究

Effects of small GTP-binding protein GDP dissociation stimulator on adipocyte hypertrophy and glucose metabolism disorder in mice

目的探讨小G蛋白二磷酸鸟苷解离刺激因子(SmgGDS)在肥胖发展中的作用及机制。方法(1)8周龄C57BL/6J小鼠购自扬州大学比较医学中心,随机分为正常饮食组和高脂饮食组,每组6只,分别喂食普通饲料和含60%脂肪的高脂饲料4个月,Western印迹检测附睾脂肪组织(eWAT)、肝脏和骨骼肌中SmgGDS表达。(2)6周龄的野生型(WT)和SmgGDS敲低(KD)小鼠分为4组,分别给予高脂饮食4个月(每组7只)和7个月(每组9只),进行葡萄糖耐量试验(GTT)和胰岛素耐受试验(ITT);记录小鼠体重、脂肪组织和肝脏重量;苏木精-伊红(HE)染色分析脂肪组织的结构改变;Western印迹检测eWAT中细胞外信号调节激酶(ERK)1/2的磷酸化水平;实时荧光定量聚合酶链式反应(RT-qPCR)检测eWAT中成脂分化相关基因CCAAT/增强子结合蛋白α(C/EBPα)、C/EBPβ和过氧化物酶体增殖物激活受体γ(PPARγ)的mRNA水平。(3)提取WT和KD小鼠的胚胎成纤维细胞(MEF)并诱导分化,通过油红O染色检测脂滴形成情况;Western印迹检测SmgGDS和ERK磷酸化水平;RT-qPCR检测C/EBPα、C/EBPβ、PPARγ的mRNA水平。(4)选取10周龄C57BL/6J小鼠随机分为2组,每组7只,分别腹腔注射SmgGDS过表达腺相关病毒(AAV-SmgGDS)和对照空载体,同时高脂饮食干预4周,进行GTT和ITT;记录小鼠体重、脂肪组织重量;HE染色分析eWAT结构改变;Western印迹检测eWAT中ERK磷酸化水平。结果(1)高脂饮食组小鼠eWAT中SmgGDS表达明显上调(正常饮食组:0.218±0.037,高脂饮食组:0.439±0.072,t=2.74,P=0.034)。(2)在高脂饮食干预4个月时,KD小鼠的葡萄糖耐量[葡萄糖注射后60 min,WT组(528±21)mg/dl,KD组(435±17)mg/dl,t=3.47,P=0.030;90 min,WT组(463±24)mg/dl,KD组(366±18)mg/dl,t=3.23,P=0.047;120 min,WT组(416±21)mg/dl,KD组(297±16)mg/dl,t=4.49,P=0.005]和胰岛素敏感性(胰岛素注射后15 min,WT组77.79%±3.45%,KD组54.30%±2.92%,t=3.49,P=0.005;30 min,WT组62.27%±5.31%,KD组42.25%±1.85%,t=2.98,P=0.024;90 min,WT组85.69%±6.63%,KD组64.71%±5.41%,t=3.12,P=0.016)较WT组明显改善,eWAT重量比增加(WT组4.19%±0.18%,KD组5.12%±0.37%,t=2.28,P=0.042),但平均脂肪细胞面积减小[WT组(5221±241)μm^(2),KD组(4410±196)μm^(2),t=2.61,P=0.026]。高脂饮食干预7个月后,KD组eWAT重量比减小(WT组5.02%±0.20%,KD组3.88%±0.21%,t=3.92,P=0.001)、平均脂肪细胞面积减小[WT组(6783±390)μm^(2),KD组(4785±303)μm^(2),t=4.05,P=0.002];eWAT中ERK1磷酸化水平升高(WT组0.174±0.056,KD组0.588±0.147,t=2.64,P=0.025),PPARγ的mRNA水平显著降低(WT组1.018±0.128,KD组0.029±0.015,t=7.70,P=0.015)。(3)在分化的MEF中SmgGDS表达显著增加(未分化:6.789±0.511,分化:10.170±0.523,t=4.63,P=0.010);敲低SmgGDS抑制MEF的脂滴形成(WT组1.00±0.02,KD组0.88±0.02,t=5.05,P=0.007),增加ERK1(WT组0.600±0.179,KD组1.325±0.102,t=3.52,P=0.025)和ERK2(WT组2.179±0.687,KD组5.200±0.814,t=2.84,P=0.047)活性,该结果可被ERK1/2抑制剂逆转。(4)SmgGDS过表达使小鼠体重增加,eWAT重量增加(对照组3.29%±0.36%,AAV-SmgGDS组4.27%±0.26%,t=2.20,P=0.048)、脂肪细胞增大[对照组(3525±454)μm^(2),AAV-SmgGDS组(5326±655)μm^(2),t=2.26,P=0.047],胰岛素敏感性受损(胰岛素注射后30 min,对照组44.03%±4.29%,AAV-SmgGDS组62.70%±2.81%,t=3.06,P=0.019),eWAT中ERK1(对照组0.829±0.077,AAV-SmgGDS组0.326±0.036,t=5.96,P=0.001)和ERK2(对照组5.748±0.287,AAV-SmgGDS组2.999±0.845,t=3.08,P=0.022)活性降低。结论SmgGDS敲低通过抑制成脂分化和脂肪细胞肥大改善肥胖相关的糖代谢紊乱,且与ERK活化有关。

Objective To explore the effect and mechanism of small GTP-binding protein GDP dissociation stimulator(SmgGDS)on the development of obesity.Methods(1)8-week-old C57BL/6J mice were randomly assigned to normal diet and high fat diet group,with 6 mice in each group.They were fed regular feed and a high fat diet containing 60%fat for 4 months,respectively.The expression of SmgGDS in epididymal adipose tissue(eWAT),liver,and skeletal muscle were measured using Western-blot.(2)6-week-old wild-type(WT)and SmgGDS knockdown(KD)mice were divided into four groups,each receiving high fat diet for 4 months(7 in each group)and 7 months(9 in each group).Glucose tolerance test(GTT)and insulin tolerance test(ITT)were conducted;the weight,adipose tissue,and liver weight of mice were recorded;HE staining examined adipose tissue structural changes;Western-blot determined extracellular signal-regulated kinase(ERK)1/2 phosphorylation levels in eWAT;real time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect mRNA levels of CCAAT/enhancer binding proteinα(C/EBPα),C/EBPβand peroxisome proliferator activated receptorγ(PPARγ)in eWAT.(3)Mouse embryonic fibroblasts(MEFs)extracted from WT and KD mice were induced for differentiation.Oil red O staining and Western-blot were used to detect lipid droplet and expression of SmgGDS and phospho-ERK;C/EBPα,C/EBPβand PPARγmRNA levels were measured using RT-qPCR.(4)10-week-old C57BL/6J mice were randomly assigned into two groups,with 7 mice in each group.Mice were infected with SmgGDS overexpressing adeno-associated virus(AAV-SmgGDS)or empty vector intraperitoneally,then fed with high fat diet.After 4 weeks,performed GTT and ITT;recorded the weight and adipose tissue weight of mice;HE staining was used to analyze structural changes of eWAT;Western-blot was used to detect the phosphorylation level of ERK in eWAT.Results(1)The expression of SmgGDS was significantly upregulated in eWAT of high fat diet fed mice(normal diet group:0.218±0.037,high fat diet group:0.439±0.072,t=2.74,P=0.034).(2)At 4 months of high fat diet intervention,the glucose tolerance(60 minutes after glucose injection,WT group:528 mg/dl±21 mg/dl,KD group:435 mg/dl±17 mg/dl,t=3.47,P=0.030;90 minutes,WT group:463 mg/dl±24 mg/dl,KD group:366 mg/dl±18 mg/dl,t=3.23,P=0.047;120 minutes,WT group:416 mg/dl±21 mg/dl,KD group:297 mg/dl±16 mg/dl,t=4.49,P=0.005)and insulin sensitivity(15 minutes after insulin injection,WT group:77.79%±3.45%,KD group:54.30%±2.92%,t=3.49,P=0.005;30 minutes,WT group:62.27%±5.31%,KD group:42.25%±1.85%,t=2.98,P=0.024;90 minutes,WT group:85.69%±6.63%,KD group:64.71%±5.41%,t=3.12,P=0.016)of KD mice were significantly improved compared to the WT group,with an increase in eWAT weight ratio(WT:4.19%±0.18%,KD:5.12%±0.37%,t=2.28,P=0.042),but a decrease in average adipocyte area(WT group:5221μm^(2)±241μm^(2),KD group:4410μm^(2)±196μm^(2),t=2.61,P=0.026).After 7 months of high fat diet,the eWAT weight ratio of KD mice decreased(WT:5.02%±0.20%,KD:3.88%±0.21%,t=3.92,P=0.001)and adipocyte size decreased(WT group:6783μm^(2)±390μm^(2),KD group:4785μm^(2)±303μm^(2),t=4.05,P=0.002).The phospho-ERK1 in eWAT increased(WT group:0.174±0.056,KD group:0.588±0.147,t=2.64,P=0.025),and mRNA level of PPARγsignificantly decreased(WT group:1.018±0.128,KD group:0.029±0.015,t=7.70,P=0.015).(3)The expression of SmgGDS was significantly increased in differentiated MEF(undifferentiated:6.789±0.511,differentiated:10.170±0.523,t=4.63,P=0.010);SmgGDS knock-down inhibited lipid droplet formation in MEF(WT group:1.00±0.02,KD group:0.88±0.02,t=5.05,P=0.007)and increased ERK1(WT group:0.600±0.179,KD group:1.325±0.102,t=3.52,P=0.025)and ERK2(WT group:2.179±0.687,KD group:5.200±0.814,t=2.84,P=0.047)activity,which can be reversed by ERK1/2 inhibitor.(4)SmgGDS over expression resulted in weight gain,increased eWAT weight(control group:3.29%±0.36%,AAV-SmgGDS group:4.27%±0.26%,t=2.20,P=0.048)and adipocyte size(control group:3525μm^(2)±454μm^(2),AAV-SmgGDS group:5326μm^(2)±655μm^(2),t=2.26,P=0.047),impaired insulin sensitivity(30 minutes after insulin injection,control group:44.03%±4.29%,AAV-SmgGDS group:62.70%±2.81%,t=3.06,P=0.019),and decreased ERK1(control group:0.829±0.077,AAV-SmgGDS group:0.326±0.036,t=5.96,P=0.001)and ERK2(control group:5.748±0.287,AAV-SmgGDS group:2.999±0.845,t=3.08,P=0.022)activity in eWAT.Conclusion SmgGDS knockdown improves obesity related glucose metabolism disorder by inhibiting adipogenesis and adipose tissue hypertrophy,which is associated with ERK activation.