敲除MSTN基因可上调骨骼肌胰岛素信号通路并减轻2型糖尿病小鼠胰岛素抵抗

Knockout of MSTN gene up-regulates insulin signaling pathway in skeletal muscle and reduces insulin resistance of mice with type 2 diabe⁃tes mellitus

目的:探究肌肉生长抑制素(myostatin,MSTN)基因敲除对2型糖尿病(type 2 diabetes mellitus,T2DM)小鼠胰岛素抵抗及骨骼肌胰岛素信号通路的影响。方法:将12只野生型(wild type,WT)、12只杂合型(MSTN^(+/−))和12只纯合型(MSTN^(−/−))雄性小鼠随机各分为2组,每组6只,分别为WT组、MSTN^(+/−)组、MSTN^(−/−)组、WT+DM组、MSTN^(+/−)+DM组和MSTN^(−/−)+DM组,前3组小鼠给予普通饮食,后3组小鼠给予高脂饮食及小剂量链脲佐菌素注射,构建T2DM模型。造模后检测小鼠抓力、转棒力竭时间;测定小鼠体质量、体长、腹围、腓肠肌质量、白色脂肪质量并计算腓肠肌比重、白色脂肪比重,测定空腹血糖(fasting blood glucose,FPG)、空腹血清胰岛素(fasting serum insulin,FIns)水平并计算胰岛素敏感指数(insulin sensitivity index,ISI)、稳态模型胰岛素抵抗指数(homeostasis mod-el assessment-insulin resistance index,HOMA-IR);进行葡萄糖耐量实验(glucose tolerance test,GTT)和胰岛素耐量实验(insulin tolerance test,ITT)并测定曲线下面积(area under curve,AUC);HE染色分析腓肠肌细胞形态及横截面积;Western blot法测定胰岛素受体(insulin receptor,InsR)、葡萄糖转运体4(glucose transporter 4,GLUT4)水平及胰岛素受体底物1(insulin receptor substrate 1,IRS1)、磷脂酰肌醇3激酶(phosphatidylinositol 3 kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)和糖原合成酶激酶3β(glycogen synthetase kinase 3β,GSK3β)的蛋白及磷酸化水平。结果:造模后,与WT组比较,WT+DM组小鼠体质量、腹围、腓肠肌比重、抓力、转棒力竭时间、FIns水平、ISI及腓肠肌细胞横截面积均降低(P<0.05),白色脂肪比重、FPG水平、HOMA-IR、GTT-AUC、ITT-AUC均升高(P<0.05),InsR和GLUT4蛋白水平及IRS1、PI3K、Akt和GSK3β磷酸化水平均降低(P<0.05)。与WT组比较,造模后的MSTN^(−/−)组小鼠体质量、体长、腹围、腓肠肌比重、抓力、ITT-AUC、腓肠肌细胞横截面积增大(P<0.05),GTT-AUC减小(P<0.05),GLUT4蛋白水平及PI3K和Akt、GSK3β磷酸化水平升高(P<0.05)。与WT+DM组比较,MSTN^(−/−)+DM组体质量、体长、腹围、腓肠肌比重、抓力、ISI、腓肠肌细胞横截面积增大(P<0.05),白色脂肪比重、HOMA-IR、GTT-AUC和ITT-AUC减小(P<0.05),InsR、GLUT4蛋白水平及IRS1、PI3K、Akt、GSK3β磷酸化水平升高(P<0.05)。结论:抑制MSTN表达可引起肌量、肌力增加以及肌细胞肥大,上调胰岛素信号通路相关分子表达,减轻胰岛素抵抗,可在一定程度上拮抗T2DM对小鼠肌量、肌力及胰岛素信号通路的负性影响,改善糖耐量及胰岛素抵抗。

AIM:To analyze the effect of myostatin(MSTN)gene knockout on insulin resistance and insulin-signaling pathway in skeletal muscle of mice with type 2 diabetes mellitus(T2DM).METHODS:Wild type(WT)mice,MSTN heterozygous(MSTN^(+/−))mice and MSTN homozygous(MSTN^(−/−))mice were randomly divided into WT group,MSTN^(+/−)group,MSTN^(−/−)group,WT+DM group,MSTN^(+/−)+DM group and MSTN^(−/−)+DM group,with 6 in each group.The mice in the first 3 groups were fed with standard chow and the rest were fed with high-fat diet combined with low-dose strep-tozotocin to induce T2DM.Body mass(BM),body length(BL),abdominal circumference(AC),gastrocnemius muscle mass(BMM),white adipose mass(WAM),gripping force,rotating time,fasting plasma glucose(FPG),fasting serum insulin(FIns),tolerance test(GTT)and insulin tolerance test(ITT)in all groups were measured after modeling,and GMM/BM,WAW/BM,insulin sensitivity index(ISI),homeostasis model assessment-insulin resistance index(HOMA-IR)and area under curve(AUC)were calculated.HE staining was used to analyze the gastrocnemius fiber morphology and cross-sectional area.The protein or/and phosphorylated protein levels of insulin receptor(InsR),insulin receptor sub-strate 1(IRS1),phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt),glucose transporter 4(GLUT4),glyco-gen synthase kinase-3β(GSK3β)on insulin signaling pathway in gastrocnemius muscle were measured by Western blot.RESULTS:After modeling,compared with WT group,BM,AC,GMM/BM,gripping force,rotating time,FIns,ISI,cross-sectional area of gastrocnemius fibers,protein levels of InsR and GLUT4,phosphorylation ratios of IRS1,PI3K,Akt and GSK3βwere decreased in WT+DM group(P<0.05),while WAM/BM,FPG,HOMA-IR,GTT-AUC and ITT-AUC were increased in WT+DM group(P<0.05).Compared with WT group,BM,BL,AC,GMM/BM,gripping force,ITT-AUC and cross-sectional area of gastrocnemius fibers,protein level of GLUT4 and phosphorylation ratios of PI3K,Akt,GSK3βwere increased in MSTN^(−/−)group(P<0.05),while GTT-AUC was decreased in MSTN^(−/−)group after modeling(P<0.05).Compared with WT+DM group,BM,BL,AC,GMM/BM,gripping force,ISI and cross-sectional area of gas-trocnemius fibers,protein levels of InsR and GLUT4,phosphorylation ratios of IRS1,PI3K,Akt,GSK3βwere increased in MSTN^(−/−)+DM group(P<0.05),while WAM/BM,HOMA-IR,GTT-AUC and ITT-AUC was decreased in MSTN^(−/−)+DM group(P<0.05).CONCLUSION:Inhibition of MSTN expression results in increases of muscle mass,muscle strength and myocyte hypertrophy,up-regulates the insulin signaling pathway related-molecules and reduces insulin resistance,and antagonizes the negative effect of T2DM on muscle mass,muscle strength and insulin signaling pathway,thus im-proves glucose tolerance and insulin resistance.