SLC16A11调控小鼠骨骼肌Akt/GLUT4通路的研究及运动对SLC16A11和GLUT4表达的影响

The Mechanism for SLC16A11-medilated Akt/GLUT4 Pathway and The Effect of Exercise on Expressions of SLC16A11 and GLUT4

目的:探讨在高脂饮食条件下,腺相关病毒(AAV)介导的SLC16A11表达抑制对小鼠骨骼肌Akt/GLUT4通路的影响,并探讨运动对骨骼肌SLC16A11蛋白表达的影响。方法:将60只4周龄C57BL/6小鼠随机分为普通饮食对照组(Control组)、高脂饮食组(HFD组)和高脂饮食加AAV注射组(HFD+AAV组),每组20只。小鼠喂养8周后经尾静脉注射AAV,其中Control组和HFD组注射阴性对照AAV,HFD+AAV组小鼠注射靶向SLC16A11基因的干扰AAV。AAV注射后两周取材,并在取材前从每组小鼠中随机抽取10只经腹腔注射胰岛素(1 IU/kg),每组剩余10只小鼠注射1×PBS缓冲液。将另外20只4周龄C57BL/6小鼠随机分为安静对照组(Sed组)和运动组(Ex组),每组10只。Ex进行8周跑台运动,20 m/min,1小时/天,5天/周。在小鼠运动干预结束前一周,葡萄糖耐量试验(IPGTT)检测Sed组和Ex组小鼠的葡萄糖耐受性。每周称量各组小鼠体重,采用试剂盒检测Control组、HFD组和HFD+AAV组小鼠骨骼肌内甘油三酯含量,Western Blot检测小鼠骨骼肌内SLC16A11、Akt、p-Aktser473和GLUT4的蛋白表达水平。结果:1)与Control组相比,HFD组小鼠体重显著升高(P<0.05),但HFD组和HFD+AAV组之间无显著差异。与HFD相比,HFD+AAV组小鼠骨骼肌内甘油三酯含量显著减少(P<0.05)。2)与Control组胰岛素注射小鼠相比,HFD组胰岛素注射小鼠骨骼肌内胰岛素刺激的p-Aktser473/Akt比值显著降低(P<0.05);与Control组小鼠相比,HFD组小鼠骨骼肌内GLUT4蛋白表达显著减少(P<0.01);与HFD组相比,HFD+AAV组小鼠骨骼肌内GLUT4蛋白表达水平显著上调(P<0.05)。3)与Sed组相比,Ex组小鼠体重在运动后的第7周显著降低(P<0.05)。此外,与Sed组相比,Ex组小鼠骨骼肌内SLC16A11蛋白表达显著降低(P<0.05),而GLUT4蛋白表达则显著升高(P<0.01)。结论:采用AAV抑制SLC16A11表达可显著改善高脂饮食导致的小鼠骨骼肌Akt/GLUT4通路传导障碍。此外,长期中等强度运动显著下调小鼠骨骼肌内SLC16A11蛋白表达并上调GLUT4蛋白表达。这提示SLC16A11是介导骨骼肌Akt/GLUT4通路的一个潜在靶点,而适度运动可能通过降低SLC16A11表达来改善Akt/GLUT4通路,进而缓解肥胖、糖尿病等代谢性疾病。

Objective To explore the effect of adeno-associated virus(AAV)—mediated inhibition o SLC16A11 expression on the protein kinase B(AKT)/glucose transporter 4(GLUT4) pathway in the skeletal muscle of mice on high-fat diet and the effect of exercise on SLC16A11 protein expression.Methods Sixty four-week-old C57BL/6 mice were randomly divided into a normal diet Control group(Control,n=20),a high-fat diet group(HFD,n=20) and a high-fat diet combined AAV injection group(HFD+AAV,n=20).After eight-week feeding,via tail vein,the Control and HFD groups were injected with negative Control AAV,and HFD+AAV group was injected with positive AAV targeting SLC16A11 gene.Two weeks after the injection,ten mice in each group were randomly selected for intraperitoneal injection of insulin(1 IU/kg) before being sacrificed,and the remaining ten mice were injected with 1×PBS buffer.Still other twenty four-week-old C57BL/6 mice were randomly divided into a sedentary group(Sed,n=10) and an exercise group(Ex,n=10).The Ex mice performed treadmill running for eight weeks,20 m/min,1 h/day,5 days/week.One week before the end of the exercise intervention,the glucose tolerance test(IPGTT) was performed for both the Sed and Ex mice.The body weights were measured for all groups weekly.The triglyceride (TAG) levels of the Control group,HFD group and HFD+AAV group were detected by the kit.The protein expressions of SLC16A11,Akt,p-Akt;and GLUT4 were detected by Western blotting.Results 1) The body weight of HFD mice increased significantly compared with the Control group(P<0.05),while there was no significant difference in that between HFD and HFD+AAV groups.Compared with HFD group,the average TAG level in the skeletal muscle of the HFD+AAV group decreased significantly(P<0.05).2) Compared with the Control group,the insulin stimulated p-Akt;/Akt ratio and the protein expression of GLUT4 of the HFD group decreased significantly(P<0.05).By contrast,the GLUT4 protein expression of the HFD+AAV group increased significantly compared with the HFD group(P<0.05).3) Compared with Sed group,the body weight of Ex group decreased significantly after seven-week running(P<0.05).Moreover,compared with Sed group,the average SLC16A11 protein expression of Ex group decreased significantly(P<0.05),while the average GLUT4 protein expression increased significantly(P<0.01).Conclusion Inhibition of SLC16A11 expression by AAV treatment significantly alleviates the impaired Akt/GLUT4 pathway in the skeletal muscle of mice induced by high-fat diet.Additionally,chronic moderate-intensity exercise inhibits SLC16A11 protein expression and promotes GLUT4 protein expression significantly.Therefore,it is suggested that SLC16A11 is a potential target for the mediation of the Akt/GLUT4 pathway in the skeletal muscle of mice,and exercise may improve the Akt/GLUT4 pathway by decreasing SLC16A11 expression,thus relieving metabolic diseases such as obesity and diabetes.