目的:研究不同强度跑台运动对AMPKα2三种不同基因状态鼠MEF2/GLUT4DNA结合活性及GLUT4基因表达的影响,以探讨运动提高骨骼肌MEF2/GLUT4DNA结合活性及GLUT4基因表达可能机制。方法:野生小鼠和AMPKα2高表达转基因小鼠,AMPKα2基因敲除小鼠各30只,分别随机分为安静对照组、低强度(12m/min)跑台运动组和高强度(20/min)跑台运动组。运动时间均为1h。运动后3h取材。Westernblot法测定AMPK(THr72)磷酸化水平。CHIP法测定MEF2/GLUT4DNA结合活性。Real-TimePCR法测定GLUT4mRNA含量。结果:1)野生鼠1h不同强度跑台运动后,GLUT4基因表达量显著增加的同时,伴随着骨骼肌核内MEF2/GLUT4DNA结合活性的显著增加;2)AMPKα2高表达转基因鼠1h不同强度跑台运动后,其骨骼肌细胞核内MEF2/GLUT4DNA结合活性及GLUT4基因表达量,均比野生鼠增加更为显著;3)AMPKα2敲除鼠1h不同强度跑台运动后,骨骼肌核内MEF2/GLUT4DNA结合活性显著低于野生鼠,但GLUT4基因表达量与野生鼠相比没有显著差异。结论:1)运动通过提高MEF2/GLUT4DNA结合活性而提高GLUT4表达;2)AMPKα2参与调解了运动诱导的MEF2/GLUT4DNA结合活性及GLUT4基因表达量的升高;3)虽然AMPKα2参与调节了运动诱导的MEF2/GLUT4DNA结合活性的提高,但机体还可以募集其他的信号通路代偿AMPKα2对GLUT4表达的调节作用。
Purpose:The role of AMPK in regulating the exercise induced MEF2A binding to the GLUT4 promoter,and GLUT4 mRNA expression was investigated.Methods:Wild-type(WT;n=30),AMPKα2 over-expression(OE;n=30),and AMPKα2 knockout(KO;n=30) C57/BL mice were randomly divided into three groups:control group,low-intensity(12m/min) group and high-intensity(20m/min) group.Mice were killed after 3h of treadmill running completed by 60 min of continuous running on the experimental day.1) AMPKα-Thr172 phosphorylation by western blot.2) Total and nuclear MEF2A by western bolt.3) Bound MEF2A at the GLUT4 MEF2 cis-e,m lement by CHIP.4) GLUT4 expression by Real Time-PCR and Western blot.Results:Over-expression of AMPKα2,which was associated with higher AMPKα-P in exercise muscles,heightens the exercise-induced increase in MEF2A binding to the GLUT4 promoter and GLUT4 mRNA expression.Exercise-induced increases of GLUT4 mRNA expression,were normal in α2-KO muscles despite the reduced AMPK signaling and GLUT4 promoter-bound MEF2A.Conclusion:1)Exercise increases Glut4 expression by increase the binding of MEF2A to the Glut4 promoter.2)Exercise increases the binding of MEF2A to their binding sites on the Glut4 gene by an AMPK-dependent mechanism.3)AMPK activity during exercise is not indispensable for expression of GLUT4.