CaMK Ⅱ参与调解运动诱导的MEF2/GLUT4 DNA结合活性及GLUT4表达研究

The Effects of Exercise in the Binding of MEF2A to the Binding Sites on the Glut4 Gene and Glut4 Expression by CaMK Ⅱ-dependent Mechanism

研究运动激活的CaMK Ⅱ对骨骼肌细胞核内MEF2/GLUT4 DNA结合活性及骨骼肌细胞内GLUT4基因表达量的调节作用。方法:两月龄C57BL/6J小鼠40只,按体重随机分为安静对照(control,C)、运动组(exercise,E)、运动+KN93组(KN93,93)、运动+KN92(KN92,92)组。Western Blotting法测CaMK II THR286磷酸化水平。EMSA法测MEF2/GLUT4 DNA结合活性。实时荧光定量PCR法测骨骼肌GLUT4 mRNA表达量。结果:1)运动+KN93组小鼠1 h跑台运动后,其骨骼肌细胞核内MEF2/GLUT4 DNA结合活性及骨骼肌细胞内GLUT4基因表达量,均显著低于单纯运动组。运动+KN92组小鼠1 h跑台运动后,骨骼肌CaMKⅡ活性,MEF2/GLUT4 DNA结合活性及骨骼肌细胞内GLUT4基因表达量与单纯跑台运动组相比均无显著差异。2)虽然运动+KN93组小鼠1 h跑台运动后骨骼肌GLUT4基因表达量显著低于单纯运动组,但与安静对照组相比,仍显著增加。结论:虽然CaMK Ⅱ参与调节运动诱导的MEF2/GLUT4 DNA结合活性及骨骼肌细胞内GLUT4基因表达量的升高,但CaMKⅡ并不是调节运动诱导的GLUT4基因和蛋白表达增多的唯一信号通路,机体还存在其他的调节信号机制。

Objective：The role of CaMK Ⅱ in regulating the exercise induced MEF2A binding to the GLUT4 promoter,and GLUT4 mRNA expression was investigated.Methods： Forty C57BL/6J mice,two months old,were randomly divided into four groups： control group（C,n=10）,treadmill exercise group（E,n=10）,treadmill exercise with KN93 injection group（93,n=10）,treadmill exercise with KN92 injection group（92,n=10）.CaMK Ⅱ-Thr286 phosphorylation by western blot.Bound MEF2A at the GLUT4 MEF2 cis-element by EMSA.GLUT4 expression by Real Time-PCR.Results：1）Compared with controls,exercise caused a twofold increase in CaMK II phosphorylation.GLUT4 mRNA increased 1.8-fold after exercise.2）The amount of MEF2A that bind to the site increased approximately twofold post-exercise.The exercise-induced increases in CaMK II phosphorylation,MEF2A binding,and GLUT4 expression were attenuated or abolished when KN93 was administered to mice prior to exercise.KN92 did not affect the increases in pCaMK II and GLUT4.Conclusion： These data support the hypothesis that exercise increases the binding of MEF2A to their binding sites on the Glut4 gene and Glut4 expression by CaMK Ⅱ-dependent mechanism.