电刺激促进大鼠骨骼肌细胞GLUT4转位并激活Akt信号通路
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摘要
目的:利用L6细胞模型研究电脉冲刺激对骨骼肌细胞GLUT4转位及其信号通路的影响。方法:将稳定过表达GLUT4myc-AS160的L6-GLUT4myc-AS160大鼠骨骼肌细胞体外培养于6孔培养板中,用含2%胎牛血清的细胞分化液诱导分化为肌管,将6孔板随机分成对照组、胰岛素组和电脉冲刺激(EPS)组,用终浓度为100 nmol/L的胰岛素或电刺激仪对细胞刺激,对照组不做任何处理。ELISA测定细胞膜上的GLUT4myc含量,免疫印记法测定蛋白激酶B(Akt)和Rab-GTPase激活蛋白AS160的磷酸化。结果:与对照组相比,1 h电刺激显著增加细胞膜表面GLUT4myc水平并磷酸化Akt及Rab-GTPase激活蛋白AS160。结论:电刺激促进L6骨骼肌细胞GLUT4myc转位,激活Akt信号通路。
Objective: To study the electric pulse stimulation(EPS)-stimulated signal transduction and GLUT4myc translocation in L6-GLUT4myc-AS160 myotubes. Methods: L6-GLUT4myc-AS160 myoblasts were incubated in 6-well plates, differentiated to myotubes with 2% FBS differentiation media, after which they were divided into 3 groups(EPS, insulin, control) with the first two treated with 100 nmol/L insulin or contract cell and the control group receiving no treatment. The amount of GLUT4myc on the cell surface was measured by ELISA and the phosphorylation of protein kinase B(Akt)and Rab-GTPase AS160 were obtained by immunoblotting. Results: Compared to the control group, EPS stimulated GLUT4myc translocation significantly in 1h and the Akt and AS160 were both phosohorylted by EPS. Conclusion: EPS-induced GLUT4myc can be translocated to the cell surface and activate the Akt signaling pathway.
Objective: To study the electric pulse stimulation(EPS)-stimulated signal transduction and GLUT4myc translocation in L6-GLUT4myc-AS160 myotubes. Methods: L6-GLUT4myc-AS160 myoblasts were incubated in 6-well plates, differentiated to myotubes with 2% FBS differentiation media, after which they were divided into 3 groups(EPS, insulin, control) with the first two treated with 100 nmol/L insulin or contract cell and the control group receiving no treatment. The amount of GLUT4myc on the cell surface was measured by ELISA and the phosphorylation of protein kinase B(Akt)and Rab-GTPase AS160 were obtained by immunoblotting. Results: Compared to the control group, EPS stimulated GLUT4myc translocation significantly in 1h and the Akt and AS160 were both phosohorylted by EPS. Conclusion: EPS-induced GLUT4myc can be translocated to the cell surface and activate the Akt signaling pathway.
引文
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[2]Farmawati A,Kitajima Y,Nedachi T,et al.Characterization of contraction-induced IL-6 up-regulation using contractile C2C12 myotubes[J].Endocr J,2013,60(2):137
[3]Roberts C K,Little J P,Thyfault J P.Modification of insulin sensitivity and glycemic control by activity and exercise[J].Med Sci Sports Exerc,2013,45(10):1868
[4]Lee S,Kim Y.Effects of exercise alone on insulin sensitivity and glucose tolerance in obese youth[J].Diabetes Metab J,2013,37(4):225
[5]Burch N,Arnold AS,Item F,et al.Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle[J].PLoS One,2010,5(6):e10970
[6]Raney M A,Turcotte L P.Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle[J].Am J Physiol Endocrinol Metab,2006,291(6):E1220
[7]Hardie D G.AMPK:A target for drugs and natural products with effects on both diabetes and cancer[J].Diabetes,2013,62(7):2164
[8]Wadley G D,Konstantopoulos N,Macaulay L,et al.Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and shortterm training[J].J Appl Physiol,2007,102(4):1624
[9]Osorio-Fuentealba C,Contreras-Ferrat A E,Altamirano F,et al.Electrical stimuli release ATP to increase GLUT4 translocation and glucose uptake via PI3Kγ-Akt-AS160 in skeletal muscle cells[J].Diabetes,2013,62(5):1519
[10]Wojtaszewski J F,Hansen B F,Gade J,et al.Insulin signaling and insulin sensitivity after exercise in human skeletal muscle[J].Diabetes,2000,49(3):325
[11]Sakamoto K,Hirshman M F,Aschenbach W G,et al.Contraction regulation of Akt in rat skeletal muscle[J].JBC,2002,277(14):11910
[12]Cartee G D,Funai K.Exercise and insulin:Convergence or divergence at AS160 and TBC1D1[J].Exerc Sport Sci Rev,2009,37(4):188
[13]Jensen T E,Schjerling P,Viollet B,et al.AMPK alphal activation is required for stimulation of glucose uptake by twitch contraction,but not by H2O2,in mouse skeletal muscle[J].PLoS one,2008 3(5):e2102