Study The Role Of Axin1 In Electrical Pulse Regulated-Akt Phosphorylation In Skeletal Muscle Cells

Objective:The skeletal muscle is the largest insulin-sensitive human organ, and plays an important role in regulation of the whole body metabolism. GLUT4 mediated glucose uptake in skeletal muscle cells. The skeletal muscle insulin resistance is an early defect in the pathogenesis of type 2 diabetes. Insulin and exercise/muscle contraction are the major stimulators of glucose uptake in skeletal muscle, but the molecular mechanism of contraction stimulated skeletal muscle glucose uptake is unclear.Our team wants to study the molecular mechanism of contraction stimulated skeletal muscle glucose uptake. We have found that EPS increased Akt phosphorylation via AMPK-Rac1 signaling pathways. Axin1 is a multi-domain scaffold protein that coordinates several different protein complexes,and may play a part in regulation of AMPK activity. In this study, we investigate the effect of Axin1 on the regulation of Akt by electrical pulse in mouse skeletal muscle cells. Content:The first part: The 3T3-L1 cells as a positive control, to detect whether L6 myoblasts?C2C12-GLUT4 HA myotubes and mouse skeletal muscle could express Axin1, and to detect whether EPS could increase Axin1 content.The second part: The expression of Axin1 was down-regulated by small interfering RNA, and to study the effect of Axin1 in the regulation of Akt by electrical pulse in mouse skeletal muscle cells.The third part: 12 weeks old C57BL/6 male mice were treated with different stimulus to test the expression of Axin1, and to study the relationship between AMPK and Axin1 in skeletal muscle. Methods:Cultured C2C12-GLUT4 HA cells were differentiated into mature contractile myotubes. C2C12GLUT4 HA myotubes were treated with EPS, and Western Blot detected the level of Axin1 protein in cells. The expression of Axin1 was down-regulated by small interfering RNA, Western Blot detected AMPK ?Akt phosphorylation and Rac1 activity in C2C12-GLUT4 HA cells. 12 weeks old C57BL/6 male mice were treated with different stimulus, Western Blot detected the expression of Axin1, and co-immunoprecipitation was used to study the interaction between AMPK and Axin1 Results: 1. L6?C2C12-GLUT4 HA and skeletal muscle cells in mice expressed Axin1, and EPS increased Axin1 content in C2C12-GLUT4 HA myotubes. 2. SiAxin1 inhibited EPS induced Akt and AMPK phosphorylation, and decreased Rac1 activity.EPS enhanced the interaction between AMPK and Axin1 3. Exercise increased Axin1 content, and enhanced the interaction between AMPK and Axin1 in skeletal muscle. Conclusion:In C2C12GLUT4 HA myotubes, Axin1 may formed a complex with AMPK to increase Akt phosphorylation in EPS.Therefore, Axin1 can be used as a new point for improving insulin resistance.