Creation Of Overexpressing GLUT4HA Skeletal Muscle Cell Line To Study The Mechanism Of EPS-regulated GLUT4 Translocation

Objective:To create a HA-tagged glucose transporter 4(GLUT4HA)-overexpressing C2C12 cell line with contractile activity and to study the mechanism of electrical pulse stimulation(EPS)-regulated GLUT4 translocation in this skeletal muscle cell line.Methods:In Part 1,C2C12-GLUT4HA cell line stably overexpressing GLUT4HA was created,and 23 clones were selected.The differentiation and contraction ability were observed by light microscope.The expression level of GLUT4HA protein was detected by Western blotting.The clones with good myotubes,contraction ability and high GLUT4HA protein level were tested the response to insulin and EPS.The amount of GLUT4HA on the cell surface was measured by enzyme-linked immunosorbent assay(ELISA)under basal,insulin and EPS conditions.In Part 2,C2C12-GLUT4HA skeletal muscle cells were treated by EPS.The effect of EPS on GLUT4HA translocation was investigated.Then,the roles of AMPK,PI3K and Akt in EPS-stimulated GLUTHA translocation were explored by using AMPK inhibitor Compound C(CC),ATPase inhibitor BTS,PI3K inhibitorWortmmanin and Akt inhibitor Akti.Results:1.Among the selected 23 clones,clone No.17 showed higher GLUT4HA protein level,good myotubes and contraction ability.In addition,GLUT4HA in clone No.17 myotubes translocates to cell surface under insulin and EPS stimulation.2.EPS-stimulated GLUT4HA translocation in C2C12-GLUT4HA skeletal muscle cells was partly inhibted by Compound C,BTS,Wortmmanin and Akti,respectively.Conclusion:1.A contractile C2C12 muscle cell line stably overexpressing GLUT4HA was created.This cell line will be useful to study the mechanism of insulin-and contraction stimuli-regulated GLUT4 traffic.2.EPS increases GLUT4HA translocation in C2C12 musle cells.The mechanism may involve AMPK,PI3K and Akt.