Amira Klip, Yingying Yue, Zhi Yao, Maolong Fu, Fang Hu, Wenyan Niu, Liming Chen, Lihong Qiu, Chang Zhang, Xiaofang Ma, Philip J Bilan, Nana Li, Zhu Li
The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated C2C12 myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4HA EPS (60min, 20V, 1Hz, 24ms pulses at 976ms intervals) elicited a gain in surface GLUT4HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by siRNA-mediated AMPKα1/α2 knockdown, Compound C, siRNA-mediated CaMKIIδ knockdown or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13 or Rab14 reduced in part GLUT4 translocation induced by EPS, whereas only Rab8a or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13 and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.