Electrical pulse stimulation induces GLUT4 translocation in C2C12 myotubes that depends on Rab8A, Rab13, and Rab14

Electrical pulse stimulation induces GLUT4 glucose transporter translocation in C2C12 myotubes that depends on Rab8A, Rabl3. and Rabl4. Am J Physiol Endocrinol Metab 314: K478-E493, 2018. First published November 14, 2017; doi: 10.1152/ajpendo.00103.2017.-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 GEUT4HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GEUT4H4 (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 small interfering (si)RNA-mediated AMPK alpha l/alpha 2 knockdown, compound C, siRNA-mediated Ca2+ /calmodulin-dependent protein kinase (CaMKII)8 knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS 160 and TBC1D1 were phosphorylaled in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulinstimulated GLUT4 translocation, suggesting regulation of GLEJT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a. Rabl3. or Rab 14 reduced, in part. GLUT4 translocation induced by EPS, whereas only RabSa, or Rab 14 knockdown reduced the AICAR response. In conclusion, EPS involves RabSa. Rab 13, and Rab 14 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.