Electrical pulse stimulation induces GLUT4 translocation in C₂C₁₂myotubes that depends on Rab8A, Rab13, and Rab14

Abstract

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 CC myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (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 AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca/calmodulin-dependent protein kinase (CaMKII)δ 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-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

Electrical pulse stimulation induces GLUT4 translocation in a Rac‐Akt‐dependent manner in C2C12 myotubes

“…With our in vitro model of EPS‐induced contraction mouse C2C12‐GLUT4 HA skeletal muscle cells, we previously showed AMPK and CaMKII were activated and required for EPS‐stimulated GLUT4 translocation . However, a portion of the EPS‐stimulated GLUT4 response remained with simultaneous inhibition of both kinases, suggesting additional pathways were needed for the full response.…”

In vitro experimental models for examining the skeletal muscle cell biology of exercise: the possibilities, challenges and future developments

“…Additionally, whilst GLUT4 translocation, a key process which contributes to the glucoregulatory effects of exercise References are provided within the text of the relevant section ↑ indicates values increase following in vivo exercise, ↓ indicates values decrease following in vivo exercise, ✓ indicates the same direction of change induced by exercise-like treatment compared to in vivo exercise, × indicates different direction of change induced by exercise-like treatment compared to in vivo exercise, ✓ × indicates contradictory findings, ? indicates evidence lacking regarding the effect of exercise-like treatment in vivo, has been demonstrated in C2C12 myotubes [52,68,83], such effects have not yet been reported in human primary muscle cell-based EPS models. Additionally, whilst C2C12 cells are responsive to EPS in regard to contractility, primary human myotubes reportedly vary in their contractility depending on culture conditions and cell donor meaning that the degree to which EPS induces exercise-like contraction can be variable.…”

“…Such EPSinduced sarcomeric structure formation reportedly relies upon calpain-mediated proteolysis [37]. EPS has also been demonstrated to increase lactate production, reduce intracellular glycogen content [68] and protect against lipid-induced insulin resistance [84]. However, some key changes seen following exercise in vivo have not been reproduced in vitro.…”

Caveolin-3 promotes glycometabolism, growth and proliferation in muscle cells

“…We examined total and phosphorylated AMPK, and we found that AMPK was activated, further confirming our previous confocal results: CAV3 increased GLUT4 localization at the cell membrane. And Rab GTPases had be reported to affect translocation of GLUT4 to the cell membrane, so we will include it in our further exploration [ 29 ].…”