Glycogen Enhancement Augments Basal and Leucine‐Stimulated Protein Synthesis in C2C12 Myotubes

Abstract

Age‐related skeletal muscle wasting is associated with elevated 5′‐AMP‐Activated Protein Kinase (AMPK) activity, which inhibits overload‐induced skeletal muscle protein synthesis (MPS) and growth. Glycogen, an inhibitor of AMPK, is reduced in aged skeletal muscle. We recently found that glycogen enhancement augments 21‐day overload‐induced MPS and growth in aged rat skeletal muscle. Unexpectedly, these findings were largely independent of AMPK and other signaling regulating MPS. However, the effects of glycogen enhancement on the skeletal muscle response to other anabolic stimuli, such as leucine, are largely unknown. Therefore, the purpose of this investigation was to examine the effects of enhanced glycogen on AMPK phosphorylation status and activity, MPS, and MPS‐related signaling in response to leucine in C2C12 myotubes. We hypothesized that glycogen enhancement in myotubes would enhance the anabolic effect of leucine by inhibiting AMPK and increasing anabolic signaling and MPS, and that this effect would be reduced by rapamycin‐induced mechanistic target of rapamycin (mTOR) blockade. Four days after the onset of differentiation, myotubes were transfected (via Lipofectamine 3000) for 48 hrs with a mutant glycogen synthase plasmid (mutant‐GS; designed to enhance skeletal muscle glycogen content) or an empty‐vector plasmid. At six days post‐differentiation, transfected myotubes were serum‐starved for 4 hrs prior to an amino acid starvation for 1 hr. Myotubes were then treated with either a vehicle control (CT), 10 mM leucine (LEU), 100 nM rapamycin (RAP), or leucine/rapamycin co‐treatment (LEU+RAP) for 30 min. All treatment conditions contained 100 μM puromycin for measurement of MPS rate. All values were normalized to the empty‐vector CT condition. As expected, mutant‐GS expression and glycogen content were significantly (p ≤ 0.05) higher in all four mutant‐GS myotube conditions vs. corresponding empty‐vector conditions. Within both the mutant‐GS conditions and the empty‐vector conditions, MPS was stimulated with LEU treatment and suppressed with RAP treatment as expected; however, MPS was significantly (p ≤ 0.05) higher in all four mutant‐GS conditions vs. corresponding empty‐vector conditions. LEU treatment increased the phosphorylation status of mTOR (Ser2448), p70S6K (Thr389), and 4EBP1 (Thr37/46), and this effect was blocked with RAP treatment within both the mutant‐GS myotubes or the empty‐vector myotubes. However, there were no significant (p > 0.05) differences between mutant‐GS or empty‐vector myotubes in this signaling. Additionally, markers of AMPK phosphorylation status (Thr172) and activity (ACC at Ser79) and other MPS signaling intermediates (TSC2 Ser1387 & Thr1462) were not significantly different between mutant‐GS vs. empty‐vector conditions and were unaffected by LEU or RAP treatments. Although the stimulation of MPS by leucine is mTOR‐dependent regardless of glycogen enhancement in myotubes, glycogen enhancement augments MPS independent of changes in AMPK, mTOR, or other MPS‐related signaling markers assessed. Even though the signaling mechanism(s) remain to be identified, these findings along with our recent in vivo data support the translatable potential to enhance human skeletal muscle glycogen content to augment leucine‐stimulated MPS or augment MPS and growth under conditions of chronic overload (such as resistance exercise training).Support or Funding InformationThis research was partially supported by an ACSM Foundation Doctoral Student Research Grant from the American College of Sports Medicine Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.