The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Abstract

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 µM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the 'slow' type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for 'intermediate' and 'faster' IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48-72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring 'slower' Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction.

Keywords: AMPK; Atrophy; Dietary restriction; Hypertrophy; MYHC; Myoblasts; P70S6K; SIRT1.

Fig. 1

a Glucose Restriction (GR) blocked differentiation in differentiating myoblasts with significant reductions in; b myogenin and increases in Sirt1 gene expression. Resveratrol improved, c CK activity in GR conditions # (albeit non-significantly p = 0.069), without improvements in differentiation. Resveratrol also improved, d, e myotube area when glucose was more readily available in DM (differentiation media control) conditions at 7 days post induction of differentiation. This was associated with increases in f Myhc7 and Myhc4 coding for the slow type I and fast IIb MYHC protein isoforms. Further, EX-527 administration in DM conditions resulted in reductions in g Myhc1 coding for IIx MYHC protein isoform with this condition displaying the smallest mean myotube size versus all other conditions. *Significantly different (p ≤ 0.05) versus DM conditions. **Significantly, (p ≤ 0.05) different versus EX-527 conditions. All experiments are at least n = 3 in duplicate

Fig. 2

Loss of myotubes and reduced myotube growth occurred when existing myotubes were placed in Glucose Restriction (GR) conditions for 72 h, demonstrated a morphologically and via reductions in b myotube number, c myotube diameter and d myotube area. This corresponded with reductions in gene expression of myotube maturation genes e Myhc1, 2, 4, 7, reductions in genes associated with myofibre hypertrophy Igf-I, Igf-Ir and Igfbp2 and increased expression of genes associated with myotube atrophy/protein degradation, Mafbx and Musa1. Resveratrol was unable to improve myotube survival or reduced myotube growth in GR conditions at 72 h however, resveratrol evoked increases in f, h myotube area at 72 h when glucose was more readily available (DM/differentiation media control conditions). This was associated with increases in i Igf-I and Myhc7 that codes for the slow type I Myhc protein isoform. EX-527 treatment also reduced g myotube number and h myotube area. This was associated with reductions in gene expression of j Myhc 1, 2 and 4 coding for intermediate and IIx, IIa and IIb MYHC protein isoforms, respectively. It is worth noting EX-527 administration evoked a similar 65–68% loss of myotubes experienced in GR conditions alone. *Significantly different (p ≤ 0.05) versus DM 72-h conditions. All experiments are at least n = 3 in duplicate

Fig. 3

Resveratrol was able to improve a, b myotube area for a period of 24 h after dosing in Glucose Restriction (GR) conditions. *Significantly different (p ≤ 0.05) versus GR conditions alone. All experiments are at least n = 3 in duplicate

Fig. 4

Resveratrol treatment did not alter the activity of energy-dependent sensing protein AMPK or protein translation initiator P70s6K in Glucose Restriction (GR) conditions over 24 h. However, EX-527 treatment on average increased a, b (approached significance) AMPK activity at 30 min and 24 h in DM (differentiation media control) conditions (level of significance depicted on Fig. 2b). All experiments are at least n = 3