Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Abstract

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.

Figure 1

Resveratrol inhibits dexamethasone-induced expression of atrogin-1 and MuRF1 in cultured L6 myotubes. Myotubes were treated for 24 h with 1 μM dexamethasone, 100 μM resveratrol, both drugs in combination or solvent (0.1% ethanol) followed by measurement of mRNA levels for (A) atrogin-1 and (B) MuRF1 and protein levels for (C) atrogin-1 and (D) MuRF1. Results are means ± SEM with n=8 per group. *p<0.05 vs all other groups by ANOVA.

Figure 2

Resveratrol blocks dexamethasone-induced acetylation of FOXO1 in cultured L6 myotubes. Myotubes were treated with dexamethasone and resveratrol as described in Fig 1. Nuclear levels of acetylated FOXO1 were determined by co-immunopecipitation as described in Materials and Methods. Results are means ± SEM with n=6 per group. *p<0.05 vs all other groups by ANOVA.

Figure 3

Resveratrol inhibits dexamethasone-induced protein degradation and atrophy in cultured L6 myotubes. Myotubes were treated with dexamethasone and resveratrol as described in Fig 1. (A) Protein degradation, (B) myotube morphology, and (C) myotube diameter were determined as described in Materials and Methods. Results in (A) and (B) are means ± SEM with n=8 per group. *p<0.05 vs all other groups by ANOVA.

Figure 4

SIRT1 is involved in the protective effects of resveratrol in dexamethasone-treated L6 myotubes. (A) Nuclear levels of acetylated PGC-1α were determined by co-immunoprecipitation in dexamethasone-treated myotubes in the absence or presence of resveratrol. (B) SIRT1 mRNA levels were determined in myotubes after transfection with non-targeting (NT) or SIRT1 siRNA. In other experiments, myotubes were treated with dexamethasone and resveratrol as described in Fig 1 following transfection with non-targeting (NT) or SIRT1 siRNA and atrogin-1 (C and D) and MuRF1 (E and F) mRNA levels were determined by real-time PCR. Results are means ± SEM with n=6 (A) or 8 per group. *p<0.05 by Student’s t-test (A and B); *p<0.05 vs all other groups; +p<0.05 vs Dex by ANOVA (C-F).