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. 2013 Jul;11(7):e1001603.
doi: 10.1371/journal.pbio.1001603. Epub 2013 Jul 9.

Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

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Free PMC article

Effects of Resveratrol and SIRT1 on PGC-1α Activity and Mitochondrial Biogenesis: A Reevaluation

Kazuhiko Higashida et al. PLoS Biol. .
Free PMC article

Erratum in

  • PLoS Biol. 2014 Jan;12(1). doi:10.1371/annotation/900c9397-eeb9-4d4e-9a05-ff18d657be79

Abstract

It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C₂C₁₂ myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Feeding rodents resveratrol does not induce mitochondrial biogenesis in skeletal muscle.
(A) Feeding rats a chow diet containing 4 g resveratrol (RSV) per kg diet for 8 wk had no effect on expression of PGC-1α or mitochondrial enzyme proteins in soleus muscle. (B) Feeding rats a high fat diet containing 4 g RSV per kg diet for 8 wk had no effect on expression of PGC-1α or mitochondrial enzyme proteins in triceps muscle. (C) Feeding mice a high fat diet containing 4 g resveratrol per kg diet had no effect on expression of PGC-1α or mitochondrial proteins in triceps muscle. Values are means ± SE for 6–8 muscles per group.
Figure 2
Figure 2. 50 µM resveratrol is toxic to C2C12 myotubes.
(A) Exposure to 50 µM resveratrol (RSV) for 24 h is toxic to C2C12 myotubes resulting in a decrease in viable cells and (B) a decrease in ATP content. Values are means ± SE for 4–6 experiments. (C) PGC-1α overexpression in C2C12 myotubes protects against the toxic effect of 24 h exposure to 50 µM RSV on viability and (D) ATP content. Values are means ± SE for six experiments. *p<0.05 versus basal.
Figure 3
Figure 3. High concentrations of resveratrol induce an increase in mitochondrial proteins in C2C12 myotubes.
(A) Treatment with 50 µM resveratrol (RSV) for 24 h resulted in an increase in mitochondrial proteins in C2C12 myotubes in which PGC-1α was overexpressed by infection with an adenovirus expressing PGC-1α (AdPGC-1α). Values are means ± SE for 7–8 experiments. (B) Treatment of wild-type C2C12 myotubes with 20 µM RSV for 6 h, followed by 18 h recovery, for 3 d induced increases in PGC-1α and mitochondrial proteins. Values are means ± SE for 6–8 experiments. *RSV versus control, p<0.05.
Figure 4
Figure 4. Resveratrol activates AMPK.
(A) Treatment of C2C12 myotubes with 20 µM resveratrol (RSV) for 3 h results in increased phosphorylation of AMPK and acetyl CoA carboxylase (ACC). (B) Blocking AMPK activity by expression of dominant-negative AMPK (DN AMPK) in C2C12 myotubes prevents AMPK and ACC phosphorylation in response to RSV treatment. Nicotinamide had no effect on AMPK or ACC phosphorylation. (C) Blocking AMPK activity (DN AMPK) prevents induction of an increase in mitochondrial proteins by resveratrol. In experiments in which C2C12 myotubes were treated with 50 µM resveratrol, PGC-1α was overexpressed in the myotubes (see Figure 3 and text). Values are means ± SE for 6–8 experiments. *RSV versus other groups, p<0.05.
Figure 5
Figure 5. Suppression of SIRT1 activity increases PGC-1α acetylation and coactivator activity.
(A) Nicotinamide (Nic) decreases SIRT1 activity in C2C12 myotubes as evidenced by increased p53 acetylation and prevention of p53 deacetylation in response to resveratrol (RSV). *p<0.05 versus control; # p<0.05 versus RSV and RSV+Nic. (B) Inhibition of SIRT1 with nicotinamide (Nic) does not prevent the resveratrol (RSV)-induced increase in mitochondrial proteins. *p<0.05 versus control. (C) Suppression of SIRT1 activity with nicotinamide or with dominant-negative SIRT1 H355A results in increased acetylation of PGC-1α. SIRT1 H355A reduces PGC-1α deacetylation in response to resveratrol (RSV) treatment, while overexpression of wild-type (WT) SIRT1 results in PGC-1α deacetylation. Values are means ± SE for 6–8 experiments. *p<0.05 versus control; # p<0.05 versus other groups. (D) PGC-1α coactivator activity, measured in C2C12 myotubes co-transfected with a PGC-1α GAL4 fusion product and a luciferase reporter, was increased by treatment with 20 µM resveratrol (RSV). Overexpression of wild-type (WT) SIRT1 resulted in reduced PGC-1α coactivator activity. Suppression of SIRT1 activity with dominant-negative SIRT1 H355A or knockdown of SIRT1 with SIRT1 shRNA resulted in increases in PGC-1α coactivator activity and potentiation of the effect of resveratrol on PGC-1α activity. In the experiments in which C2C12 myotubes were treated with 50 µM resveratrol, PGC-1α was overexpressed in the myotubes (see Figure 3 and text). Values are means ± SE for 6–7 experiments. *p<0.05 versus control; # p<0.05 versus basal.
Figure 6
Figure 6. Reducing SIRT1 activity increases, and increasing SIRT1 expression decreases, mitochondrial enzymes in C2C12 myotubes and rat skeletal muscle.
(A) Suppression of SIRT1 activity with dominant-negative SIRT1 H355A in C2C12 myotubes increases mitochondrial proteins. Values are means ± SE for 6–10 experiments per group. p<0.5 versus control. (B) Suppression of SIRT1 activity by expression of dominant-negative SIRT1 H355A in rat triceps muscle by electroporation resulted in increases in mitochondrial proteins. Values are means ± SE for 5–7 muscles per group. *p<0.05 versus control. (C) Knockdown of SIRT1 with a SIRT1 shRNA in C2C12 myotubes resulted in increases in mitochondrial proteins. Values are means ± SE for 6–8 experiments per group. *p<0.05 versus control. (D) Overexpression of wild-type (WT) SIRT1 in C2C12 myotubes resulted in decreased cytochrome c protein expression and inhibited the resveratrol (RSV)-induced increase in cytochrome c. Values are means ± SE for 6 experiments. *p<0.05 versus control. # p<0.05 versus other groups. (E) Overexpression of wild-type (WT) SIRT1 in rat triceps muscle by electroporation resulted in decreased expression of mitochondrial proteins. Values are means ± SE for 7–8 muscles per group. *p<0.05 versus empty vector.

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