Sirtuin 1-mediated effects of exercise and resveratrol on mitochondrial biogenesis

Abstract

The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.

FIGURE 1.

A and B, acute effect of RSV (1 h treatment) versus vehicle on P-p38 and P-AMPK protein expression. C and D, longer term (total 24 h) effect of RSV or NAM/RSV versus vehicle on AMPK and p38 (phospho/total) phosphorylation in quiescent or contracting (3 h followed by 21 h of recovery) myotubes (n = 9, arbitrary scanner units (A.U.) corrected for loading using GAPDH; *, p < 0.05, versus vehicle; ‡, p < 0.05, overall effect of treatment with no stimulation (Stim) versus control (Con)).

FIGURE 2.

A, SirT1 and PGC-1α protein expression in vehicle and RSV-treated C2C12 myotubes, with or without stimulation using the 4-day protocol. B and C, SirT1 and PGC-1α protein expression data shown graphically. D, immunofluorescence of SirT1 (red) and PGC-1α (green) with DAPI (blue)-stained nuclei. Images examine SirT1 and PGC-1α nuclear localization in C212 myotubes treated with vehicle or RSV, with and without CCA using the 4-day protocol (n = 9, arbitrary scanner units (A.U.) corrected for loading using α-tubulin; *, p < 0.05, overall effect of stimulation versus control (Con); ‡, p < 0.05, overall effect of RSV versus control).

FIGURE 3.

A, ROS production measured using 2′,7′-dichlorofluorescein (DCF) fluorescence with and without CCA and treated with vehicle, RSV, or quercetin using the 4-day protocol. Con, control. B, COX-IV protein expression in vehicle and RSV-treated C2C12 myotubes, with or without CCA, using the 4-day protocol is shown graphically. C, COX activity in vehicle, RSV, quercetin- or NAM-treated myotubes, with or without the 4-day protocol of CCA. D, mitochondrial mass measured using MTGFM in a 6-well cell culture plate. E, MTGFM-stained C2C12 myotubes that were treated with CCA, vehicle, or RSV using the 4-day protocol. Images were taken using a fluorescent microscope (n = 9–12, arbitrary scanner units (A.U.) corrected for loading using GAPDH; *, p < 0.05, overall effect of CCA; ‡, p < 0.05, overall effect of treatment versus vehicle-treated control; †, p < 0.05, interaction of treatment versus vehicle-treated myotubes; ϵ, p < 0.05, versus myotubes treated with CCA/RSV).

FIGURE 4.

A, COX and SDH histochemical staining of serial sections of gastrocnemius muscle from WT and SirT1-KO mice. B, fatigue response from the in situ stimulation of WT or SirT1-KO gastrocnemius muscle as a percent of initial twitch force. TPS, tetanic contraction/s. C, intraperitoneal glucose tolerance test was performed on WT and SirT1-KO animals. Mice were given an intraperitoneal injection of d-glucose (2 g/kg of body weight) following 6 h of fasting. D, voluntary wheel running distance by mice over 9 weeks fed control or RSV diets. E, total number of pellets of RSV or control diet consumed throughout the treatment. Con, control. F, body weight of animals. G, total wet muscle weight (tibialis anterior, gastrocnemius, and triceps)/g of body weight (b.w.) for WT and SirT1-KO animals. H, heart weight/g of body weight (n = 9–12, *, p < 0.05, effect of RSV versus control; ¶, p < 0.05, overall effect of each treatment under the line versus control).

FIGURE 5.

A, skeletal muscle COX activity following training, RSV, and combined treatments in skeletal muscle from WT and SirT1-KO mice. B, cytochrome c (Cyt C) protein expression. C and D, state 4 and state 3 respirations/nanoatom oxygen consumed in subsarcolemmal mitochondria. E, PGC-1α and Nampt protein expression following training, RSV, and combined treatments in skeletal muscle from WT and SirT1-KO mice. F, and G, PGC-1α and Nampt protein expression is shown graphically (n = 7–12, arbitrary scanner units (A.U.) corrected for loading using GAPDH; ¶, p < 0.05, overall effect of each treatment under the line versus control; *, p < 0.05, versus control (Con) WT mice; †, p < 0.05, interaction of each treatment under the line versus control; ††, p < 0.05, interaction of RSV/Trained versus the trained mouse).

FIGURE 6.

A and B, state 4 and state 3 ROS production per nanoatom oxygen consumed in SS mitochondria (n = 7–12, *, p < 0. 05, versus control WT mice, ¶, p < 0.05, overall effect of treatment versus control mice, †, p < 0.05, interaction of each treatment under the line versus control mice).