Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD + levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

This study was aimed at testing whether the ability of resveratrol to activate AMPK and increase mitochondrial function requires SIRT1 in vivo, and whether SIRT1 overexpression is sufficient to mimic these effects. The main impediment to studying SIRT1 in vivo has been the poor survival, impaired growth, and developmental defects of germline SIRT1 knockout mice (). While studies performed in outbred mice suggest that the effects of resveratrol treatment on cancer may be impaired in SIRT1 knockout mice (), the knockout mice used in these studies were small, sterile, and suffered from craniofacial abnormalities and eyelid inflammatory conditions, making interpretation of the data extremely difficult. To circumvent these issues, we have developed an adult-inducible SIRT1 knockout mouse. This mouse shows efficient deletion of SIRT1 across a variety of tissues and appears grossly normal and healthy beyond 1 year of age. Using these animals, as well as cell culture models, we observe that the stimulation of AMPK activity and increase in NADlevels, mitochondrial biogenesis, and mitochondrial function in skeletal muscle by a moderate dose of resveratrol are entirely dependent upon SIRT1. Further, overexpression of SIRT1 mimics the effects of resveratrol on both mitochondria and AMPK activation. Interestingly, an ∼10-fold higher dose of resveratrol activates AMPK in a SIRT1-independent manner, though improvements in mitochondrial function are SIRT1 dependent. Taken together, these data highlight the differences resveratrol treatment can have at different doses and demonstrate an important role for SIRT1 in activating AMPK and mediating the benefits of resveratrol in vivo.

Unfortunately, studies to date have been unable to determine which model is most relevant under physiological conditions. We and others have shown that resveratrol activates AMPK in cell culture and in vivo (), and a study of AMPK knockout mice established that AMPK is required for many of the beneficial effects of resveratrol on metabolic function (). On the other hand, recent enzymological studies have presented evidence for direct SIRT1 activation by small molecules (), and there is a growing literature of cell culture studies in which the effects of resveratrol are lost after knocking down or inhibiting SIRT1 (). Moreover, resveratrol's central effects on liver gluconeogenesis () are abrogated when SIRT1 activity is impaired in the hypothalamus (), and treatment of mice with a SIRT1 activator that is structurally unrelated to resveratrol, SRT1720, increases mitochondrial capacity in skeletal muscle () and liver in a SIRT1-dependent manner (), while improving the health and survival of mice on a high-fat diet, similar to what has been observed with resveratrol ().

Effects of resveratrol on NO secretion stimulated by insulin and its dependence on SIRT1 in high glucose cultured endothelial cells.

Induction of manganese superoxide dismutase by nuclear translocation and activation of SIRT1 promotes cell survival in chronic heart failure.

SIRT1 activation by small molecules: kinetic and biophysical evidence for direct interaction of enzyme and activator.

One of the most robust and reproducible effects of resveratrol treatment is an increase in mitochondrial mass (). SIRT1 promotes mitochondrial biogenesis through deacetylation and activation of PGC-1α (), a master regulator of mitochondrial biogenesis that coactivates the nuclear respiratory factors (NRF-1 and NRF-2), which induce the transcription of genes involved in mitochondrial biogenesis (). PGC-1α is also activated by another important metabolic sensor, the AMP-activated protein kinase (AMPK) (). Though the effects of resveratrol and SIRT1 on PGC-1α are well established, there is considerable debate about the mechanism by which this regulation is achieved. One school of thought is that the direct activation of SIRT1 by resveratrol is an in vitro artifact () and that resveratrol works primarily by activating AMPK (), potentially by inhibition of phospodiesterases (PDEs), ATPase, or complex III (). It has been proposed that AMPK then activates SIRT1 indirectly by elevating intracellular levels of its cosubstrate, NAD). Alternatively, resveratrol may first activate SIRT1 in vivo, leading to AMPK activation via deacetylation and activation of the AMPK kinase LKB1 ().

SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation.

In obese rodents, treatment with resveratrol produces a variety of health benefits including improved metabolic and vascular function, decreased hepatic steatosis, reduced inflammation, greater endurance, and a gene expression pattern resembling CR (). Recent clinical studies show that resveratrol also confers metabolic benefits in humans (). Understanding how resveratrol exerts its effects is important, not only for the potential insights into the biological causes of age-related diseases, but also to allow the development of more potent and specific molecules.

The polyphenol resveratrol (2,3,4′-trihydroxystilbene) first attracted scientific attention when it was linked to the cardiovascular benefits of red wine and was subsequently found to possess potent antitumor activity (). In 2003, a screen for small molecule activators of SIRT1 identified 21 different SIRT1-activating molecules, the most potent of which was resveratrol (). In the majority of studies to date, resveratrol has been found to increase life span in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster in a sirtuin-dependent manner, although the life span extension in yeast and flies, and the Sir2 dependence in worms, has been challenged (). In addition, resveratrol extends life and delays the onset of age-related phenotypes in a short-lived species of fish ().

The mammalian sirtuins (SIRT1–SIRT7) are a conserved family of NAD-dependent deacetylases and ADP-ribosyltransferases involved in numerous fundamental cellular processes including gene silencing, DNA repair, and metabolic regulation (). Deletion of SIRT1 in outbred strains of mice abrogates the effect of caloric restriction (CR) on physical activity () and life span extension (), whereas overexpression of SIRT1 mimics many of the salutary effects of CR, including a reduced incidence of cardiovascular and metabolic diseases (), cancer (), and neurodegeneration (). Recent human genetic studies also support a role for SIRT1 in maintaining human health status with age ().

SIRT1 mRNA expression may be associated with energy expenditure and insulin sensitivity.

SIRT1 is associated with a decrease in acute insulin secretion and a sex specific increase in risk for type 2 diabetes in Pima Indians.

Neuronal SIRT1 activation as a novel mechanism underlying the prevention of Alzheimer disease amyloid neuropathology by calorie restriction.

To further elucidate the epistasis of SIRT1 and AMPK, we treated primary hepatocytes and myoblasts isolated from SIRT1 KO mice with AICAR, an AMP mimetic that directly activates AMPK and promotes its phosphorylation by LKB1. Phosphorylation of AMPK by AICAR was blunted in both the primary hepatocytes and primary myoblasts lacking SIRT1 ( Figure 7 G and Figure S7 J). Moreover, the ability of AICAR to increase mitochondrial DNA copy number and ATP in the liver and muscle KO cells was completely blocked ( Figures 7 H and 7I and Figures S7 H and S7I). In further support of SIRT1 acting upstream of AMPK, treatment of C2C12 cells with 25 μM resveratrol resulted in a SIRT1-dependent decrease in LKB1 acetylation ( Figure 7 J). These findings are consistent with previous work done in C2C12 cells () and support previous findings that resveratrol-stimulated, SIRT1-mediated deacetylation of LKB1 plays a direct role in the activation of AMPK (). Taken together, these findings show that treatment of mice on a high-fat diet with moderate doses of resveratrol results in increased phosphorylation of AMPK, induction of mitochondrial biogenesis, increased ATP and NADlevels, and a shift toward more oxidative muscle fibers, all of which are SIRT1-dependent effects.

SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation.

Treatment with 25 μM resveratrol elevated ATP levels at 4, 6, and 12 hr, consistent with what we observed in vivo ( Figure 7 D). In contrast, the 50 μM dose significantly decreased ATP levels early as 1 hr after treatment. At the 25 μM dose, activation of AMPK occurred in a SIRT1-dependent manner, while the 50 μM dose activated AMPK independently of SIRT1 ( Figure 7 E). Importantly, the increase in ATP was evident before any changes in cellular NADlevels were detected ( Figure 7 F), indicating that improvements in mitochondrial function and elevation of cellular ATP levels are both dependent upon SIRT1 and occur prior to increases in cellular NAD

Resveratrol has been implicated in the direct modulation of numerous targets (), but it has been difficult to discern which of these targets are physiologically relevant. Part of the difficulty has arisen from the fact that doses of resveratrol given to animals are wildly variable and concentrations used on cells vary greatly as well (). To provide some clarity, we performed a series of dose and time course experiments with resveratrol. Treatment of C2C12 cells with a moderate dose of resveratrol (25 μM) activated AMPK in a SIRT1-dependent manner, while at a higher dose of resveratrol (50 μM) AMPK was activated in a SIRT1-independent manner ( Figure 7 A ). Similarly, treatment with the lower dose of resveratrol for 24 hr mimicked the effects on muscle in vivo by increasing ATP and mitochondrial membrane potential. In contrast, the 50 μM dose reduced mitochondrial membrane potential and cellular ATP levels, indicative of mitochondrial dysfunction, an effect of resveratrol that was not observed in vivo ( Figures 7 B and 7C).

(K) Moderate doses of resveratrol activate AMPK and stimulate mitochondrial biogenesis in a SIRT1-dependent manner that results in improvement of mitochondrial function.

(J) C2C12 cells infected with SIRT1 or nontargeting shRNA and expressing Flag-LKB1 were treated with resveratrol 25 μM for 24 hr, and LKB1 acetylation was tested in Flag immunoprecipitates. Total LKB1 was evaluated in total extracts as input.

(I) ATP content in primary myoblasts isolated from wild-type and SIRT1 knockout mice and treated with 500 μM AICAR for 24 hr (n = 3, ∗ p < 0.05 versus DMSO).

(H) Mitochondrial DNA content analyzed by quantitative PCR in primary myoblasts isolated from wild-type and SIRT1 knockout mice and treated with 500 μM AICAR for 24 hr. Relative expression values were normalized to control (n = 3, ∗ p < 0.05 versus empty DMSO).

(G) Representative immunoblot for for p-AMPK (Thr172), and total AMPK in primary myoblasts isolated from wild-type and SIRT1 knockout mice and treated with 500 μM AICAR for 24 hr.

(F) NAD + content in C2C12 cells treated with 25 or 50 μM resveratrol for 1, 4, 6, and 12 hr (n = 4, ∗ p < 0.05 versus 50 μM DMSO, #p < 0.05 versus 25 μM DMSO).

(E) Representative immunoblot for for p-AMPK (Thr172), and total AMPK in C2C12 cells treated with 25 or 50 μM resveratrol for 1, 4, 6, and 12 hr.

(D) ATP content in C2C12 cells treated with 25 or 50 μM resveratrol for 1, 4, 6, and 12 hr (n = 4, ∗ p < 0.05 versus DMSO).

(C) Mitochondrial membrane potential in C2C12 cells treated with 25 or 50 μM resveratrol for 24 hr (n = 4, ∗ p < 0.05 versus DMSO).

(A) Representative immunoblot for p-AMPK (Thr172) and total AMPK in C2C12 cells infected with SIRT1 or nontargeting shRNA and treated with 10, 25, or 50 μM resveratrol for 24 hr.

Treatment with a moderate dose of resveratrol increased the levels of phosphorylated AMPK and NADin gastrocnemius of WT mice ( Figures 6 F–6I). Strikingly, none of these changes were observed in SIRT1 KO mice. Consistent with these findings, the regulation of AMPK-regulated genes involved in fatty acid metabolism (LCAD, MCAD, CPT1b, FAS, and Scd1) was also dependent upon SIRT1. Interestingly, at the higher dose of resveratrol, many of these effects were SIRT1 independent, demonstrating that the dose is critical to the outcome ( Figures 6 F and 6G, Figures S7 F and S7G). SIRT1-overexpressing mice had significantly increased levels of AMPK phosphorylation ( Figures 6 J and 6K). Together these data demonstrate that SIRT1 is necessary for moderate doses of resveratrol to activate AMPK and increase NADand that SIRT1 acts upstream of AMPK. Interestingly, despite the different requirements for resveratrol to activate AMPK in the SIRT1 KO mice, neither dose of resveratrol improved mitochondrial function in the absence of SIRT1 ( Figure 2 ).

Given the complex interplay between SIRT1 and AMPK, it has been difficult to untangle their roles in mediating the effects of resveratrol, but the inducible SIRT1 KO mouse presented us with an opportunity to test their epistasis. Our previous work showed that resveratrol increases levels of activated (phosphorylated) AMPK in vivo (), indicating that AMPK may play a role in mediating resveratrol's benefits. This was supported by a subsequent study demonstrating that AMPK is required for many of the metabolic effects of resveratrol in vivo (). Consistent with this, the ability of resveratrol and SIRT1 overexpression to boost cellular ATP and mtDNA copy number was prevented by knockdown of the AMPKα1 subunit or treatment with Compound C, an AMPK inhibitor ( Figures 6 A–6E and Figures S7 A–S7E).

(K) Quantification of AMPK activity evaluated by the ratio of quantification of p-AMPK and AMPK in gastrocnemius of WT and SIRT1 Tg mice (n = 6, ∗ p < 0.05 versus WT).

(J) Representative immunoblot for p-AMPK (Thr172), and total AMPK in gastrocnemius of WT and SIRT1 Tg mice.

(G) Quantification of AMPK activity evaluated by the ratio of p-AMPK and AMPK in gastrocnemius of WT and SIRT1 KO mice on experimental diets (n = 8).

(F) Representative immunoblot for p-AMPK (Thr172) and total AMPK in gastrocnemius of WT and SIRT1 KO mice on experimental diets.

(E) Mitochondrial DNA content analyzed by quantitative PCR in C2C12 cells infected with AMPKα or nontargeting shRNA and with adenovirus overexpressing SIRT1 or empty vector. Relative expression values were normalized to control (n = 5, ∗ p < 0.05 versus empty DMSO).

(D) ATP content in C2C12 cells infected with AMPKα or nontargeting shRNA and treated with adenovirus overexpressing SIRT1 or empty vector (n = 5, ∗ p < 0.05 versus empty DMSO).

(C) Mitochondrial DNA content analyzed by quantitative PCR in C2C12 cells infected with AMPKα or nontargeting shRNA and treated with 25 μM resveratrol for 24 hr. Relative expression values were normalized to WT (n = 5, ∗ p < 0.05 versus DMSO).

(B) ATP content in C2C12 cells infected with AMPKα or nontargeting shRNA and treated with 25 μM resveratrol for 24 hr (n = 5, ∗ p < 0.05 versus DMSO).

(A) Representative immunoblot for AMPKα and tubulin in C2C12 cells infected with AMPKα or nontargeting shRNA.

In contrast to the knockout mouse, treatment of primary hepatocytes isolated from liver-specific SIRT1 KO (Alb-Cre; SIRT1mice or in HepG2 cells demonstrated that the ability of resveratrol (25 μM) to induce changes in gene expression, AMPK activation, and mitochondrial function of hepatocytes is SIRT1 dependent ( Figures S6 H–S6L and Figure S7 J). These data indicate that the benefits of resveratrol on liver physiology in the SIRT1 KO mice may be due to signaling from other tissues or the incomplete deletion of SIRT1 in the livers of these mice. As further evidence of the important role signaling between tissues can play, we have observed that overexpression of SIRT1 exclusively in SF1 neurons is sufficient to induce expression of genes involved in mitochondrial biogenesis in skeletal muscle ( Figure S6 M).

As resveratrol has previously been found to improve metabolic function in mice on a high-fat diet () and mitochondrial dysfunction is known to play an important role in the development of glucose imbalance in diabetes (), we sought to determine whether the ability of resveratrol to improve glucose homeostasis was lost in our inducible SIRT1 knockout mice. As expected, blood glucose levels were elevated in high-fat-diet-fed mice under both fed and fasted conditions. Resveratrol treatment partially prevented the increase in both fed and fasted blood glucose levels induced by high-fat diet in both WT and SIRT1 KO mice, suggesting that these effects are at least partially independent of SIRT1 ( Figures S5 A and S5B). Similarly, resveratrol-treated WT and SIRT1 KO mice performed better in a glucose tolerance test ( Figures S5 D and S5E). As the liver is known to play a key role in regulating glucose homeostasis and resveratrol was previously shown to improve liver function (), we evaluated liver metabolism in response to resveratrol treatment. Liver weight and triglycerides were reduced in both WT and SIRT1 KO mice ( Figures S5 C and S5F). Analysis of gene expression in the livers of these mice revealed an overall shift toward utilization of fatty acids ( Figure S5 G) and increased mitochondrial biogenesis ( Figure S6 D), which in many cases occurred in both WT and SIRT1 KO mice. Resveratrol-treated mice also showed a significant increase in COX activity and cellular ATP levels in both WT and SIRT1 KO mice, while citrate synthase activity was unaltered ( Figures S6 A–S6C).

In a striking recapitulation of the effects of resveratrol treatment, mitochondria isolated from the skeletal muscle of the SIRT1-Tg mice had significantly greater mitochondrial membrane potential, state 3 respiration, and maximal respiration ( Figures 5 B–5E). Additionally, mtDNA copy number ( Figure 5 F) as well as mRNA levels of ETC components and regulators of mitochondrial biogenesis that were induced by resveratrol treatment were similarly upregulated in mice overexpressing SIRT1, while those unaffected by resveratrol were also unchanged in SIRT1-Tg mice ( Figures 5 G and 5H). Together these data show that overexpression of SIRT1 in skeletal muscle is sufficient to induce mitochondrial biogenesis and improve mitochondrial function. When coupled with the SIRT1 KO data establishing that SIRT1 is required for the beneficial effects of resveratrol, these results provide strong evidence that the ability of resveratrol to improve mitochondrial function in skeletal muscle is due to SIRT1-mediated stimulation of mitochondrial biogenesis.

These data are consistent with the hypothesis that resveratrol acts via SIRT1 to increase mitochondrial function in vivo. This hypothesis also predicts that SIRT1 overexpression should be sufficient to mimic the effects of resveratrol. To test this, we generated a SIRT1 transgenic mouse (SIRT1-Tg) that constitutively expresses high levels of SIRT1 in skeletal muscle and other tissues ( Figure 5 A , Figure S4 A). This mouse differs from previous whole-body SIRT1 transgenics that overexpress SIRT1 either at lower levels (1.5- to 2-fold) () or predominately in brain and adipose tissue (). Interestingly, despite the high levels of SIRT1 expression in multiple tissues (>5×), there was no detectable difference in overall appearance, body weight, or home cage behavior.

(H) PGC-1α, PGC-1β, NRF-1, NRF-2, TFAM, TFB1M, and TFB2M mRNA analyzed by quantitative RT-PCR in gastrocnemius of WT and SIRT1 Tg mice. Relative expression values were normalized to WT mice (n = 6).

(G) NDUFS8, SDHb, Uqcrc1, COX5b, and ATP5a1 mRNA analyzed by quantitative RT-PCR in gastrocnemius of WT and SIRT1 Tg mice. Relative expression values were normalized to WT mice (n = 6).

(F) Mitochondrial DNA content analyzed by means of quantitative PCR in skeletal muscle of WT and SIRT1 Tg mice. Relative expression values were normalized to WT mice (n = 6).

(C) State 4 respiration of isolated mitochondria from skeletal muscle of WT and SIRT1 Tg mice (n = 6).

(A) Representative immunoblot for SIRT1 and tubulin in gastrocnemius of 6-month-old WT and SIRT1 Tg mice.

Consistent with the results in C2C12 cells, the decrease in mtDNA copy number in HFD mice was prevented by resveratrol treatment in the gastrocnemius of WT mice, while the SIRT1 KO mice showed no response to resveratrol treatment ( Figure 4 C). Measurement and quantification of mitochondrial mass by electron microscopy showed that resveratrol treatment induced an increase in mitochondrial area in WT but not in SIRT1 KO mice ( Figure 4 D, Figure S2 ). Additionally, transcript levels of PGC-1α, PGC-1β, and the mitochondrial transcription factors TFAM and TFB2M were increased in resveratrol-treated mice in a SIRT1-dependent manner ( Figure 4 E). Importantly, these effects were not exclusive to gastrocnemius, as similar changes in citrate synthase activity, mtDNA copy number, and the transcription of both ETC components and mitochondrial biogenesis factors were also seen in the soleus and cardiac tissue of WT mice in response to resveratrol treatment but were absent in SIRT1 KO mice ( Figure S3 ). Overall, our findings from adult-inducible SIRT1 KO mice demonstrate that resveratrol increases mitochondrial biogenesis, induces a shift toward more oxidative muscle fibers, and improves mitochondrial function in mice on a high-fat diet, and that all of these beneficial effects require SIRT1.

We next sought to understand more precisely the mechanisms by which resveratrol increases mitochondrial function and ATP production, and to investigate their relationship to SIRT1. Based on previous work, we expected that resveratrol treatment would increase mitochondrial biogenesis, and we hypothesized that these effects would be dependent upon SIRT1. We first assessed citrate synthase activity, a commonly used marker of mitochondrial content. Consistent with the changes observed in fiber type and mitochondrial function, the high-fat diet decreased citrate synthase activity in the gastrocnemius of WT mice, and resveratrol treatment completely prevented this decrease. Similarly, mRNA levels of components of the mitochondrial ETC were increased by resveratrol treatment. Interestingly, none of these effects were observed in SIRT1 KO mice ( Figures 4 A and 4B ).

(E) PGC-1α, PGC-1β, NRF-1, NRF-2, TFAM, TFB1M, and TFB2M mRNA analyzed by means of quantitative RT-PCR in gastrocnemius of WT and SIRT1 KO mice on experimental diets. Relative expression values were normalized to WT SD mice (n = 4, ∗ p < 0.05 versus WT HFD, #p < 0.05 versus WT SD, +p < 0.05 versus WT SD).

(D) Electronic microscopy analysis of gastrocnemius from WT and SIRT1 KO mice on experimental diets and the respective mitochondrial area quantification (n = 4) ( ∗ p < 0.05 versus WT HFD).

(C) Mitochondrial DNA content analyzed by quantitative PCR in gastrocnemius of WT and SIRT1 KO mice on experimental diets. Relative expression values were normalized to WT SD mice (n = 8 experiments, ∗ p < 0.05 versus WT SD, #p < 0.05 versus WT HFD).

(B) NDUFS8, SDHb, Uqcrc1, COX5b, and ATP5a1 mRNA analyzed by quantitative RT-PCR in gastrocnemius of WT and SIRT1 KO mice on experimental diets. Relative expression values were normalized to WT SD mice (n = 4 experiments, ∗ p < 0.05 versus WT HFD).

Interestingly, as with the measures of mitochondrial function, these changes in muscle type were entirely dependent upon SIRT1 ( Figure 3 A). The shift toward more oxidative fibers and the SIRT1 dependence of these effects was not exclusive to gastrocnemius, as similar gene expression changes were observed in the soleus muscle as well ( Figure 3 B). This induction of oxidative type II fibers in response to resveratrol treatment in WT but not SIRT1 KO mice was confirmed by western blot and histological analysis ( Figure 3 C and 3D). These data demonstrate that the ability of resveratrol to induce a shift toward more oxidative muscle fibers, improve mitochondrial function, and increase cellular ATP requires SIRT1.

In addition to impairment in mitochondrial function, feeding of a high-fat diet is known to cause an increased abundance of glycolytic muscle fibers. Thus, we tested whether treatment with resveratrol counteracted these changes and, if so, whether SIRT1 was required. Gene expression analysis of myosin heavy-chain genes from gastrocnemius muscle indicated that while the number of type I muscle fibers was not altered by resveratrol, the abundance of highly glycolytic fast twitch (type IIb) muscle fibers was lower, and more oxidative fast twitch (type IIa and IIx) fibers were more abundant in resveratrol-treated mice ( Figure 3 A ), consistent with the findings of. Together, these changes indicate an overall shift toward more oxidative fiber types in response to resveratrol treatment.

(C) Representative immunoblot for MyHCIIa, MyHCIIb, and tubulin in gastrocnemius of WT and SIRT1 KO mice on experimental diets.

(A and B) MyHCI, MyHCIIa, MyHCIIb, and MyHCIIx mRNA analyzed by quantitative RT-PCR in gastrocnemius (A) and soleus (B) of WT and SIRT1 KO mice on experimental diets. Relative expression values were normalized to WT SD mice (n = 4, ∗ p < 0.05 versus WT HFD, #p < 0.05 versus WT SD, +p < 0.05 versus WT SD).

The function of mitochondria isolated from skeletal muscle was significantly impaired by feeding of a high-fat diet, while treatment with both high and low doses of resveratrol prevented these deleterious effects. At both low and high doses, resveratrol produced substantial increases in ADP-induced respiration (state 3), maximal respiration (FCCP-induced), mitochondrial membrane potential, and cellular ATP levels ( Figures 2 C–2G). Strikingly, none of the significant increases in mitochondrial function seen in the WT mice treated with resveratrol were observed in SIRT1 KO mice ( Figure 2 C–2G). While the beneficial effects of resveratrol were clearly evident in animals treated with both doses of resveratrol, the variability between animals receiving the higher dose was considerably greater. For this reason, the majority of our subsequent analyses focused on the cohorts receiving the lower dose of resveratrol (i.e., 25–30 mg/kg per day).

Resveratrol Improves Mitochondrial Function in Skeletal Muscle of WT Mice but Has No Effect on Adult-Inducible SIRT1 KO Mice

Deletion of the catalytic region of SIRT1 in S1-ERT2 (SIRT1 KO) mice was evaluated by western blot analysis. Full-length SIRT1 protein was nearly undetectable in skeletal and cardiac muscle of SIRT1 KO mice ( Figure 2 B) and substantially reduced (>85%) in all other tissues examined ( Figure S1 D). In contrast to the much smaller size and developmental abnormalities reported for the germline knockouts (), induction of SIRT1 knockout in adult mice did not result in any overt phenotype ( Figures S1 A and S1B). Similarly, whole-body measurements of various metabolic parameters did not reveal any obvious differences between SIRT1 KO and WT mice, although weight gain was slightly lower in KO mice fed the SD ( Table S1 ).

To test whether these findings were relevant in vivo, we generated an adult-inducible whole-body SIRT1 knockout mouse designed to circumvent the developmental issues commonly observed in germline SIRT1 knockouts. We backcrossed tamoxifen-inducible cre-ERT2 mice () to C57BL/6J and combined this with floxed SIRT1mice (). Tamoxifen treatment did not result in detectable liver damage, as reflected by serum aminotransferase levels 7 or 30 days after the final dose (see Figures S1 E–S1H online). Following tamoxifen treatment, S1ERT2 and control mice were placed on one of four different diets: a standard diet (SD), a high-fat diet (HF; 60% FDC), a high-fat diet supplemented with 400 mg resveratrol/kg of food (HFLR), or a high-fat diet supplemented with a high dose of 4 g resveratrol/kg of food (HFHR). The former is a relatively low dose used in our laboratories' previous studies, while the latter dose is similar to the concentrations used by other groups () ( Figure 2 A ). Feeding of these diets resulted in an approximate daily dose of 25–30 mg/kg per day and 215–230 mg/kg of body weight per day, respectively.

(F) Mitochondrial membrane potential of isolated mitochondria from skeletal muscle of WT and SIRT1 KO mice on experimental diets (n = 8, ∗ p < 0.05 versus WT SD, #p < 0.05 versus WT HFD).

(E) FCCP-induced respiration of isolated mitochondria from skeletal muscle of WT and SIRT1 KO mice on experimental diets (n = 8, ∗ p < 0.05 versus WT SD, #p < 0.05 versus WT HFD).

(D) State 4 respiration of isolated mitochondria from skeletal muscle of WT and SIRT1 KO mice on experimental diets (n = 8).

(C) State 3 respiration of isolated mitochondria from skeletal muscle of WT and SIRT1 KO mice on experimental diets (n = 8) ( ∗ p < 0.05 versus WT SD, #p < 0.05 versus WT HFD).

(B) Representative immunoblot for SIRT1 and tubulin in skeletal muscle and heart of WT and SIRT1 KO mice.

(A) Schematic representation of induction of SIRT1 KO and treatment with the different diets.

Generation of Adult-Inducible SIRT1 KO Mice Revealed that Ability of Resveratrol to Improve Mitochondrial Function Requires SIRT1 In Vivo

Figure 2 Generation of Adult-Inducible SIRT1 KO Mice Revealed that Ability of Resveratrol to Improve Mitochondrial Function Requires SIRT1 In Vivo

Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss.

Further analysis revealed that resveratrol treatment increased mtDNA copy number in control cells ( Figure 1 F and 1G), suggesting that increased mitochondrial biogenesis may underlie the ability of resveratrol to improve mitochondrial function. Similar to the results with mitochondrial membrane potential and ATP, knockdown of SIRT1 or EX-527 treatment completely blocked the ability of resveratrol to increase mtDNA copy number. Immunoprecipitation of the SIRT1 target PGC-1α showed a substantial decrease in PGC-1α acetylation in resveratrol-treated cells, consistent with previous reports (). Importantly, resveratrol treatment had no effect on PGC-1α acetylation in cells lacking SIRT1 ( Figure 1 H). Consistent with these findings, resveratrol treatment increased mRNA expression of a number of genes downstream of PGC-1α, including transcription factors responsible for stimulating mitochondrial biogenesis (NRF-1, NRF-2) and components of the mitochondrial electron transport chain (NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1). All of the increases in gene expression were absent in cells treated with EX-527 or in which SIRT1 expression was knocked down ( Figure 1 I, data not shown).

While both overexpression of SIRT1 and treatment with resveratrol have been shown to increase mitochondrial content via activation of PGC-1α, it remains to be established whether SIRT1 is required for resveratrol to improve mitochondrial function in skeletal muscle. Our initial investigation was performed using C2C12 myoblasts, a murine skeletal muscle cell line. Following treatment with 25 μM resveratrol, C2C12 cells showed a significant increase in mitochondrial membrane potential ( Figure 1 A ) and cellular ATP content ( Figure 1 B). Treatment with the SIRT1 inhibitor EX-527 or shRNA knockdown of SIRT1 consistently reduced mitochondrial membrane potential and ATP content and completely abolished the ability of resveratrol to improve these parameters ( Figures 1 A–1E).

(I) PGC-1α, NRF-1, NDUFS8, SDHb, Uqcrc1, COX5b, and ATP5a1 mRNA were analyzed by means of quantitative RT-PCR in C2C12 cells infected with SIRT1 or nontargeting shRNA after 24 hr treatment with 25 μM resveratrol. Relative expression values were normalized to untreated cells.

(H) C2C12 cells infected with SIRT1 or nontargeting shRNA, and expressing Flag-HA-PGC-1α, were treated with resveratrol 25 μM for 24 hr, and PGC-1α acetylation was tested in Flag immunoprecipitates. Total PGC-1α was evaluated on total extracts as input.

(F and G) (F) Mitochondrial DNA content analyzed by means of quantitative PCR in C2C12 cells treated with 10 μM EX-527 or (G) infected with SIRT1 or nontargeting shRNA and treated with 25 μM resveratrol. Relative expression values were normalized to untreated cells.

(D and E) (D) Mitochondrial membrane potential and (E) ATP content in C2C12 cells infected with SIRT1 or nontargeting shRNA and treated with 25 μM resveratrol for 24 hr.

(C) Representative immunoblot for SIRT1 and tubulin in C2C12 cells infected with SIRT1 or nontargeting shRNA.

(A and B) (A) Mitochondrial membrane potential and (B) ATP content in C2C12 cells treated with 25 μM resveratrol and 10 μM EX-527 for 24 hr.

Discussion

The adult-inducible SIRT1 knockout mouse strain described here has allowed us to directly assess the effects of resveratrol treatment in otherwise healthy adult animals lacking functional SIRT1. Using this model, we clearly demonstrate that the ability of resveratrol to stimulate mitochondrial biogenesis, increase mitochondrial function, and raise both ATP and NAD+ levels in skeletal muscle is dependent on SIRT1 in vivo. While further work is needed to fully determine the importance of SIRT1 in the ability of resveratrol to prevent metabolic syndrome and other age-related diseases, this study provides in vivo evidence that beneficial effects of resveratrol on mitochondrial function require SIRT1.

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Gorospe M.

de Cabo R.

Sinclair D.A. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Pearson et al., 2008 Pearson K.J.

Baur J.A.

Lewis K.N.

Peshkin L.

Price N.L.

Labinskyy N.

Swindell W.R.

Kamara D.

Minor R.K.

Perez E.

et al. Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. Smith et al., 2009 Smith J.J.

Kenney R.D.

Gagne D.J.

Frushour B.P.

Ladd W.

Galonek H.L.

Israelian K.

Song J.

Razvadauskaite G.

Lynch A.V.

et al. Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo. Given our observation that resveratrol increases the transcript levels of PGC-1α, mitochondrial transcription factors, and components of the electron transport chain, the beneficial effects of resveratrol on skeletal muscle are likely due to PGC-1α-mediated increases in mitochondrial biogenesis and a shift toward more oxidative muscle fibers. Consistent with this, overexpression of PGC-1α was recently reported to result in a similar shift toward more oxidative fiber types (). This model is also consistent with the data from our SIRT1-Tg mouse, which showed changes in mitochondrial biogenesis, mitochondrial function, and cellular energy status similar to those in mice treated with resveratrol. This mouse further corroborates the striking parallels between resveratrol treatment, CR, and increased SIRT1 activity ().

Gledhill et al., 2007 Gledhill J.R.

Montgomery M.G.

Leslie A.G.

Walker J.E. Mechanism of inhibition of bovine F1-ATPase by resveratrol and related polyphenols. Hawley et al., 2010 Hawley S.A.

Ross F.A.

Chevtzoff C.

Green K.A.

Evans A.

Fogarty S.

Towler M.C.

Brown L.J.

Ogunbayo O.A.

Evans A.M.

et al. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Zini et al., 1999 Zini R.

Morin C.

Bertelli A.

Bertelli A.A.

Tillement J.P. Effects of resveratrol on the rat brain respiratory chain. + levels were not significantly increased until 12 hr of treatment. Moreover, we found no evidence of a decrease in ATP or increase in AMP in animals treated with either a low or high dose of resveratrol. Thus, when moderate doses are used, it seems unlikely that resveratrol activates AMPK by altering AMP and/or ATP levels. In contrast, 50 μM resveratrol caused a dramatic decline in mitochondrial membrane potential and ATP levels. Thus, SIRT1-independent activation of AMPK by high concentrations of resveratrol may be secondary to inhibition of mitochondrial respiration and ATP synthesis. In light of the data generated using our adult-inducible SIRT1 KO mice, we can reassess the physiologic relevance of proposed models of resveratrol's action. If resveratrol induces AMPK by acting as an ATPase or complex III inhibitor (), then ATP levels should be lower in the resveratrol-treated mice, and AMPK activation should occur independently of SIRT1. We do not observe such effects using moderate doses of resveratrol in vitro or in vivo. In time course cell culture experiments, ATP levels were not altered after 1 hr and steadily increased at the 4, 6, and 12 hr time points, while NADlevels were not significantly increased until 12 hr of treatment. Moreover, we found no evidence of a decrease in ATP or increase in AMP in animals treated with either a low or high dose of resveratrol. Thus, when moderate doses are used, it seems unlikely that resveratrol activates AMPK by altering AMP and/or ATP levels. In contrast, 50 μM resveratrol caused a dramatic decline in mitochondrial membrane potential and ATP levels. Thus, SIRT1-independent activation of AMPK by high concentrations of resveratrol may be secondary to inhibition of mitochondrial respiration and ATP synthesis.

In a recent study, a new model was presented in which resveratrol increases mitochondrial biogenesis by inhibiting PDE, leading to increased levels of cAMP. This increases cellular calcium levels, thereby stimulating phosphorylation of AMPK by CamKKβ. As cellular NAD+ levels were found to be elevated under these conditions, this was proposed as a mechanism by which resveratrol activates SIRT1. This model is not consistent with the effects we have observed with moderate doses of resveratrol in vitro or in vivo, as increases in p-AMPK, NAD+, LKB1 acetylation, and mitochondrial function were entirely SIRT1 dependent. Inhibition of PDE may, however, provide an explanation for some of the effects seen in animals treated with a higher dose of resveratrol where phosphorylation of AMPK and increased levels of NAD+ are observed independently of SIRT1.

In experiments using pharmacological agents, it is well recognized that care should be taken to use the lowest effective dose to minimize the chances of off target effects. Our study exemplifies the risk of using high doses of resveratrol: we clearly show that the ability of resveratrol treatment to increase phosphorylation of AMPK both in vivo and in vitro was dependent upon SIRT1, but only at moderate doses. Moreover, treatment of cells with low doses of resveratrol mimicked the in vivo situation, but high doses of resveratrol (≥50 μM) resulted not only in SIRT1-independent activation of AMPK but also in toxic effects that included a dramatic reduction in mitochondrial membrane potential and cellular ATP levels.

Ramadori et al., 2009 Ramadori G.

Gautron L.

Fujikawa T.

Vianna C.R.

Elmquist J.K.

Coppari R. Central administration of resveratrol improves diet-induced diabetes. Knight et al., 2011 Knight C.M.

Gutierrez-Juarez R.

Lam T.K.

Arrieta-Cruz I.

Huang L.

Schwartz G.

Barzilai N.

Rossetti L. Mediobasal hypothalamic SIRT1 is essential for resveratrol's effects on insulin action in rats. In this study we chose to test the SIRT1 dependence of resveratrol in cardiac and skeletal muscle, two organs with high energetic demands requiring efficient mitochondrial function. Cell culture experiments performed with hepatocytes from liver-specific SIRT1 knockout mice demonstrated that SIRT1 is required for resveratrol and AICAR to induce phosphorylation of AMPK and increase mitochondrial biogenesis and function. In the mouse, however, resveratrol improved glucose homeostasis and liver function in both WT and SIRT1 KO mice. It remains unclear whether the SIRT1-independent effects we observed are due to an accumulation of resveratrol in specific organs, the inefficiency of SIRT1 knockdown in liver, or differences in the actions of resveratrol between cell types. The discrepancy may also be due to contributions from other tissues. For example, direct administration of resveratrol to the brain of rodents has been found to improve whole-body glucose homeostasis (), and these effects were lost when SIRT1 function in the hypothalamus was impaired (). Additionally, overexpression of SIRT1 in SF1 neurons is sufficient to induce the expression of genes involved in mitochondrial biogenesis in skeletal muscle, further demonstrating the complex interplay between tissues.