skeletal muscle regulates mitochondrial biogenesis in

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle

Brendon J. Gurd, Yuko Yoshida, Jay T. McFarlan, Graham P. Holloway, Chris D. Moyes, George J. F. Heigenhauser, Lawrence Spriet and Arend Bonen Am J Physiol Regul Integr Comp Physiol 301:R67-R75, 2011. First published 4 May 2011; doi:10.1152/ajpregu.00417.2010 You might find this additional info useful... This article cites 45 articles, 18 of which can be accessed free at: http://ajpregu.physiology.org/content/301/1/R67.full.html#ref-list-1 Updated information and services including high resolution figures, can be found at: http://ajpregu.physiology.org/content/301/1/R67.full.html

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Am J Physiol Regul Integr Comp Physiol 301: R67–R75, 2011. First published May 4, 2011; doi:10.1152/ajpregu.00417.2010.

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Mitochondrial Function/Dysfunction in Health and Disease

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle Brendon J. Gurd,1 Yuko Yoshida,3 Jay T. McFarlan,3 Graham P. Holloway,3 Chris D. Moyes,2 George J. F. Heigenhauser,4 Lawrence Spriet,3 and Arend Bonen3 1

School of Kinesiology and Health Studies, 2Department of Biology, Queen’s University, Kingston, Ontario, Canada; Department of Human Health and Nutritional Science, University of Guelph, Guelph, Ontario, Canada; and 4Department of Medicine, McMaster University, Hamilton, Ontario, Canada 3

Submitted 24 June 2010; accepted in final form 26 April 2011

exercise training

(SIRT1) is a class three deacetylase that is implicated in a wide range of cellular function, including cellular maturation and differentiation, aging, neural and cardio-protection, and hepatic and skeletal muscle metabolism (2, 15). Within skeletal muscle, SIRT1 appears to contribute in the chronic regulation of

SILENT MATING TYPE INFORMATION REGULATOR 2 HOMOLOG 1

Address for reprint requests and other correspondence: B. Gurd, School of Kinesiology and Health Studies, Queen’s Univ., Kingston, Canada (e-mail: [email protected]). http://www.ajpregu.org

metabolism through a pathway in which it deacetylates and activates peroxisome proliferator-activated receptor gamma coactivator-1␣ (PGC-1␣) (38). PGC-1␣ is a coactivator involved in activating both nuclear and mitochondrial transcription, resulting in mitochondrial biogenesis and upregulation of genes involved in lipid metabolism and oxidative phosphorylation (4, 28, 46). The ability of SIRT1 in activating PGC-1␣ has been demonstrated elegantly in selected cell lines (C2C12 cells and FaO hepatocyctes) where changes in SIRT1 protein content resulted in corresponding changes in the expression of mitochondrial genes, enzymes activity, and lipid metabolism (19, 39). However, reports surrounding SIRT1 function in mammalian muscle are less clear (12, 22, 42). Skeletal muscle is capable of undergoing dramatic changes in mitochondrial content. This biogenic process is already initiated after a single bout of exercise in rats (45), and a pronounced increase in muscle mitochondria is observed after a period of exercise training in humans (20, 37), as well as after chronic electrical stimulation in rats (9, 22, 31). Given the plasticity of skeletal muscle mitochondrial content, understanding the association between SIRT1 and PGC-1␣ in this tissue is important. While one report has demonstrated a positive association between SIRT1 protein and exercise training (42), a number of others have failed to observe a positive relationship between SIRT1 mRNA and/or protein and mitochondrial biogenesis. For example, there was an inverse relationship between mitochondrial content and SIRT1 mRNA (32, 41), as well as SIRT1 protein content (22) in heart and skeletal muscle. Moreover, interventions that promoted mitochondrial biogenesis, namely, chronic muscle contraction (7 days), 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) administration (5 days) (22), and exercise training (12) induced a reduction in SIRT1 protein content (12, 22). In addition, SIRT1 overexpression reduced mitochondrial content in PC12 cells (35) and in skeletal muscle (22). In contrast to the unexpected inverse relationship between SIRT1 protein content and mitochondrial biogenesis, increased SIRT1 deacetylase activity is associated with the upregulation of mitochondrial biogenesis in chronically stimulated and AICAR-treated mammalian skeletal muscle (12, 22), and in hearts of exercise-trained rats (17). Taken together, in contrast to initial observations in C2C12 cells and FaO hepatocyctes (19, 39), these findings indicate that in vivo SIRT1 protein content per se does not appear to contribute to PGC-1␣-mediated mitochondrial biogenesis in mammalian

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Gurd BJ, Yoshida Y, McFarlan JT, Holloway GP, Moyes CD, Heigenhauser GJ, Spriet L, Bonen A. Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle. Am J Physiol Regul Integr Comp Physiol 301: R67–R75, 2011. First published May 4, 2011; doi:10.1152/ajpregu.00417.2010.—Silent mating type information regulator 2 homolog 1 (SIRT1)-mediated peroxisome proliferator-activated receptor gamma coactivator-1␣ (PGC-1␣) deacetylation is potentially key for activating mitochondrial biogenesis. Yet, at the whole muscle level, SIRT1 is not associated with mitochondrial biogenesis (Gurd, BJ, Yoshida Y, Lally J, Holloway GP, Bonen A. J Physiol 587: 1817–1828, 2009). Therefore, we examined nuclear SIRT1 protein and activity in muscle with varied mitochondrial content and in response to acute exercise. We also measured these parameters after stimulating mitochondrial biogenesis with chronic muscle contraction and 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) administration in rodents and exercise training in humans. In skeletal and heart muscles, nuclear SIRT1 protein was negatively correlated with indices of mitochondrial density (citrate synthase activity, CS; cytochrome oxidase IV, COX IV), but SIRT1 activity was positively correlated with these parameters (r ⬎ 0.98). Acute exercise did not alter nuclear SIRT1 protein but did induce a time-dependent increase in nuclear SIRT1 activity. This increase in SIRT1 activity was temporally related to increases in mRNA expression of genes activated by PGC-1␣. Both chronic muscle stimulation and AICAR increased mitochondrial biogenesis and muscle PGC-1␣, but not nuclear PGC-1␣. Concomitantly, muscle and nuclear SIRT1 protein contents were reduced, but nuclear SIRT1 activity was increased. In human muscle, training-induced mitochondrial biogenesis did not alter muscle or nuclear SIRT1 protein content, but it did increase muscle and nuclear PGC-1␣ and SIRT1 activity. Thus, nuclear SIRT1 activity, but not muscle or nuclear SIRT1 protein content, is associated with contractionstimulated mitochondrial biogenesis in rat and human muscle, possibly via AMPK activation.

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NUCLEAR SIRT1 ACTIVITY REGULATES MUSCLE OXIDATIVE CAPACITY

METHODS

Experiments were performed with female Sprague-Dawley rats (3– 6 mo, 250 –300 g) that were bred on site and housed at 22.5°C on a 12:12-h light-dark cycle (0700 –1900, light; 1900 – 0700, dark) cycle. At the end of each experiment, animals were anesthetized with Somnotol (60 mg/kg), and the selected tissues were harvested and either processed immediately for nuclear extraction or flash frozen (liquid nitrogen) and stored at ⫺80°C until analyzed. Immediately after the harvesting of tissues, the animals were killed with an overdose of Somnotol. The procedures for the experimental treatments, the harvesting of the muscle tissue, and the killing of animals were approved by the animal care committee at the University of Guelph. Oxidative Capacity in Heart and Skeletal Muscle To compare nuclear SIRT1 activity with oxidative capacity in muscles tissues with different mitochondrial content, the red tibialis anterior (RTA) and white tibialis anterior (WTA) muscles, along with the heart (HRT), were harvested from anesthetized rats. Separation of the red and white compartments of the tibialis anterior muscle has been described previously (22). Following this procedure and all experiments described below, whole muscle lysates were prepared from frozen tissue, while isolated nuclei were prepared from fresh tissue samples. Acute Exercise To examine the acute effects of exercise on nuclear SIRT1 content and activity, rats ran on a rodent treadmill for 2 h at 15 m/min, followed by an increase in speed of 5 m/min every 5 min until

volitional cessation of exercise. Prior to the exercise day, animals were familiarized to the treadmill at slow speeds for three consecutive days followed by a full 24-h rest before the start of the exercise bout. We examined the red portion of the gastrocnemius, as this muscle is recruited during running exercise in rats (14). The red gastrocnemius muscle was harvested, following anesthetization, from nonexercised animals (control), immediately following cessation of exercise (t ⫽ 0 h), and after 3 h of recovery from exercise (t ⫽ 3 h). Chronic Muscle Stimulation In an attempt to examine the role of nuclear SIRT1 activity in the upregulation of muscle oxidative capacity, mitochondrial biogenesis was induced by 7 days of electrical stimulation. The RTA and WTA tibialis muscles from rats were chronically stimulated in one hindlimb, as described previously (9, 22, 31). Briefly, stainless-steel electrodes were sutured to muscles on either side of the peroneal nerve and passed subcutaneously from the thigh to the back of the neck, where they were attached to an external electronic stimulator. Animals recovered from surgery for 7 days before stimulation (12 Hz, 50-ms duration) was initiated. Stimulation of the peroneal nerve was administered 24 h a day for 7 days. Twenty-four hours following cessation of the stimulation, chronically contracting muscles (RTA and WTA) were removed. Muscles from the sham-operated contralateral limb were also removed and used as control. AICAR Treatment Mitochondrial biogenesis was also induced via chronic AICAR administration, as described previously (22). Briefly, rats were injected subcutaneously with a bolus of AICAR (1 mg/g AICAR) dissolved in saline solution (4.5% saline) for 5 days. Control animals received an equivalent volume of saline solution alone (subcutaneously). Twenty-four hours after the final AICAR or saline injection, the RTA and WTA muscles were removed. Human Exercise Training We also examined the effect of exercise training on SIRT1 in human skeletal muscle. Exercise training was performed by seven volunteers (4 females and 3 males) who were recreationally active. The experimental protocol utilized for human exercise training was approved by the Research Ethics Boards of the University of Guelph and McMaster University. Prior to, and following (between 24 and 48 h following the last training bout) the training period, subjects completed a continuous incremental cycling test to exhaustion on an electromagnetically braked cycle ergometer (Lode Instrument, Groningen, The Nether˙ O2peak using a metabolic measurelands) to determine pulmonary V ment system (Vmax Series 229; Sensormedics, Yorba Linda, CA). Subjects trained 7 times over a 2-wk period on a cycle ergometer (Monark 894 E; Vasbro, Sweden) at a power output that elicited ˙ O2peak. Subjects completed 10 exercise intervals per session, ⬃90% V with each interval lasting 4 min and separated by 2 min of rest, as described previously (37). Pretraining and posttraining muscle biop˙ O2peak test, using sies were obtained at rest and ⬃48 h after the final V the needle biopsy technique (6). Nuclear Extraction Nuclei were isolated from muscle using a commercially available kit (Pierce Biotechnology, Rockford, IL). Briefly, harvested muscles were immediately placed in 750 ␮l of PBS, where they were minced and briefly homogenized (⬃3 s at 24,000 rpm). Cytosolic and nuclear extraction was performed using the cytosolic and nuclear extraction reagents supplemented with 1 mM sodium orthovanadate, 1 mM PMSF, and 10 ␮g/ml of pepstatin A, aprotinin, and leupeptin. Isolated nuclei were washed 15⫻ in PBS and PBS supplemented with 0,1% Nonidet P-40 alternatively before nuclear extraction. To confirm the

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skeletal muscle. Instead, SIRT1 activity appears to be the critical determinant. Interestingly, we have unexpectedly observed an inverse relationship between SIRT1 activity and protein expression (22). The lack of a relationship between SIRT1 protein expression and activity may suggest that SIRT1 protein is localized to specific subcellular compartments, raising the possibility that SIRT1 protein and/or its activity can be independently regulated within these compartments. SIRT1 is present in the nucleus (32) and can be localized to the cytoplasm in HeLa cells (24), and can be actively translocated between the nucleus and cytosol in C2C12 cells (43). The location of SIRT1 in the nucleus in these cells increased its ability to deacetylate nuclear targets (43). Similarly, the translocation of PGC-1␣ to the nucleus has recently been shown to be important for stimulating mitochondrial biogenesis (45), a process that is already evident after a single bout of exercise (45) and that is accompanied by PGC-1␣ deacetylation (10). Thus, to impact PGC1␣-mediated transcription in skeletal muscle, SIRT1 would presumably need to be localized and activated in the nucleus. Whether the subcellular localization and activation of SIRT1 in mammalian skeletal muscle are central to inducing mitochondrial biogenesis remains to be determined. Therefore, we have examined the content and the activity of SIRT1 in the nucleus in rats across metabolically heterogeneous muscle tissues with differing oxidative capacities and after treatments designed to stimulate mitochondrial biogenesis, including acute exercise, chronic muscle stimulation, and chronic AICAR treatment. In addition, we have also examined in human skeletal muscle changes in SIRT1 protein content and activity in the nucleus of following 2 wk of exercise training, which is known to stimulate mitochondrial biogenesis (20).


nuclear extract purity, all muscle extracts were analyzed using Western blotting for the presence of the cytosolic protein lactate dehydrogenase (LDH). LDH, a highly abundant nonnuclear protein, was typically not detected in our highly purified nuclear extracts or, if present at all, constituted much less than 5% contamination (Fig. 1). Furthermore, our ability to detect the nuclear proteins PGC-1␣ and SIRT1 confirm that we were able to obtain a pure nuclear extract. The sample shown (Fig. 1) is from a series of isolations in RTA muscle; this control experiment yielded similar purity in all experiments for all muscle tissues examined (⬍5% contamination). Western Blot Analysis

mRNA Content RNA was extracted using the Qiagen RNA mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions for muscle tissue. One microgram of resulting RNA was reverse transcribed using the Qiagen Omniscript reverse transcription kit (Qiagen). Fifty micrograms of resulting cDNA was used for real-time PCR with Promega’s Go Taq quantitative PCR SYBRgreen master mix (Promega, Madison, WI) with the following cycles 95°C for 15 min, 40 cycles of 95°C 15 s, 60°C for 30 s and 72°C 36 s, followed by a dissociation curve to assess specificity of the reaction. Tata binding protein (TBP) was used as an endogenous control. The following primer sets were used: ␦-aminolevulinate synthase (ALAS) forward, 5=AAGAAACCCCTCCAGCCAATG-3= and ALAS reverse, 5=GGAGTCTGTGCCATCTGGGA-3=; citrate synthase forward, 5=GTACTATGGCATGACGGAGATG-3= and citrate synthase reverse, 5=-TCCGTGCTCATGGACTTG-3=; cytochrome c forward, 5=TGTGGAAAAAGGAGGCAAGCA-3= and cytochrome c reverse, 5=CGCCCAAACAGACCATGGAG-3=; PGC-1␣ forward, 5=-CAATGAGCCCGCGAACATAT-3= and PGC-1␣ reverse 5=-CAATCCGTCTTCATCCACCG-3=; and TBP forward 5=-TGAGTTGCTTGCTCTGTGCT-3= and TBP reverse, 5=-ACTTGCTTGTGTGGGAAAGG-3=. SIRT1 Activity Nuclear SIRT1 activity was measured using a SIRT1 fluorometric assay kit (BIOMOL, Plymouth Meeting, PA), as described by the manufacturer. Twenty-five microliters of nuclear extract was incubated with 15 ␮l of Fluor de Lys-SIRT1 substrate (100 ␮M) and

NAD⫹ (100 ␮M) for 30 min at 37°C. The reaction was stopped by the addition of 50 ml of developer reagent and nicotinamide (2 mM), and the fluorescence was subsequently monitored for 30 min at 360 nm (excitation) and 460 nm (emission). The change in fluorescence [arbitrary fluorescence units (AFU)] per minute was normalized to the amount of total muscle (mg wet weight) used for the nuclear extraction procedure. To validate the specificity of the Fluor de Lys substrate, we compared the relationship between increasing recombinant SIRT1 protein and SIRT1 activity in vitro. These experiments yielded a linear relationship between SIRT1 protein and SIRT1 deacetylase activity (data not shown). In addition, we have previously shown an increase in whole muscle SIRT1 activity in vivo following acute overexpression of SIRT1 protein via transfection (22). Citrate Synthase and ␤-Hydroxyacyl-CoA Dehydrogenase Activity A small portion of muscle (⬃10 mg) from HRT, RTA, and WTA muscle was used for determination of citrate synthase (CS) and ␤-hydroxyacyl-CoA dehydrogenase (␤-HAD) activity. Total CS and ␤-HAD activity were measured in Tris·HCl buffer (50 mM Tris·HCl, 2 mM EDTA, and 250 ␮M NADH pH 7.0) and 0.04% Triton-X. The CS reaction was started by the addition of 10 mM oxaloacetate, and activity was measured spectrophotometrically at 37°C by measuring the disappearance of NADH at 412 nm, while the ␤-HAD reaction was started by the addition of 100-␮m acetoacetyl-CoA, and the absorbance was measured at 340 nm over a 2-min period (37°C) (5). Statistics In rat experiments, two-way ANOVA were used to compare the effects of muscle type and either chronic stimulation, AICAR treatment, or acute exercise on enzyme activity and protein expression. Post hoc tests were conducted using the Bonferroni test. Paired t-tests were used to compare the effects of exercise training in humans on enzyme activity and protein expression. Correlation coefficients were determined using least-squares linear regression. Throughout, statistical significance was accepted at a P ⬍ 0.05, unless otherwise noted. RESULTS

Oxidative Capacity and Nuclear SIRT1 Protein and Activity Muscle and nuclear SIRT1 protein. Whole muscle SIRT1 protein was highest in the WTA (100%) followed by RTA (86%) and heart (36%), respectively (Fig. 2A). However, nuclear SIRT1 protein content was similar in the WTA and RTA (100%), but was significantly lower in the heart (28%) (Fig. 2B). Nuclear SIRT1 activity. In contrast to SIRT1 protein, the nuclear activity of SIRT1 differed among the muscle tissues and was highest in the heart (100%), followed by the RTA (31%) and WTA (20%) (Fig. 2C). This pattern was also observed for indices of muscle tissue mitochondrial content, namely, COX IV protein, CS activity, and ␤-HAD activity (data not shown). Across the muscle tissues examined, there was a strong positive relationship between nuclear SIRT1 activity, and all these indices of oxidative capacity (COX IV, r ⫽ 0.99, Fig. 3A; CS activity, r ⫽ 0.98, Fig. 3B; ␤-HAD activity, r ⫽ 0.99, Fig. 3C). Adaptive Responses of SIRT1 to Acute Exercise, Chronic Muscle Stimulation, and AICAR in Rats

Fig. 1. Nuclear extracts are clear of cytosolic contamination. Lactate dehydrogenase (LDH), both muscle lysates (MS), and nuclear extracts (NE) obtained from RTA muscles as an index of contamination of the nuclear extract.

Acute exercise and nuclear SIRT1 activity. After a single bout of exercise, there was no change, relative to control, in muscle protein contents of SIRT1, PGC-1␣, or COX IV, either immediately after exercise, or 3 h after exercise. Similarly, the nuclear SIRT1 (Fig. 4A) protein was not altered. There was a graded increase in nuclear SIRT1 activity immediately (0 h;


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Protein contents were determined on either the nuclear extract or a whole muscle lysate isolated from the tissues, as previously described (7, 8, 30). Proteins were separated by SDS-PAGE using a 7.5% (SIRT1, PGC-1␣, LDH) or 12.5% cytochrome oxidase subunit IV (COX IV) polyacrylamide gel and were subsequently transferred to a polyvinylidene difluoride membrane. For the detection of proteins, commercially available antibodies were used for SIRT1 (Upstate Biotechnology, Temecula, CA), PGC-1␣ (Calbiochem, San Diego, CA), COX IV (Molecular Probes, Carlsbad, CA), and LDH (Abcam). Proteins were visualized by chemiluminescence detection, according to the manufacturer’s instructions (Perkin Elmer Life Sciences, Boston, MA). Blots were quantified using the ChemiGenius 2 Bioimaging System (Syngene, Cambridge, UK). Equal amounts of protein were added for all Western blots, and Ponceau staining was used to control for loading differences.

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Fig. 2. Nuclear silent mating type information regulator 2 homolog 1 (SIRT1) activity but not whole muscle or nuclear SIRT1 protein is increased in heart muscle compared with skeletal muscle. Whole muscle (A) and nuclear protein content of SIRT1 (B) are shown along with nuclear SIRT1 activity (C). (n ⫽ 4). *Significantly different (P ⬍ 0.05) from white tibialis anterior (WTA) muscle. †Significantly different (P ⬍ 0.05) from red tibialis anterior (RTA) muscle.

Chronic Muscle Stimulation and Nuclear SIRT1 Activity As chronically increased muscle activity induces mitochondrial biogenesis and the oxidative capacity of skeletal muscle, we examined the nuclear content and activity of SIRT1 after 7 days of chronic electrical stimulation of the RTA and WTA. As we have observed previously (22), there was an increase in whole muscle COX IV protein (RTA, ⫹23%; WTA, ⫹70%) and whole muscle PGC-1␣ protein (RTA, ⫹26%; WTA, ⫹87%), consistent with an increase in mitochondrial biogenesis. PGC-1␣ protein content in the nucleus was not altered in either the RTA or WTA (data not shown).

Muscle and nuclear SIRT1 protein and activity. After 7 days of chronic muscle stimulation, there was a marked decrease in SIRT1 protein, both at the whole muscle level (RTA ⫺26%, WTA ⫺40%) (Fig. 5A) and in the nucleus (RTA ⫺24%, WTA ⫺53%) (Fig. 5B). In marked contrast, nuclear SIRT1 activity was increased in both the RTA (⫹84%) and WTA (⫹127%) (Fig. 5C). AICAR Administration Following 5 days of AICAR treatment, whole muscle COX IV protein, an index of mitochondrial biogenesis, was increased (RTA, ⫹14%; WTA, ⫹25%). Similarly, whole muscle PGC-1␣ protein increased (RTA, ⫹17%; WTA, ⫹47%). Nuclear PGC-1␣ protein was not altered (data not shown). Muscle and nuclear SIRT1 protein and activity. While whole muscle SIRT1 protein was decreased in the RTA but not the WTA (Fig. 6A), there was no change in nuclear SIRT1 protein content (Fig. 6B). However, there was a modest increase in SIRT1 activity in the RTA (⫹8%) and a larger increase in the WTA (⫹30%) (Fig. 6C). Effects of Exercise Training on SIRT1 Activity in Human Muscle ˙ O2peak (⫹7%), markers of Exercise training increased V oxidative capacity (CS activity; ⫹9%), COX IV protein (⫹35%), and fatty acid oxidation ␤-HAD activity (⫹19%; Fig. 7A). Muscle and nuclear PGC-1␣. Muscle PGC-1␣ protein was increased after 2 wk of training (⫹36%; 7B). Similarly,

Fig. 3. Nuclear SIRT1 activity is correlated with oxidative capacity in skeletal muscle and heart. Nuclear SIRT1 activity was positively correlated with cytochrome oxidase subunit (COX IV) (r ⫽ 0.99, P ⬍ 0.05; A), citrate synthase (CS) activity (r ⫽ 0.98, P ⬍ 0.05; B), and ␤-hydroxyacyl-CoA dehydrogenase (␤-HAD) activity (r ⫽ 0.99, P ⬍ 0.05; C). (n ⫽ 4). AJP-Regul Integr Comp Physiol • VOL

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⫹17%) and 3 h after exercise (⫹33%) (Fig. 4B). Linear regression analysis indicated that the nuclear SIRT1 activity increased progressively (slope⫽1.26, P ⬍ 0.05; Fig. 4B). Nuclear PGC-1␣ (Fig. 4C) increased immediately after exercise (⫹32%) and at 3 h postexercise (⫹51%). There was also an increase in mRNA content for several genes targeted by PGC-1␣ and associated with mitochondrial biogenesis (Fig. 4D). PGC-1␣ ALAS and citrate synthase all increased immediately following exercise and further increased during the 3-h recovery period. Cytochrome c was elevated immediately following exercise but did not increase further following the 3-h recovery period. The increases in PGC-1␣ (r ⫽ 0.98), ALAS (r ⫽ 0.95), and citrate synthase (r ⫽ 0.99) were all positively associated with the observed linear increase in nuclear SIRT1 activity. A somewhat weaker positive relationship was also observed between increases in nuclear PGC-1␣ protein and increases in mRNA (PGC-1␣ r ⫽ 0.93; ALAS, r ⫽ 0.89; citrate synthase, r ⫽ 0.95).


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there was also an increase in nuclear PGC-1␣ protein (⫹34%; Fig. 7C). Muscle and nuclear SIRT1 protein and activity. Exercise training did not alter muscle SIRT1 protein (Fig. 7D) or nuclear SIRT1 protein (Fig. 7E). In contrast, nuclear SIRT1 activity demonstrated a trend toward an increase (P ⫽ 0.08; ⫹12%; Fig. 7F). DISCUSSION

We have determined SIRT1 protein content and the activity of SIRT1 within the nucleus of skeletal muscle through a series of experiments aimed at examining the role of SIRT1 in regulating the oxidative capacity of skeletal muscle. These experiments have demonstrated 1) that nuclear SIRT1 activity, rather than nuclear SIRT1 protein content, is correlated with the oxidative capacity of heart and skeletal muscle, 2) that increased nuclear SIRT1 activity in skeletal muscle accompanies the mitochondrial biogenesis induced by chronic muscle

stimulation and AICAR administration in rat muscle, 3) that nuclear SIRT1 activity tended to be higher following exercise training in humans, and 4) that the induction of nuclear SIRT1 activity in rats was already evident after a single exercise bout, and this increase was associated with an increase in the mRNA expression of genes targeted by PGC-1␣. However, unexpectedly, in all experiments, in both rats and humans, nuclear SIRT1 activity was inversely related to nuclear SIRT1 protein content. Role of Nuclear SIRT1 Activity in the Regulation of Oxidative Capacity We have confirmed our previous findings (22) of an inverse relationship between SIRT1 protein expression in whole muscle homogenates and markers of oxidative capacity across a range of muscles in rats. This result has recently been corroborated by Chabi et al. (12). Indeed, these studies have demonstrated a negative relationship between SIRT1 protein and

Fig. 5. Nuclear SIRT1 activity and mitochondrial biogenesis are increased following chronic electrical stimulation. Both whole muscle (A) and nuclear (B) SIRT1 protein were reduced following stimulation, while nuclear SIRT1 activity was increased (C). (n ⫽ 6). *Significantly different (P ⬍ 0.05) from control. †Significantly different (P ⬍ 0.05) from red muscle within same condition. AJP-Regul Integr Comp Physiol • VOL

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Fig. 4. Acute exhaustive exercise results in activation of nuclear SIRT1 and induction of mitochondrial biogenesis. Following exercise in rats, the nuclear content of SIRT1 (A) was unchanged. The nuclear activity of SIRT1 increased in a linear fashion (B), while the nuclear content of peroxisome proliferatoractivated receptor gamma coactivator-1␣ (PGC-1␣) was increased immediately following and 3 h after exercise (C). Acute exercise also induced an increase in mRNA expression of genes both targeted by PGC-1␣ and associated with mitochondrial biogenesis (D). (n ⫽ 6). *Significantly different (P ⬍ 0.05) from control. †Significantly different (P ⬍ 0.05) 0 h.

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Fig. 6. Chronic AICAR administration results in elevated nuclear SIRT1 activity and mitochondrial biogenesis. Whole muscle SIRT1 (A) decreased while nuclear SIRT1 (B) was unchanged. Nuclear SIRT1 activity was increased (C) (n ⫽ 5). *Significantly different (P ⬍ 0.05) from control. †Significantly different (P ⬍ 0.05) from red muscle within same condition.

geted by PGC-1␣, Fig. 4). Interestingly there was also a linear relationship between the apparent increase in PGC-1␣ transcriptional activity and nuclear PGC-1␣ protein content. While this latter relationship was slightly weaker than the relationship observed with nuclear SIRT1 activity, our data indicate that the increase in PGC-1␣ transcriptional activity following acute exercise in rats is temporally related to increases in both nuclear SIRT1 activity and nuclear translocation of PGC-1␣ protein. These findings are consistent with work from cell lines (C2C12 cells, FaO hepatocytes) in which increased SIRT1 deacetylase activity was associated with increased expression of mitochondrial genes, enzyme activity, and lipid metabolism (1, 19, 39). In addition, activation of SIRT1 by resveratrol (3, 16, 25) or SRT 1720 (16, 33) increased mitochondrial content of both liver (3) and muscle (25), and the maximal activity of citrate synthase (25, 33) and palmitate oxidation (16) in muscle. These improvements have been linked to increases in SIRT1-mediated deacetylation, and activation, of PGC-1␣ (3, 16, 25) leading to the proposed model of control of mitochondrial content, whereby activation of SIRT1 increases deacety-

Fig. 7. Effects of exercise training on nuclear SIRT1 and PGC-1␣ in human skeletal muscle. Exercise training induced increases in oxidative capacity, as evidenced by increases in COX IV protein content (A). Whole muscle (B) and nuclear PGC-1␣ were increased following training, while there was no change in whole muscle (D) or nuclear (E) SIRT1 protein. There was a trend for SIRT1 activity being increased (P ⫽ 0.08; F). *Significantly different (P ⬍ 0.05) from control, P ⫽ 0.08 for nuclear SIRT1 activity. AJP-Regul Integr Comp Physiol • VOL

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PGC-1␣ protein content in rat skeletal muscle (12, 22). The novel finding from the current set of experiments is the positive relationship between nuclear SIRT1 activity and several markers of oxidative capacity in skeletal and heart muscle. These data are consistent with previous findings of increased nuclear SIRT1 activity following training in rat heart muscle (17) and highlights the premise that nuclear SIRT1 activity rather than whole muscle or nuclear SIRT1 protein is an important determinant of oxidative capacity in muscle in vivo. The results in the present study suggest that an increase in nuclear SIRT1 activity, whether by acute or chronic exercise/ contraction, occurs in response to stimuli that increase mitochondrial biogenesis in both rat and human skeletal muscle Indeed, increases in mitochondrial protein content observed following chronic contractile activity (Fig. 5) and AICAR injections (Fig. 6) were both accompanied by an elevated nuclear SIRT1 activity. Further, the linear increase in nuclear SIRT1 activity observed following acute exercise was positively associated with an increase in PGC-1␣ transcriptional activity (as evidenced by increased expression of genes tar-


Nuclear SIRT1 in Human Skeletal Muscle Following Exercise Training There are limited and contradictory data surrounding the role of SIRT1 in exercise-induced mitochondrial biogenesis in humans. In obese subjects and in healthy individuals, aerobic training (combined with calorie restriction) (13) and interval training (29) increased SIRT1 mRNA and protein, respectively. In contrast, following 6 wk of interval training, we have observed a decrease in whole muscle homogenate SIRT1 protein (21). This finding was not repeated in the current study following only 2 wk of interval training, suggesting that the effect may only manifest after a period of training that is in excess of 2 wk. This decrease in SIRT1 protein observed previously (21) paralleled chronic contraction-induced reductions in whole muscle SIRT1 protein (22). Importantly, we have observed that SIRT1 activity and SIRT1 protein are not correlated, whether in whole muscle (21, 22) or in the nucleus (present study). However, the findings of increased whole muscle (21, 22) and a trend for increased nuclear SIRT1 activity (Fig. 7) after a period of training are consistent with the proposed model (39) of SIRT1 participating in activation of PGC-1␣ and the regulation of mitochondrial biogenesis in human skeletal muscle. SIRT1 Protein, Activity, and Intracellular Localization The current study demonstrates that increases in oxidative capacity (Figs. 2 and 3) and mitochondrial biogenesis (Figs. 5–7) were accompanied by increased nuclear SIRT1 activity but not SIRT1 protein, as whole muscle SIRT1 protein expression was either decreased (Figs. 2 and 3) or unchanged (Fig. 7). This lack of a relationship, between SIRT1 protein and activity in vivo, as well as the decreased oxidative capacity and reduction of PGC-1␣ in skeletal muscle in which SIRT1 was overexpressed (22), are suggestive of an inhibition of mitochondrial biogenesis when whole muscle SIRT1 protein is increased. This raises the specter that the biological effects mediated by cytosolic SIRT1 differ from the effects mediated by SIRT1 that is localized to the nucleus. There is some evidence for this suggestion, since increases in cytosolic SIRT1 protein are linked to increased apoptotic signaling in

HeLa cells (24, 36). Thus, if the increases in SIRT1 activity following its overexpression were confined largely to the cytosol (a question we are currently examining), it would be possible that the resulting fall in muscle oxidative capacity was a result of increased cytosolic apoptotic signaling. This remains to be determined. Regulation of Nuclear SIRT1 Activity We have demonstrated that nuclear SIRT1 is activated by both chronic muscle stimulation and acute muscle contraction in rats and exercise training in humans (Figs. 4, 5, and 7). This activation appears to be mediated, in part, by an AMPK-linked mechanism as chronic AICAR also increased nuclear SIRT1 activity (Fig. 6). Recent work has demonstrated that the SIRT1 response following exercise is dependent on functional AMPK in mice (11), and a positive relationship between AMPK and SIRT1 activity in skeletal muscle is gaining general acceptance (18). While changes in the NAD⫹/NADH ratio may also contribute to the observed increases in SIRT1 activity (10), there is also evidence that SIRT1 can be modified posttranslationally by reversible phosphorylation (34, 40). Our results demonstrating increases in nuclear SIRT1 activity, independent from changes in nuclear SIRT1 protein content are consistent with the activity of SIRT1 being regulated by posttranslational mechanisms in skeletal muscle in vivo. Perspectives and Significance We have observed a positive association between oxidative capacity and the activity of SIRT1 in the nucleus across a range of muscle tissues. We have also observed increases in nuclear SIRT1 activity in concert with increases in mitochondrial biogenesis in both rat (chronic electrical stimulation, AMPK activation) and human (exercise training) skeletal muscle. In almost all instances, increases in nuclear SIRT1 activity were associated with a decrease in whole muscle SIRT1 protein content and either no change or a decrease in nuclear SIRT1 protein content. A single acute bout of exercise also increased nuclear SIRT1 activity in muscle, and this increase was associated with an increase in the apparent transcriptional activity of PGC-1␣ (as indicated by increased mRNA content of target genes). These results support a positive role for nuclear SIRT1 activity in mitochondrial biogenesis, likely via deacetylation and activation of nuclear PGC-1␣. These findings also underscore the importance of determining both SIRT1 protein content and activation status and the intracellular localization of activated SIRT1. ACKNOWLEDGMENTS A. Bonen is the Canada Research Chair in Metabolism and Health. GRANTS These studies were supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Ontario, and the Canada Research Chair program. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. REFERENCES


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lation of PGC-1␣ within the nucleus, increasing transcriptional activity of PGC-1␣ and subsequently mitochondrial gene expression (38). Thus, our results suggest that increases in PGC1␣-mediated mitochondrial biogenesis in skeletal muscle are mediated, at least in part, by increased nuclear SIRT1 activity and suggest that activation of nuclear SIRT1 is likely a key step in a complex pathway that activates PGC-1␣-mediated transcription. Other factors that are likely involved in this pathway include p38 MAP kinase, AMPK kinase (23, 44, 45), and Akt/PKB (27). It is important to note that we have been unable to confirm that the observed increase in SIRT1 activity was accompanied by a decrease in PGC-1␣ acetylation. This remains an important question that should be addressed by future research in this area. The role of GCN5 in regulating PGC-1␣ acetylation in mature skeletal muscle also represents an important area of future study as GCN5 acetylates and represses PGC-1␣ transcriptional activity in human embryonic kidney 293 (26), C2C12, and primary muscle cells (19).

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