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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (May 4, 2011). doi:10.1152/ajpregu.00417.2010
Nuclear SIRT1 activity regulates oxidative capacity
Nuclear SIRT1 activity but not protein content regulates mitochondrial biogenesis in rat and
human skeletal muscle
Brendon J. Gurd1, Yuko Yoshida3, Jay T. McFarlan3, Graham P Holloway3, Chris D Moyes2,
George J.F. Heigenhauser4, Lawrence Spriet3 and Arend Bonen3
1
School of Kinesiology and Health Studies
2
Department of Biology
Queen’s University, Kingston, Ontario, K7L 3N6, Canada
3
Department of Human Health and Nutritional Science
University of Guelph, Guelph, Ontario, N1G 2W1, Canada
4
Department of Medicine
McMaster University, Hamilton, Ontario, L8N 3Z5, Canada
Running Head: Nuclear SIRT1 activity regulates muscle oxidative capacity
Address for correspondence:
Brendon Gurd, PhD
Assistant Professor
School of Kinesiology and Health Studies
Queen’s University
Kingston, Canada
Ph: 613-533-6000 x 79023
Email: [email protected]
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Copyright © 2011 by the American Physiological Society.
Nuclear SIRT1 activity regulates oxidative capacity
Abstract
SIRT1-mediated PGC-1α deacetylation is potentially key for activating mitochondrial biogenesis.
Yet, at the whole muscle level SIRT1 is not associated with mitochondrial biogenesis (Gurd et al.
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 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
(CS, COXIV), 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-1α. Both chronic muscle stimulation and AICAR
increased mitochondrial biogenesis and muscle PGC-1α, but not nuclear PGC-1α.
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 did increase muscle and nuclear PGC-1α and
SIRT1 activity. Thus, nuclear SIRT1 activity, but not muscle or nuclear SIRT1 protein content, is
associated with contraction-stimulated mitochondrial biogenesis in rat and human muscle,
possibly via AMPK activation.
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Nuclear SIRT1 activity regulates oxidative capacity
Introduction
SIRT1 (silent mating type information regulator 2 homolog 1) 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 metabolism through a pathway in which it deacetylates and activates peroxisome
proliferator-activated receptor gamma co-activator-1α (PGC-1α) (38). PGC-1α is a co-activator
involved in activating both nuclear and mitochondrial transcription resulting in mitochondrial
biogenesis and upregulation of genes involved in lipid metabolism and oxidative phosporylation
(4; 28; 46). The ability of SIRT1 in activating PGC-1α 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-1α in this tissue is important.
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Nuclear SIRT1 activity regulates oxidative capacity
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 SIRT 1
protein content (22) in heart and skeletal muscle. Moreover, interventions that promoted
mitochondrial biogenesis, namely, chronic muscle contraction (7 days), 5-aminoimidazole-4carboxamide-1-β-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 altogether, in contrast to initial observations
in C2C12 cells and fao hepatocyctes (19; 39), these findings indicate that in vivo SIRT1 protein
content per sé does not appear to contribute to PGC-1α-mediated mitochondrial biogenesis in
mammalian 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
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Nuclear SIRT1 activity regulates oxidative capacity
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-1α 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 which is accompanied by PGC-1α deacetylation (10). Thus, to impact
PGC-1α 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, a) across
metabolically heterogeneous muscle tissues with differing oxidative capacities, and b) after
treatments designed to stimulate mitochondrial biogenesis including, i) acute exercise, ii)
chronic muscle stimulation and iii) chronic AICAR treatment. In addition, we have also
examined, in human skeletal muscle, c) changes in SIRT1 protein content and activity in the
nucleus of following 2 weeks of exercise training, which is known to stimulate mitochondrial
biogenesis (20).
Methods
Experiments were performed with female Sprague-Dawley rats (3-6 months, 250-300 g)
that were bred on site and housed at 22.5°C on a 12 hour light (7:00-19:00) and 12 hour dark
(19:00-7:00) cycle. At the end of each experiment animals were anaesthetized with Somnotol
(60 mg/kg) and the selected tissues were harvested and either processed immediately for
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nuclear extraction or flash frozen (liquid nitrogen) and stored at –80oC 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 (RTA) and white tibialis anterior (WTA) muscles, and
the heart (HRT), were harvested from anaesthetized rats. Separation of the red and white
compartments of the TA muscle has been described previously (22). Following this procedure
and all experiments described below, whole muscle lysates where 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 hours at 15 m/min followed by an increase in speed of 5 m/min
every 5 minutes 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
hours 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 anesthetisation, from non-exercised animals
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(control), immediately following cessation of exercise (t=0 h) and after 3 hours 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 red (RTA) and white (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 hours 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.
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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 hours 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 Netherlands) to
determine pulmonary VO2peak using a metabolic measurement system (Vmax Series 229,
Sensormedics Corporation, Yorba Linda, CA). Subjects trained 7 times over a 2 week period on
a cycle ergometer (Monark 894 E, Vasbro, Sweden) at a power output that elicited ~90%
VO2peak. Subjects completed ten exercise intervals per session, with each interval lasting 4 min
and separated by 2 min of rest as described previously (37). Pre- and post-training muscle
biopsies were obtained at rest and ~48 hours after the final VO2peak test, using 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 uL of
phosphate buffered saline (PBS) where they were minced and briefly homogenized (~3 sec at
24 000 rpm). Cytosolic and nuclear extraction was performed using the cytosolic and nuclear
extraction reagents supplemented with 1mM sodium orthovanadate, 1mM PMSF, and 10ug/mL
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Nuclear SIRT1 activity regulates oxidative capacity
of pepstatin A, aprotinin, and leupeptin. Isolated nuclei were washed 15X in PBS and PBS
supplemented with 0,1% Nonidet P-40 alternatively before nuclear extraction. To confirm the
nuclear extracts 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 les than %5 contamination (Fig 1). Furthermore, our ability to detect the
nuclear proteins PGC-1α 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 Blotting
Proteins 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-1α, H2B, LDH) or 12.5% (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-1α (Calbiochem, San Diego, CA), COX IV (Molecular Probes, Carlsbad, CA),
Histone H2B (Abcam Inc., Cambridge, MA) and LDH (Abcam Inc., Cambridge, MA). Proteins
were visualized by chemiluminescence detection, according to the manufacteurer’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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mRNA Content
RNA was extracted using the Qiagen RNA mini Kit (Quigen Inc., Valencia, Ca) according
to the manufacturer’s instructions for muscle tissue. 1μg of resulting RNA was reverse
transcribed using the Qiagen Omniscript reverse transcription Kit (Quigen Inc., Valencia, Ca).
50μg of resulting cDNA was used for Realtime PCR with Promega’s Go Taq QPCR sybergreen
master mix (Promega, Madison, WI) with the following cycles 95oC for 15 minutes, 40 cycles of
95oC 15 sec, 60oC for 30 sec and 72oC 36 sec followed by a dissociation curve to assess specify
of the reaction. Tata binding protein (TBP) was used as an endogenous control. The following
primer sets were used: 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-1α forward, 5’-CAATGAGCCCGCGAACATAT-3’, and PGC-1α reverse 5’CAATCCGTCTTCATCCACCG-3’; 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. 25 uL of nuclear extract was
incubated with 15 uL of Fluor de Lys-SIRT1 substrate (100 uM) and NAD+ (100 uM) for 30
minutes 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 minutes at 360
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Nuclear SIRT1 activity regulates oxidative capacity
nm (excitation) and 460 nm (emission). The change in fluorescence (arbitrary fluorescence
units (AFU)) per minutes was normalized to the amount of total muscle (mg wet weight) used
for the nuclear extraction procedure. In order 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 β-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 β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity.
Total CS and β-HAD activity were measured in Tris-HCl buffer (50 mM Tris-HCl, 2 mM EDTA, and
250 uM 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 β-HAD reaction was started by the addition of 100
μm acetoacetyl-CoA and absorbance was measured at 340 nm over a 2 min period (37°C) (5).
Statistics
In rat experiments, two-way analyses of variance 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
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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 β-HAD activity (data not shown). Across the
muscles 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; β-HAD activity, r = 0.99, Fig 3C).
ADAPTIVE RESPONSES OF SIRT1 TO ACUTE EXERCISE, CHRONIC MUSCLE STIMULATION AND AICAR IN RATS
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-1α, or COXIV either immediately after exercise, or 3 h after exercise.
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Similarly, the nuclear SIRT1 (Fig 4A) protein was not altered. There was a graded increase in
nuclear SIRT1 activity immediately (0 hours; +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-1α (Fig 4C) increased immediately after exercise (+32%) and at 3h
hours post exercise (+51%). There was also an increase in mRNA content for several genes
targeted by PGC-1a and associated with mitochondrial biogenesis (Fig 4D). PGC-1α, ALAS and
citrate synthase all increased immediately following exercise and further increased during the 3
hours recovery period. Cytochome C was elevated immediately following exercise but did not
increase further following the 3 hour recovery period. The increases in PGC-1α (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-1α protein and increases in mRNA (PGC-1α, r = 0.93; ALAS, r
= 0.89; citrate synthase, r = 0.95).
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-1α protein (RTA, +26%; WTA, +87%), consistent with an increase in mitochondrial
biogenesis. PGC-1α protein content in the nucleus was not altered in either the RTA or WTA
(data not shown).
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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-1α protein
increased (RTA, +17%; WTA, +47%). Nuclear PGC-1α 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
Exercise training increased VO2peak (+7%), markers of oxidative capacity (CS activity (+9%), COX
IV protein (+35%) and fatty acid oxidation β-HAD activity (+19%; Fig 7A).
Muscle and nuclear PGC-1α: Muscle PGC-1α protein was increased after 2 weeks of training
(+36%; 7B). Similarly, there was also an increase in nuclear PGC-1α 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 towards an increase (P = 0.08; +12%; Fig 7F).
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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) 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-1α. 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
PGC-1α 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
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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-1α transcriptional activity (as evidence by increased
expression of genes targeted by PGC-1α, Fig 4). Interestingly there was also a linear
relationship between the apparent increase in PGC-1α transcriptional activity and nuclear PGC1α protein content. While this later relationship was slightly weaker than the relationship
observed with nuclear SIRT1 activity, our data indicate that the increase in PGC-1α
transcriptional activity following acute exercise in rats is temporally related to increases in both
nuclear SIRT1 activity and nuclear translocation of PGC-1α protein.
These findings are consistent with work from cell lines (C2C12 cells, Fao Hepatocytes)
where 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
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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-1α (3; 16; 25) leading to the proposed model of control of
mitochondrial content whereby activation of SIRT1 increases deacetylation of PGC-1α within
the nucleus, increasing transcriptional activity of PGC-1α and subsequently mitochondrial gene
expression (38). Thus, our results suggest that increases in PGC-1α 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-1α 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-1α acetylation. This remains an
important question that should be addressed by future research in this area. The role of GCN5
in regulating PGC-1α acetylation in mature skeletal muscle also represents an important area of
future study as GCN5 acetylates and represses PGC-1a transcriptional activity in HEK293 (26),
C2C12 and primary muscle cells (19).
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 weeks of interval training, we
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have observed a decrease in whole muscle homogenate SIRT1 protein (21). This finding was
not repeated in the current study following only 2 weeks on interval training suggesting that
the effect may only manifest after a period of training that is in excess of 2 weeks. 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-1α 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 (Fig 2 and 3) and
mitochondrial biogenesis (Fig 5-7) were accompanied by increased nuclear SIRT1 activity but
not SIRT1 protein, as whole muscle SIRT1 protein expression was either decreased (Fig 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-1α 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 spectre that the biologic 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 is
linked to increased apoptotic signalling in HeLa cells (24; 36). Thus, if the increases in SIRT1
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activity following its overexpression were confined largely to the cytosol (a question we are
currently examining) it is possible that the resulting fall in muscle oxidative capacity was a result
of increased cytosolic apoptotic signalling. 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 (Fig 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 post-translationally 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 post-translational 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
Page 19
Nuclear SIRT1 activity regulates oxidative capacity
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-1α (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-1α. These findings also underscore the importance of determining both SIRT1
protein content and activation status and the intracellular localization of activated SIRT1.
Page 20
Nuclear SIRT1 activity regulates oxidative capacity
Acknowledgements
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.
A Bonen is the Canada Research Chair in Metabolism and Health.
Page 21
Nuclear SIRT1 activity regulates oxidative capacity
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Nuclear SIRT1 activity regulates oxidative capacity
Figures
Figure 1. Nuclear extracts are clear of cytosolic contamination.
Lactate dehydrogenase (LDH) both muscle lysates and nuclear extracts obtained from RTA
muscles as an index of contamination of the nuclear extract. MS; muscle lysate, NE; nuclear
extract.
Figure 2. Nuclear SIRT1 activity but not whole muscle or nuclear SIRT1 protein is increased in
heart muscle compared to skeletal muscle.
Whole muscle (A) and nuclear (B) protein content of SIRT1 is shown along with nuclear SIRT1
activity (C). (n = 4)
* Significantly different (p < 0.05) from white (WTA) muscle.
† Significantly different (p < 0.05) from red (RTA) muscle.
Figure 3. Nuclear SIRT1 activity is correlated with oxidative capacity in skeletal muscle and
heart.
Nucelar SIRT1 activity was positively correlated with COX IV (r = 0.99, p < 0.05; A), CS activity (r
= 0.98, p < 0.05; B) and β-HAD activity (r = 0.99, p < 0.05; C). (n = 4)
Figure 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 PGC-1α was increased
immediately following and 3 hours after exercise (C). Acute exercise also induced an increase in
mRNA expression of genes both targeted by PGC-1a and associated with mitochondrial
biogenesis (D). (n = 6)
* Significantly different (p < 0.05) from control.
† Significantly different (p < 0.05) 0 Hrs.
Figure 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.
Page 30
Nuclear SIRT1 activity regulates oxidative capacity
Figure 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.
Figure 7. Effects of exercise training on nuclear SIRT1 and PGC-1α 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-1α 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
Page 31
Figure 1
MS NE MS NE MS NE
LDH
Figure 2
SIRT1
(a.u./Pg protein)
80
60
*
60
40
40
*†
20
20
0
White
Red
Heart
B
Nuclear SIRT1 Protein
C
60
Nuclear SIRT1 Activity
25
40
*†
20
0
SIRT1 Activity
(AFU / mg ww)
Whole Muscle SIRT1 Protein
SIRT1
(a.u./Pg nuclear protein)
A
*†
20
15
10
*
5
0
White
Red
Heart
White
Red
Heart
Figure 3
A
B
SIRT1 vs. COX IV
r = 0.99
100
50
0
150
100
r = 0.98
50
0
0
5
10
15
20
Nuclear SIRT1 Activity
(AFU / mg ww)
25
SIRT1 vs. EHAD
150
E HAD activity
(Pmol/min/g ww)
200
CS Activity
(Pmol / min / g ww)
COX IV
(a.u./Pg protein)
150
C
SIRT1 vs. CS
r = 0.99
100
50
0
0
5
10
15
20
Nuclear SIRT1 Activity
(AFU / mg ww)
25
0
5
10
15
20
Nuclear SIRT1 Activity
(AFU / mg ww)
25
Figure 4
B
NuclearSIRT1 Protein
NuclearSIRT1 Activity
1.5
12
SIRT1 Activity
(AFU / mg ww)
Nuclear SIRT1
(a.u./Pg nuclear protein)
A
1.0
0.5
0.0
10
8
6
Control
0 Hrs
3 Hrs
Control
Exercise
Nuclear PGC-1D Protein
1.75
D
*
1.50
0 Hrs
3 Hrs
Exercise
mRNA content
(arbitrary units)
Nuclear PGC-1D
(a.u./Pg nuclear protein)
C
*
*
1.25
1.00
mRNA content
20
15
10
5
5
4
3
2
PGC-1D
ALAS
Cyt-C
CS
*†
*
*†
*
*†
* *
*
1
0.75
0
Control
Exercise
0 Hrs
3 Hrs
Control
0 Hrs
Exercise
3 Hrs
Figure 5
SIRT1
(a.u./Pg protein)
2.0
1.5
Control
Stimulation
1.0
†
*
*
0.5
0.0
Red
White
B
C
NuclearSIRT1 Protein
2.5
NuclearSIRT1 Activity
20
†
2.0
1.5
*
*
1.0
0.5
0.0
Nuclear SIRT1 Activity
(AFU / mg ww)
Whole Muscle SIRT1 Protein
Nuclear SIRT1
(a.u./Pg nuclear protein)
A
15
*
*
10
†
5
0
Red
White
Red
White
Figure 6
Whole Muscle SIRT1 Protein
2.0
1.5
Control
AICAR
1.0
Nuclear SIRT1
(a.u./Pg nuclear protein)
SIRT1
(a.u./Pg protein)
2.5
†
*
0.5
0.0
Red
B
White
NuclearSIRT1 Protein
C
2.0
NuclearSIRT1 Activity
20
Nuclear SIRT1 Activity
(AFU / mg ww)
A
†
1.5
1.0
0.5
0.0
15
*
†
10
5
0
Red
White
Red
White
Figure 7
2.0
*
1.5
1.0
0.5
B
0.0
*
1.5
1.0
0.5
Post
1.5
E
1.0
0.5
0.0
Pre
Post
C
Nuclear PGC-1D Protein
2.0
NuclearSIRT1 Protein
80
*
1.5
1.0
0.5
0.0
Post
Pre
F
Post
NuclearSIRT1 Activity
P =*0.08
5.0
4.5
SIRT1 Activity
(AFU / mg ww)
Whole Muscle SIRT1 Protein
Pre
Nuclear SIRT1
(a.u./Pg nuclear protein)
SIRT1
(a.u./Pg protein)
2.0
0.0
Pre
D
Whole Muscle PGC-1D Protein
Nuclear PGC-1D
(a.u./Pg nuclear protein)
Whole Muscle COX IV Protein
PGC-1D
(a.u./Pg protein)
COX IV
(a.u./Pg protein)
A
60
40
20
0
4.0
3.5
3.0
1
0
Pre
Post
Pre
Post