School of Kinesiology and Health Science, and Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3
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ABSTRACT |
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The transcriptional coactivator the
peroxisome proliferator-activated receptor coactivator-1
(PGC-1
) has been identified as an important mediator of
mitochondrial biogenesis based on its ability to interact with
transcription factors that activate nuclear genes encoding
mitochondrial proteins. The induction of PGC-1
protein expression
under conditions that provoke mitochondrial biogenesis, such as
contractile activity or thyroid hormone (T3) treatment, is
not fully characterized. Thus we related PGC-1
protein expression to
cytochrome c oxidase (COX) activity in 1) tissues
of varying oxidative capacities, 2) tissues from animals treated with T3, and 3) skeletal muscle subject
to contractile activity both in cell culture and in vivo. Our results
demonstrate a strong positive correlation (r = 0.74;
P < 0.05) between changes in PGC-1
and COX
activity, used as an index of mitochondrial adaptations. The highest
constitutive levels of PGC-1
were found in the heart, whereas the
lowest were measured in fast-twitch white muscle and liver.
T3 increased PGC-1
content similarly in both fast- and
slow-twitch muscle, as well as in the liver, but not in heart.
T3 also induced early (6 h) increases in AMP-activated protein kinase (AMPK
) activity, as well as later (5 day) increases in p38 MAP kinase activity in slow-twitch, but not in fast-twitch, muscle. Contractile activity provoked early increases in PGC-1
, coincident with increases in mitochondrial transcription factor A
(Tfam), and nuclear respiratory factor-1 (NRF-1) protein expression, suggesting that PGC-1
is physiologically important in coordinating the expression of the nuclear and mitochondrial genomes.
Ca2+ ionophore treatment of muscle cells led to an
approximately threefold increase in PGC-1
protein, and contractile
activity induced rapid and marked increases in both p38 MAP kinase and
AMPK
activities. 5-Aminoimidazole-4-carboxamide-1-
-D-ribofuranoside
(AICAR) treatment of muscle cells also led to parallel increases in
AMPK
activity and PGC-1
protein levels. These data are consistent
with observations that indicate that increases in PGC-1
protein are
affected by Ca2+ signaling mechanisms, AMPK
activity, as
well as posttranslational phosphorylation events that increase PGC-1
protein stability. Our data support a role for PGC-1
in the
physiological regulation of mitochondrial content in a variety of
tissues and suggest that increases in PGC-1
expression form part of
a unifying pathway that promotes both T3- and contractile
activity-induced mitochondrial adaptations.
peroxisome proliferator-activated receptor-; peroxisome
proliferator-activated receptor-
coactivator-1
; mitochondrial
biogenesis; exercise; AMP-activated protein kinase; p38 MAP kinase; cytochrome c oxidase; mitochondrial transcription factor A; nuclear
respiratory factor-1; skeletal muscle
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INTRODUCTION |
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THE PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR (PPAR
) coactivator-1
(PGC-1
) is a recently discovered nuclear-encoded transcriptional
coactivator that plays a pivotal role in glucose metabolism,
mitochondrial biogenesis, muscle fiber specialization, and adaptive
thermogenesis (17, 19, 38). PGC-1
was originally cloned
from a brown fat cell cDNA library and was found to transcriptionally activate PPAR
and thyroid hormone receptor-
(TR
) on the
uncoupling protein-1 (UCP-1) promoter (23). In muscle
cells, forced expression of PGC-1
increased mtDNA copy number and
stimulated mitochondrial proliferation, providing the first
experimental evidence that PGC-1
was directly involved in
upregulating organelle biogenesis (36).
The process and regulation of mitochondrial biogenesis are complex, in
part because it requires the cooperative induction of gene expression
from two functionally independent genomes. The mechanisms involved are
partly ascribed to the activation and binding of transcription factors
to specific DNA sequences within the promoter regions of genes encoding
mitochondrial proteins (16). A number of important
transcription factors have been identified, including mitochondrial
transcription factor A (Tfam), the nuclear respiratory factors-1 and -2 (NRF-1 and NRF-2), PPAR and PPAR
, and specificity protein 1 (Sp1;
Refs. 16 and 25). In response to mitochondrial biogenesis
inducing-stimuli, mRNAs encoding these proteins change before, or
coincident with, increases in the expression of their target genes,
consistent with their function in mediating the characteristic
adaptations (12, 37). However, the collaborative effort
that is required between these regulatory proteins and the diversity
that exists between the promoter regions of nuclear genes encoding
mitochondrial proteins (20) calls for the presence of
unifying regulators of mitochondrial biogenesis. The preponderance of
evidence to date implicates PGC-1
as the most important of these.
Stimuli such as endurance exercise training and thyroid hormone
treatment have been shown to induce mitochondrial biogenesis (8,
14, 34). The increased mitochondrial content that is brought
about by endurance training attenuates muscle fatigue during submaximal
exercise, leading to an improved work capacity (16).
Defining the underlying cellular mechanisms resulting in this adaptive
response requires an understanding of the initial signaling events
involved, as well as their downstream targets that activate the
transcription of nuclear and mitochondrial genes (16).
Although PGC-1 is a vital component of this process, only one study
to date has investigated the regulation of PGC-1
protein expression
in response to an exercise stimulus (3). Increases in
PGC-1
mRNA as a result of exercise have also recently been reported
(13, 30, 31), but it is established that the relationship
between PGC-1
mRNA and protein expression is complex in skeletal
muscle, because the final protein level is subject to posttranslational
modifications that affect its stability (22). Thus we
investigated whether contractile activity could modify PGC-1
protein
expression, as well as some of the potential signaling mechanisms
involved. In addition, PGC-1
is a strong coactivator of TR
(23), and this is likely to be important in mediating some
of the effects of thyroid hormone (T3) on mitochondrial
biogenesis. There is some evidence that T3 can induce the
mRNA expression of PGC-1
in liver (32), suggesting that
PGC-1
may autoregulate its own expression via TR
. Thus we also
wished to investigate the role of T3 in mediating PGC-1
protein expression, the potential tissue specificity of this response,
as well as some possible common signaling mechanisms mediating both
T3- and contractile activity-induced mitochondrial
biogenesis. Here, we report that both contractile activity and
T3 treatment induce PGC-1
protein expression, which
occurs coincident with increases in transcription factor expression,
and oxidative capacity in a variety of tissues.
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METHODS |
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Materials.
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),
penicillin/streptomycin (P/S), 3,3',-5-triiodothyronine (T3), A-23187,
5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR), and cytochrome c from horse heart were purchased
from Sigma (Oakville, ON, Canada). Horse serum (HS) was purchased from Invitrogen (Burlington, ON, Canada). The PGC-1
antibody was obtained from Calbiochem (La Jolla, CA) and the phospho-p38 and p38 MAP kinase,
as well as the phospho-AMP-activated protein kinase (AMPK
) and
AMPK
antibodies, were from New England Biolabs (Mississauga, ON,
Canada). The AMPK antibody reacts with both the
1- and
2-isoforms of AMPK. The NRF-1 antibody was a kind gift
from Dr. R. C. Scarpulla. The Tfam and cytochrome c antibodies
have been previously described (12, 28). Nitrocellulose
membrane (Hybond N) and the chemiluminescence kits for Western blot
analyses were obtained from Amersham Pharmacia Biotech (Baie
D'Urfé, QC, Canada).
In vivo contractile activity. In vivo stimulation was performed as previously described (29). Briefly, male Sprague-Dawley rats were anesthetized with pentobarbital sodium (60 mg/kg body wt), and electrodes were sutured unilaterally beside the common peroneal nerve. Electrode wires were passed subcutaneously from the thigh to the base of the neck, where they were attached to an external stimulator unit secured to the back of the animal. After a 1-wk recovery period, the tibialis anterior (TA) and the extensor digitorum longus (EDL) muscles were stimulated (10 Hz, 0.1 ms duration) 3 h/day for 3, 5, 7, and 10 days. The contralateral limb was used as a nonstimulated control muscle for all animals (n = 4-8/day).
T3 treatment and tissue extraction. Male Sprague-Dawley rats were injected intraperitoneally with either T3 (0.4 mg/kg) or vehicle (0.9% NaCl-propylene glycol; 40:60 vol/vol) for 5 consecutive days, as done previously (7, 26). A subgroup of animals (n = 3) was injected with one dose, and the tissues were removed 6 h later. Twenty-four hours after the fifth T3 injection, or after the last 10 Hz stimulation time point, animals were anesthetized with pentobarbital sodium (40 mg/kg), and selected hindlimb muscles and tissues were removed, quick frozen, and stored in liquid N2. Total protein extracts from frozen tissue powders were made (29) for subsequent protein and enzyme activity measurements.
Cell culture and contractile activity in muscle cells.
Cell culture of rat L6E9 muscle cells and
electrical stimulation of murine C2C12 skeletal
muscle cells were done as previously described (5, 11).
Briefly, cells were propagated in DMEM supplemented with 10% FBS and
1% P/S and then differentiated in DMEM supplemented with 5%
heat-inactivated HS and 1% P/S. Stimulation of
C2C12 skeletal muscle cells was performed
successively for 2 and 4 days (3 h/day; 5 Hz, 55 V) with an
intermittent 21-h period of quiescence. Total protein extracts were
prepared 21 h after the last stimulation period (5).
In the experiment to measure p38 MAP kinase and AMPK activation,
cells were stimulated for 3 h only, and extracts were made
immediately following. L6E9 muscle cells were
treated continuously with A-23187 (1 µM) or AICAR (1 mM) for 48 h. Total protein extracts were prepared immediately following.
Western blotting.
Total protein extracts from cultured cells or tissues were
electrophoresed through SDS-polyacrylamide gels and electroblotted onto
nitrocellulose membranes. Blots were blocked (1 h) with 5% milk in ×1
TBST (Tris-buffered saline/Tween-20; 25 mM
Tris · HCl, pH 7.5, 1 mM NaCl, and 0.1%
Tween-20), followed by overnight incubation with antibodies diluted in
blocking buffer directed toward PGC-1 (1:1,000), Tfam (1:1,000),
NRF-1 (1:500), cytochrome c (1:750), phospho-p38 (1:400), p38 MAP
kinase (1:400 diluted in 5% BSA/TBST), phospho-AMPK
(1:400 diluted
in 5% BSA/TBST), or AMPK
(1:1,000 diluted in 5% BSA/TBST). After
3 × 5 min washes with TBST, blots were incubated at room
temperature (1 h) with the appropriate secondary antibody conjugated to
horseradish peroxidase. Blots were then washed again 3 × 5 min
with TBST, visualized with enhanced chemiluminescence, and quantified
using SigmaGel (Jandel, San Rafael, CA). To compare PGC-1
levels
among tissues, a standard heart sample was run on each gel and all
tissue values were then compared with the heart sample that was
assigned a value of 1.
Cytochrome c oxidase activity. Cytochrome c oxidase (COX) activity was measured as previously described (12). The enzyme activity was determined as the maximal rate of oxidation of fully reduced cytochrome c, measured by the change in absorbance at 550 nm on a Beckman DU-64 spectrophotometer.
Statistics. Experiments using C2C12 or L6E9 muscle cells were analyzed using a one way-ANOVA, followed by Tukey's post hoc analysis, to determine individual differences. Paired Student's t-tests were used for comparison of data obtained for the stimulated and contralateral control muscles, whereas unpaired t-tests were done to compare T3- and vehicle-treated animals. All data are expressed as means ± SE, and differences were considered significant if P < 0.05.
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RESULTS |
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Protein expression during contractile activity of muscle cells in
culture.
Stimulation of C2C12 cells in culture for 2 and
4 days resulted in a pattern of protein expression that is
consistent with the early adaptive responses (i.e., activation of
signal transduction pathways and upregulation of transcription factor
expression) within skeletal muscle that precede mitochondrial
biogenesis. Although no change in cytochrome c protein
expression was evident over 2 and 4 days, increases in NRF-1, Tfam, and
PGC-1 protein expression occurred progressively, reaching values
that were 1.9-, 1.4-, and 1.8-fold above those in nonstimulated cells,
respectively (P < 0.05; Fig. 1, A and
B). We also evaluated the
activation of the p38 MAP kinase and AMPK
pathways, which have been
implicated in PGC-1
protein expression (22, 39). Three
hours of contractile activity led to 3.0- and 2.6-fold increases in p38
MAP kinase and AMPK
activities, respectively (P < 0.05; Fig. 1C).
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Contractile activity in vivo stimulates PGC-1 protein
expression.
Chronic contractile activity in vivo results in an increase in
mitochondrial content and cellular respiration (14, 16). Thus we investigated whether PGC-1
protein expression could be induced by chronic contractile activity and whether these changes occurred coincident with increases in COX activity, a typical marker of
mitochondrial biogenesis. Contractile activity increased PGC-1
protein expression by 1.5-fold (P < 0.05) by 5 days of stimulation (Fig. 2, A and
B). This increase was
maintained at 7 days (P < 0.05) and was 1.3-fold
higher (P < 0.05) than control values at 10 days of
stimulation. The changes in PGC-1
protein expression occurred
coincident with significant increases (P < 0.05) in
COX activity between 5 and 10 days of stimulation (Fig. 2C).
To fortify a potential role for Ca2+ (35) in
mediating the contractile activity-induced increase in PGC-1
protein, we treated rat L6E9 muscle cells with
the Ca2+ ionophore A-23187, as done previously
(11). Consistent with this possibility, A-23187 treatment
resulted in an approximate 3.0-fold increase (P < 0.05) in PGC-1
protein expression (Fig. 2D).
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AICAR-induced AMPK activation mimicks the contractile
activity-induced PGC-1
protein expression.
To evaluate whether AMPK
activation is involved in PGC-1
protein
expression, we treated rat L6E9 muscle cells
for 48 h with AICAR, an established AMPK activator
(6). Coincident 2.6- and 2.5-fold increases
(P < 0.05) in PGC-1
protein expression and AMPK
kinase activation occurred in cells treated with AICAR for 48 h.
There was no effect of AICAR on p38 MAPK activity (Fig. 3, A
and B).
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PGC-1 protein expression is tissue specific.
The expression of PGC-1
was measured under steady-state conditions
in six different tissues possessing a wide range of oxidative capacities. The highest constitutive levels of PGC-1
protein were
found in the heart (Fig. 4A).
The slow-twitch red soleus muscle had a PGC-1
content that was
~60% of that found in heart (Fig. 4A). Intermediate
PGC-1
levels were measured in the fast-twitch muscles plantaris and
red gastrocnemius, whereas the lowest PGC-1
content was found in the
liver and in the fast-twitch white section of gastrocnemius (Fig.
4A).
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T3 treatment increases PGC-1 protein expression.
To investigate a role for PGC-1
in T3-induced
mitochondrial adaptations, we assessed changes in PGC-1
protein
expression in animals treated for 5 days with either T3 or
vehicle in both slow-twitch and fast-twitch muscle types, as well as in
tissues of varying metabolic activities (i.e., heart and liver).
T3-treatment did not increase PGC-1
protein expression
in the heart (Fig. 4, B and C) but induced
approximate 1.7-, 1.5-, and 1.3-fold changes (P < 0.05) in PGC-1
protein expression in the soleus, plantaris, and
liver, respectively. Changes in COX activity were also assessed in
these tissues as an index of mitochondrial adaptations. Figure 4D shows that T3 significantly increased
(P < 0.05) COX activity by 1.3- to 1.8-fold in all
tissues examined.
T3 treatment activates AMPK in a time-dependent and
tissue-specific manner.
Because PGC-1
protein expression can be induced by both contractile
activity and by T3 treatment, we investigated the
possibility that a common link between these stimuli could be mediated
by AMPK
. T3 treatment for 6 h induced a 5.4-fold
increase (P < 0.05) in AMPK
activity in
slow-twitch, but not fast-twitch, muscle (Fig.
5A). This effect of
T3 on AMPK
was an early, transient response because the
activation was no longer observed after 5 days of T3
treatment (Fig. 5B).
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The relationship between PGC-1 protein expression and COX
activity.
Given the existence of tissue-specific differences in PGC-1
protein
levels, we wanted to establish the overall relationship between
PGC-1
expression and COX activity, used as an indicator of
mitochondrial content. We examined this relationship under steady-state
conditions in a variety of tissues (Fig.
6A), as well as under
conditions in which COX activity was induced by T3 (Fig.
6B) and contractile activity in vivo (Fig. 6C).
T3- and contractile activity-evoked changes in PGC-1
protein expression were largely matched by parallel changes in COX
activity. A parallelism between COX activity and PGC-1
was also
evident among different tissues, with the exception of the slow-twitch
soleus muscle and the liver, in which deviations from the line of
identity are evident (Fig. 6A). When the 71 pairs of
observations from Fig. 6, A-C, were
combined, a strong, positive correlation of 0.74 was found (P < 0.05), indicating that over 50% of the variance
in mitochondrial content, as reflected by COX activity, can be
attributed to variations in PGC-1
protein level.
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DISCUSSION |
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The transcriptional coactivator PGC-1 has recently emerged as
an important regulator of adaptive thermogenesis in adipocytes (23), gluconeogenic capacity in hepatocytes
(38), and muscle fiber specialization in skeletal muscle
cells (19). Forced overexpression of PGC-1
in cell
culture and in vivo using transgenic animals resulted in large
increases in mitochondrial content, cellular respiration, and
transcription factor gene expression in both heart and skeletal muscle
(18, 36). Based on these studies, we hypothesized that
conditions that induce mitochondrial biogenesis, such as chronic
contractile activity and T3 treatment, could regulate PGC-1
protein expression and that this would play a pivotal role in
influencing mitochondrial content. In this study, we report that
PGC-1
protein expression can be modified by both chronic contractile
activity and T3 treatment and that the regulation of
PGC-1
protein expression in response to these stimuli occurs before,
or coincident with, increases in COX activity, which we used as an
index of mitochondrial content because of its requirement for gene
products from both the nuclear and mitochondrial genomes.
We show here that 2 and 4 days of contractile activity in cell culture
induced time-dependent, coincident elevations in Tfam, NRF-1, and
PGC-1 protein expression (Fig. 1). These changes represent a highly
favorable environment for the onset of mitochondrial biogenesis in
muscle cells because PGC-1
strongly coactivates NRF-1, which
transcriptionally activates the promoters of a wide range of nuclear
genes encoding mitochondrial proteins, including the important mtDNA
transcription factor, Tfam (25). The parallel increases in
Tfam protein ensure the coordinate induction of mtDNA transcription and
replication, leading subsequently to the enhanced expression of the 13 mitochondrial proteins that are vital for respiratory chain function.
In support of this, we have observed that mtDNA content is increased
approximately twofold by 4 days of contractile activity in this cell
culture model (Joseph and Hood, unpublished observations). Cytochrome
c, a nuclear-encoded component of the respiratory chain,
typically requires a greater length of time for its induction, compared
with early responding transcription factors such as NRF-1 and Tfam,
during chronic contractile activity in vivo (10, 12) and
also during contractile activity in cell culture (5, 37).
Our previous work has shown that cytochrome c
transcriptional activation and mRNA content are elevated by 4 days of
stimulation in cell culture (5) but that this change is
not yet reflected in detectable increases at the protein level at that
time (Fig. 1, A and B).
To assess a possible role for PGC-1 in contractile activity-induced
increases in mitochondrial content in vivo, we next investigated whether the changes in PGC-1
protein expression could be induced at
an early stage of mitochondrial adaptations produced via electrical stimulation of the peroneal nerve for 3, 5, 7, and 10 days. Five days
of chronic contractile activity was sufficient to induce PGC-1
protein expression. This increase occurs coincident with the change in
Tfam protein expression that occurs between 5 and 7 days of
stimulation, which we have previously been shown using this
experimental model (12). Moreover, we also show that the changes in PGC-1
protein expression parallel increases in COX activity (Figs. 2 and 6). These data indicate that chronic contractile activity in vivo induces the coordinated expression of transcription factors and the transcriptional coactivator PGC-1
, which are important in mediating the parallel responses of the nuclear and mitochondrial genomes required for organelle biogenesis within mammalian skeletal muscle.
To confirm a broader role for PGC-1 in regulating steady-state
mitochondrial content, we compared the levels of PGC-1
protein and
COX activity among a variety of untreated tissues, as done previously
(15). Analyses of the data from the heart, white gastrocnemius, plantaris, and red gastrocnemius muscles revealed a
close parallelism between PGC-1
protein content and COX activity (Fig. 6A). However, exceptions to this relationship were
observed in the liver and in the slow-twitch soleus muscle. Assuming
that COX activity accurately represents mitochondrial content in these tissues, the data obtained from liver suggest that PGC-1
activation via posttranslational modifications, rather than the absolute amount of
PGC-1
protein, is more important in determining the steady-state
mitochondrial content in this tissue. In contrast, because the elevated
level of PGC-1
is not matched by an equivalent COX activity in the
soleus muscle, these data suggest the possibility that a high level of
PGC-1
repressor activity (17) exists in this tissue type.
To some degree, our data at the protein level mirror the relative
tissue differences in PGC-1 mRNA expression, which have been
reported previously. In general, PGC-1
mRNA has been reported to be
highest in heart, followed by muscle and liver (2, 9, 23).
In addition, slow-twitch myofibers possess a greater PGC-1
mRNA
content compared with fast-twitch fibers (19), a profile that is reflected at the protein level (Fig. 4A; 19). The parallelism between PGC-1
protein and mRNA among a variety of tissues suggests that PGC-1
expression is regulated, at least in part, at the transcriptional level. However, recent evidence within some tissues (e.g., muscle cells) indicates that posttranslational mechanisms are
also involved in regulating the final PGC-1
protein content. For
example, in response to a hypermetabolic stimulus, increases in
PGC-1
protein can be induced in the absence of apparent increases at
the mRNA level. This occurs by increasing PGC-1
protein stability via p38 MAP kinase-mediated Ser/Thr phosphorylation (22).
Whether transcriptional or posttranslational mechanisms may be involved in determining the increase in PGC-1
protein as a result of chronic contractile activity, or T3 treatment, remains to be
determined. However, it is established that contractile activity can
lead to p38 activation (4, 24). In our muscle cell
contraction model, p38 MAP kinase was dramatically activated during a
single, 3-h bout of contractile activity (Fig. 1C). Because
PGC-1
is a direct downstream target of p38 (22), the
resulting phosphorylation and protein stabilization could be important
in mediating the increase in PGC-1
protein observed here.
Contractile activity also activates other signaling cascades, including
AMPK and Ca2+-regulated kinases and phosphatases (16,
24). Evidence that AMPK is involved in regulating PGC-1
expression was recently demonstrated in mice expressing a
muscle-specific, dominant-negative mutant of AMPK (39).
Activation of AMPK was shown to be a required signaling event
leading to an increase PGC-1
mRNA, as well as calcium/calmodulin-dependent protein kinase IV (CaMKIV)
protein expression. Our results here demonstrate that activation
of AMPK
also invokes changes in PGC-1
at the protein level. Taken
together, these data suggest that activation of the p38 MAP kinase and
AMPK
pathways may serve distinct roles in mediating the contractile activity-induced increase in PGC-1
protein expression.
Recent work has shown that transgenic animals overexpressing a
constitutively active form of CaMKIV displayed an increase in PGC-1
transcriptional activation (35). Because Ca2+
is a known activator of CaMK in vivo, and cytosolic Ca2+
increases during muscle contraction, it is reasonable to hypothesize (19) that Ca2+-mediated signaling may be
initially responsible for transcriptional activation leading to the
increase in PGC-1
expression during contractile activity, as
demonstrated here. Our observations using the Ca2+
ionophore A-23187 further support this. Because it appears that CaMKIV
is a downstream target of AMPK (39), it is possible that contraction-induced signaling events mediated by Ca2+ and
AMPK could converge on CaMK to regulate final PGC-1
protein content.
Another mechanism by which PGC-1 is reported to coactivate
transcription is via interaction with the TR
isoform, an effect that
is ligand dependent (23). This may be an important way in
which T3 exerts its effects on mitochondrial content within a variety of tissues. Although de novo PGC-1
protein synthesis is
not required for this process, we hypothesized that an induction of
PGC-1
could serve to amplify this transcriptional effect, leading to
more pronounced mitochondrial biogenesis in response to T3.
The induction in PGC-1
expression by T3 was expected on the basis of the recent finding that 6 h of T3
treatment induced increases in PGC-1
mRNA expression
(32). Although we failed to observe an increase in
PGC-1
protein expression after 6 h of T3 treatment
(Sheehan and Hood, unpublished observations), significant increases in
PGC-1
protein occurred by 5 days in a variety of tissues, with
similar increases observed in the slow-twitch soleus, as well as in the
fast-twitch plantaris muscles (Fig. 4C). Given the
approximate 50% higher endogenous levels of PGC-1
(Figs.
4A and 6), as well as greater levels of thyroid hormone receptor expression in slow-twitch muscle fibers (27, 33), the transcriptional effect of T3 and the resulting
mitochondrial adaptation are expected to be more pronounced in
slow-twitch muscle. This provides a potential explanation for the
differential effect of T3 on muscle type-type mitochondrial
adaptations, a result that has been established for many years
(34).
We sought to determine whether the effects of T3 and
contractile activity on PGC-1 protein content could be mediated via common mechanisms. Thus we examined the time-dependent effects of
T3 on AMPK
activity because 1) Park et al.
(21) have recently shown that combined T4 and
T3 treatment for 7 days could induce the levels of AMPK
isoform subunits and 2) our data clearly implicate AMPK
in mediating increases in PGC-1
protein levels. We found no change
in AMPK
protein expression in animals treated with T3
for either 6 h or 5 days. However, a dramatic increase in AMPK
activation was observed in slow-twitch, but not in fast-twitch, muscle
by 6 h (Fig. 5A). This effect was attenuated by 5 days of treatment (Fig. 5B). Thus the increase in PGC-1
protein in slow-twitch muscle at 5 days could be partly the result of
sequential and cumulative effects of 5 days of T3-induced
AMPK
signaling. Moreover, it appears that AMPK
activation is more
sensitive to changes in T3 levels in slow- vs. fast-twitch
muscle, perhaps related to the lower AMPK
isoform content in
slow-twitch muscle (1). The lack of T3 on
AMPK
activation in fast-twitch muscle suggests that the effect of
T3 on PGC-1
protein content may be mediated by more
traditional T3-induced increases in PGC-1
transcription. Further work characterizing the PGC-1
promoter is needed to verify this.
In contrast to the T3-mediated effects in skeletal muscle
type types, T3 had no effect on PGC-1 protein expression
in the heart, despite producing a 30% increase in COX activity. In
view of the very high levels of endogenous PGC-1
protein in the
heart, activation of PGC-1
, rather than its induction, may be a
means by which an increase in mitochondrial biogenesis is achieved in this tissue. The lack of increase in PGC-1
protein with
T3 treatment may be beneficial in preventing the
deleterious effects that excess PGC-1
protein can have on the
morphological and functional capabilities of the heart
(18). In addition, the lower COX adaptive response of
heart muscle to T3 compared with other tissues (see Fig.
3D) may be related to the relative proportion of
- and
-TR isoforms. Cardiac muscle expresses very high ratios of the
TR
2 repressor isoform relative to the activating
1- and
-isoforms (33), and this could
serve to reduce the transcriptional response of target genes, such as
PGC-1
, to T3 treatment.
In summary, the present study documents that both contractile activity
and T3 induce increases in PGC-1 protein content, consistent with the established effects of these stimuli on
mitochondrial biogenesis. Further, we present evidence to suggest that
AMPK activity may be a common signaling intermediate that leads to this
increase. However, PGC-1
adaptations are likely mediated by a
combination of both transcriptional and posttranscriptional mechanisms
in a fiber type- and tissue-specific manner.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Klip (University of Toronto, Toronto, Canada) for the provision of the L6E9 cells and Dr. R. C. Scarpulla (Northwestern University, Chicago, IL) for the NRF-1 antibody. We thank Farah Amarshi for help with COX activity measurements.
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FOOTNOTES |
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This work was supported by Canadian Institutes of Health Research and National Sciences and Engineering Research Council grants to D. A. Hood. I. Irrcher is funded by a National Sciences and Engineering Research Council post-graduate scholarship. P. J. Adhihetty is funded by a Heart and Stroke Foundation of Canada doctoral research award. D. A. Hood is the holder of a Canada Research Chair in cell physiology.
Address for reprint requests and other correspondence: D. A. Hood, Dept. of Biology, York Univ., Toronto, Ontario, Canada M3J 1P3 (E-mail: dhood{at}yorku.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00409.2002
Received 5 September 2002; accepted in final form 27 January 2003.
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