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Endocrinology, doi:10.1210/en.2006-1646
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Endocrinology Vol. 148, No. 7 3441-3448
Copyright © 2007 by The Endocrine Society

An Increase in Murine Skeletal Muscle Peroxisome Proliferator-Activated Receptor-{gamma} Coactivator-1{alpha} (PGC-1{alpha}) mRNA in Response to Exercise Is Mediated by ß-Adrenergic Receptor Activation

Shinji Miura, Kentaro Kawanaka, Yuko Kai, Mayumi Tamura, Masahide Goto, Tetsuya Shiuchi, Yasuhiko Minokoshi and Osamu Ezaki

Nutritional Science Program, National Institute of Health and Nutrition (S.M., Y.K., M.T., O.E.), Tokyo 162-8636, Japan; Department of Health and Nutrition, Niigata University of Health and Welfare (K.K.), Niigata 950-3198, Japan; Molecular Medicine Research Labs (M.G.), Astellas Pharma Inc., Tsukuba 305-8585, Japan; and Division of Endocrinology and Metabolism, National Institute for Physiological Sciences and the Graduate University for Advanced Studies (T.S., Y.M.), Okazaki 444-8585, Japan

Address all correspondence and requests for reprints to: Shinji Miura, Ph.D., or Osamu Ezaki, M.D., National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8636, Japan. E-mail: shinjim{at}nih.go.jp or ezaki{at}nih.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A single bout of exercise increases expression of peroxisome proliferator-activated receptor-{gamma} coactivator (PGC)-1{alpha} mRNA, which may promote mitochondrial biogenesis in skeletal muscle. In brown adipose tissue, cold exposure up-regulates PGC-1{alpha} expression via adrenergic receptor (AR) activation. Because exercise also activates the sympathetic nervous system, we examined whether exercise-induced increase in PGC-1{alpha} mRNA expression in skeletal muscle was mediated via AR activation. In C57BL/6J mice, injection of the ß2-AR agonist clenbuterol, but not {alpha}-, ß1-, or ß3-AR agonists, increased PGC-1{alpha} mRNA expression more than 30-fold in skeletal muscle. The clenbuterol-induced increase in PGC-1{alpha} mRNA expression in mice was inhibited by pretreatment with the ß-AR antagonist propranolol. In ex vivo experiments, direct exposure of rat epitrochlearis to ß2-AR agonist, but not {alpha}-, ß1-, and ß3-AR agonist, led to an increase in levels of PGC-1{alpha} mRNA. Injection of ß2-AR agonist did not increase PGC-1{alpha} mRNA expression in ß1-, ß2-, and ß3-AR knockout mice (ß-less mice). PGC-1{alpha} mRNA in gastrocnemius was increased 3.5-fold in response to running on a treadmill for 45 min. The exercise-induced increase in PGC-1{alpha} mRNA was inhibited by approximately 70% by propranolol or the ß2-AR-specific inhibitor ICI 118,551. The exercise-induced increase in PGC-1{alpha} mRNA in ß-less mice was also 36% lower than that in wild-type mice. These data indicate that up-regulation of PGC-1{alpha} expression in skeletal muscle by exercise is mediated, at least in part, by ß-ARs activation. Among ARs, ß2-AR may mediate an increase in PGC-1{alpha} by exercise.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXERCISE CAUSES MARKED activation of the sympathetic nervous system (SNS) (1, 2). Exercise increases norepinephrine at the nerve endings as well as plasma levels of epinephrine from the adrenal medullas (3). This increase in catecholamine production may play a major role in mediating cardiovascular and metabolic responses to exercise, which are responsible for increasing supplies of oxygen and fuel to working muscles (4). Increased mitochondrial biogenesis observed after exercise may also be mediated by the SNS (5); however, this hypothesis is not yet proven.

Peroxisome proliferator-activated receptor-{gamma} coactivator (PGC)-1{alpha}, which was originally identified as a nuclear receptor coactivator, is expressed in brown adipose tissue (BAT), skeletal muscle, heart, kidney, and brain; is markedly up-regulated in BAT and skeletal muscle after acute exposure to cold stress; and promotes mitochondrial biogenesis (6, 7). Cold is sensed in the central nervous system and results in increased sympathetic output to peripheral tissues, including muscle and BAT (8). Catecholamine production in response to cold triggers activation of ß-adrenergic receptors (ARs), resulting in the elevation of intracellular cAMP and stimulation of adaptive thermogenesis. In differentiated HIB1B brown fat cells, exposure to isoproterenol, a nonsubtype-selective ß-AR agonist, increased expression of PGC-1{alpha} mRNA (6). In addition to cold exposure, exercise also induced PGC-1{alpha} mRNA expression in skeletal muscles of rats and humans (9, 10, 11). Skeletal muscle PGC-1{alpha} protein levels were increased at 18 h after a single bout of swimming (9, 12). The physiological significance of the exercise-induced expression of PGC-1{alpha} was investigated in transgenic mice overexpressing PGC-1{alpha} in skeletal muscle. These mice showed a similar phenotype to that observed in exercise-trained mice, namely the reddish color characteristic of oxidative muscle and increased production of enzymes related to mitochondrial oxidative phosphorylation and fatty acid oxidation (13, 14). However, continuous increases in PGC-1{alpha} caused muscle atrophy with depletion of ATP (15).

Although it is not known whether an increase in skeletal muscle PGC-1{alpha} is beneficial or harmful and increased PGC-1{alpha} mRNA in response to exercise promotes mitochondrial biogenesis, multiple signaling pathways are thought to be involved in exercise-induced expression of PGC-1{alpha}. Because administration of 5-amino-inmidazol-4-carboxamide ribonucleoside, an activator of AMP-activated protein kinase (AMPK), increased expression of PGC-1{alpha} mRNA in isolated epitrochlearis (16) and skeletal muscle (17), it was hypothesized that the increase in AMPK with muscle contraction mediated the up-regulation of PGC-1{alpha} mRNA expression. However, a recent study showed that {alpha}2-AMPK knockout mice as well as wild-type mice showed increased cytochrome c oxidase subunit (COX) I and citrate synthase protein production in response to exercise training, and therefore, {alpha}2-AMPK may not be essential for exercise-induced mitochondrial biogenesis (18). The Ca2+-signaling (19) and p38 MAPK pathways (20) have also been reported to be activated in skeletal muscle in response to exercise and to regulate PGC-1{alpha} expression. However, the physiological significance of these pathways is not clear.

The molecular mechanism of exercise-induced up-regulation of PGC-1{alpha} was clarified by analysis of the PGC-1{alpha} gene promoter (21). In that study, the myocyte enhancing factor (MEF)-2 and cAMP response element (CRE) sequences were required for contractile-induced activation of PGC-1{alpha} promoter in skeletal muscle (21). The transducers of regulated cAMP response element-binding protein (CREB) (TORCs), coactivators of CREB, markedly activated PGC-1{alpha} transcription and mitochondrial biogenesis in muscle cells (22). Because TORC is activated by calcium and cAMP (23), AR activation might be involved in an increase in skeletal muscle PGC-1{alpha} expression in response to exercise.

In the present study, to examine whether the AR activation is involved in the exercise-induced increase in PGC-1{alpha} mRNA expression in skeletal muscle, we evaluated the effects of ß-AR agonist and antagonist in vivo and ex vivo and the effect of exercise in mice that lack the three known ß-ARs (ß-less mice).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Eight-week-old C57BL/6J mice were obtained from Japan SLC (Hamamatsu, Japan). Knockout mice that lack the three known ß-ARs (ß-less mice) and wild-type (WT) mice were kindly supplied by Dr. Bradford B. Lowell (Harvard Medical School, Boston, MA) (24, 25). Mice were exposed to a 12-h light, 12-h dark cycle and maintained at a constant temperature of 22 C. Mice were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and our institutional guidelines. All animal experiments were conducted with the approval of the National Institute of Health and Nutrition Ethics Committee on Animal Research (no. 0610).

Experimental protocols
For {alpha}- or ß-AR agonist experiments, mice were injected sc with phenylephrine, dobutamine, clenbuterol, or CL 316,243 dissolved in saline or ethanol. The amount of agonist administered is described in each figure legend. At 4 or 24 h after the injection, skeletal muscles (gastrocnemius) were isolated. Before injection and at 15, 60, and 150 min after injection, blood was collected for plasma free fatty acids (FFA) analysis using NEFA-C (Wako Pure Chemical Industries, Ltd., Osaka, Japan). For exercise experiments, mice were subjected to treadmill running at 15 m/min for 45 min. At 0 (just after exercise), 3, 6, and 9 h after exercise, skeletal muscles (gastrocnemius) were isolated. For propranolol experiments, mice were injected sc with 10 mg/kg body weight propranolol or the same volume of saline 1 h before injection of AR agonist or exercise. This dose of propranolol completely inhibits the clenbuterol (5 mg/kg body weight)-induced apoptosis in rat skeletal muscle (26). To examine the effect of a ß2-AR-specific inhibitor, mice were injected sc with 10 mg/kg body weight ICI 118,551 at 1 h before exercise. All AR agonists and antagonist were purchased from Sigma (St. Louis, MO). In all experiments, skeletal muscle was rapidly (30–60 sec) removed from mice killed by decapitation, frozen immediately in liquid nitrogen, and kept at –80 C.

Epitrochlearis preparation
Male Wistar rats were anesthetized with an ip injection of pentobarbital sodium (5 mg per 100 g body weight), and the epitrochlearis muscles were dissected out and incubated at 35 C for 5 h in 3 ml of oxygenated Krebs-Henseleit bicarbonate buffer containing 8 mM glucose and 32 mM mannitol (27). The gas phase in the flasks was 95% O2-5% CO2. Epitrochlearis muscles were exposed to 1 µM phenylephrine, dobutamine, clenbuterol, or CL 316,243 for 5 h. After the incubations, muscles were blotted and frozen at –80 C for RNA preparation.

Preparation of cDNA probe and Northern blotting
The DNA fragments for PGC-1{alpha}, COX II, and COX IV were prepared as described previously (14). Northern blotting was performed as described previously (14). 18S rRNA was analyzed as a control for loading (28). Transcript levels were quantitated with an image analyzer (BAS 1800-II; Fuji Film, Tokyo, Japan). The sum of the three different transcripts detected of PGC-1{alpha} was shown.

Real-time RT-PCR
Total RNA isolated from the muscle was reverse transcribed with ReverTra Ace (Toyobo, Osaka, Japan) with random hexamers. Reactions were performed in the 96-well format with SYBR Green PCR master mix and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA). Results were normalized to the expression of 36B4 or 18S rRNA. The following mouse-specific primer pairs were used: PGC-1{alpha} forward, 5'-CGGAAATCATATCCAACCAG-3'; PGC-1{alpha} reverse, 5'-TGAGGACCGCTAGCAAGTTTG-3'; estrogen-related receptor (ERR)-{alpha} forward, 5'-TTCGGCGACTGCAAGCTC-3'; ERR{alpha} reverse, 5'-CACAGCCTCAGCATCTTCAATG-3'; pyruvate dehydrogenase kinase 4 (PDK4) forward, 5'-CCGCTGTCCATGAAGCA-3'; PDK4 reverse, 5'-GCAGAAAAGCAAAGGACGTT-3'; medium chain acetyl-coenzyme A dehydrogenase (MCAD) forward, 5'-TGGATCTGTGCAGCGGATT-3'; MCAD reverse, 5'-GGGTCACCATAGAGCTGAAGACA-3'; mitochondrial transcription factor A (mtTFA) forward, 5'-GGAATGTGGAGCGTGCTAAAA-3'; mtTFA reverse, 5'-TGCTGGAAAAACACTTCGGAATA-3'; 36B4 forward, 5'-GGCCCTGCACTCTCGCTTTC-3'; 36B4 reverse, 5'-TGCCAGGACGCGCTTGT-3'; 18S rRNA forward, 5'-GGGAGCCTGAGAAACGGC-3'; and 18S rRNA reverse; 5'-GGGTCGGGAGTGGGTAATTT-3'. PCR primers for PGC-1{alpha} were selected to correspond to sequences in the second and third exons of the PGC-1{alpha} gene.

Statistical analysis
Comparisons of data from multiple groups were made by one-way ANOVA. When differences were significant, data were compared by Fisher’s protected least significant difference test (Statview 5.0; Abacus Concepts, Berkeley, CA). Comparisons of data between two groups were made by unpaired Student’s t test. Changes in plasma FFA concentration in each group over time were compared by two-way repeated-measures ANOVA (StatView 5.0). Statistical significance was defined as P < 0.05. All values are mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ß2-AR agonist but not {alpha}-, ß1-, or ß3-AR agonist increases expression of PGC-1{alpha} mRNA in skeletal muscles in vivo and ex vivo
Selective AR agonist (phenylephrine for {alpha}-AR, dobutamine for ß1-AR, clenbuterol for ß2-AR, and CL 316,243 for ß3-AR) was injected into mice sc. At 4 h after the injection, expression of PGC-1{alpha} mRNA in skeletal muscles was analyzed by real-time RT-PCR (Fig. 1AGo). Among these compounds, only the ß2-AR agonist clenbuterol increased PGC-1{alpha} expression in skeletal muscles. Similar results were obtained when mice were injected with a lower dose (0.1 mg/kg body weight) of each AR agonist (data not shown). Clenbuterol (1 mg/kg body weight) increased the levels of PGC-1{alpha} mRNA 1.3-, 9.7-, 47.7-, and 31.5-fold at 1, 2, 4, and 6 h after the injection, respectively. Other ß2-AR agonists (1 mg/kg body weight), salbutamol and salmeterol, also increased the levels of PGC-1{alpha} mRNA 9.7- and 23.1-fold, respectively, at 4 h after the injection. To determine whether AR agonist-induced increases in plasma FFA concentration affect the expression of PGC-1{alpha} in skeletal muscle, plasma FFA concentrations at 15, 60, and 90 min after each AR agonist injection were measured (Fig. 1BGo). Significant increases in plasma FFA concentrations were observed in ß3-AR agonist-injected mice. Discrepancies in the increases in PGC-1{alpha} mRNA and FFA concentration between AR agonist-injected mice indicated that increases in plasma FFA concentration may not be involved in ß2-AR agonist-induced PGC-1{alpha} expression in skeletal muscle. Although it has not been proven that increased gene expression related to energy metabolism is solely mediated by an increase in PGC-1{alpha}, increased expression of several of PGC-1{alpha} target genes, including ERR{alpha}, PDK4, MCAD, mtTFA, COX II, and COX IV was observed in clenbuterol-injected mice (29) (Fig. 2Go).


Figure 1
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  		FIG. 1. Regulation of skeletal muscle PGC-1{alpha} expression by {alpha}-, ß1-, ß2-, and ß3-AR agonists in vivo. C57BL/6J mice were injected sc with 1 mg/kg body weight of phenylephrine ({alpha}), dobutamine (ß1), clenbuterol (ß2), or CL 316,243 (ß3). All AR agonists except dobutamine were dissolved in saline. Dobutamine was dissolved in ethanol (EtOH). An identical volume of saline or EtOH was injected as a control. A, At 4 h after injection, skeletal muscle (gastrocnemius) was removed. The level of PGC-1{alpha} mRNA was analyzed by real-time RT-PCR and normalized to the expression of 36B4. The graph shows the percent in PGC-1{alpha} mRNA level relative to that of saline-injected mice. Each value is the mean ± SEM of three mice. ***, P < 0.001 vs. saline-injected mice. B, Plasma FFA concentrations were measured before injection and at 15, 60, and 150 min after injection. Each value is the mean ± SEM of three mice. Data were analyzed by repeated ANOVA. The FFA curve of ß3-AR-injected mice differed significantly from that of control, saline-injected mice (P < 0.05).

 

Figure 2
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  		FIG. 2. Effects of a ß2-AR agonist, clenbuterol, on expression of mRNAs for transcription factors and mitochondrial oxidative metabolism enzymes in skeletal muscles. C57BL/6J mice were injected sc with 1 mg/kg body weight of clenbuterol. An identical volume of saline was injected as a control. At 24 h after injection of saline or clenbuterol, skeletal muscle (gastrocnemius) was removed. Levels of mRNA were analyzed by real-time RT-PCR normalized to the expression of 36B4 for ERR{alpha}, PDK4, MCAD, and mtTFA, or Northern blotting normalized to the expression of 18S rRNA for COX II and COX IV. The graphs show the percent in mRNA levels relative to those of saline-injected mice. Each value is the mean ± SEM of five mice. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. saline-injected mice.

 
To examine whether an increase in PGC-1{alpha} mRNA in response to ß2-AR agonist was also observed in ex vivo experiments, isolated epitrochlearis from rat was incubated with each of the AR agonists at 1 µM for 5 h at 35 C, and expression levels of PGC-1{alpha} mRNA were measured by real-time RT-PCR (Fig. 3Go). We used rat epitrochlearis instead of mouse epitrochlearis because of its larger size and easier manipulation. Only the ß2-AR agonist clenbuterol increased PGC-1{alpha} expression in rat epitrochlearis, although the increase ex vivo was smaller than that in vivo. The reduced clenbuterol response in rat epitrochlearis may be due to species differences and/or a decreased accessibility of clenbuterol to isolated tissues. These data indicated that an increase of PGC-1{alpha} mRNA in response to systemic clenbuterol might be due, at least in part, to a direct effect of clenbuterol on skeletal muscle.


Figure 3
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  		FIG. 3. Regulation of PGC-1{alpha} expression in isolated epitrochlearis by {alpha}-, ß1-, ß2-, or ß3-AR agonists in an ex vivo experiment. Isolated rat epitrochlearis was exposed for 5 h to 1 µM of phenylephrine ({alpha}), dobutamine (ß1), clenbuterol (ß2), or CL 316,243 (ß3). All AR agonists except dobutamine were dissolved in saline. Dobutamine was dissolved in ethanol (EtOH). An identical volume of saline or EtOH was used as a control. The level of PGC-1{alpha} mRNA was analyzed by real-time RT-PCR and normalized to the expression of 18S rRNA. The graph shows the percent of PGC-1{alpha} mRNA level relative to that of saline-treated epitrochlearis. Each value is the mean ± SEM of four epitrochlearis muscles obtained from four rats. Two were obtained from one rat; one was used as a control, and the other was used for treatment. *, P < 0.05 vs. saline-treated specimens.

 
Administration of ß-AR antagonist propranolol can inhibit the clenbuterol-mediated increase in PGC-1{alpha} mRNA
To examine clenbuterol-mediated increase in PGC-1{alpha} mRNA in detail, dose-dependent effects of clenbuterol were studied. Clenbuterol was injected sc at various concentrations into mice, and PGC-1{alpha} mRNA levels in skeletal muscle were measured by Northern blotting (Fig. 4Go). Levels of all three endogenous PGC-1{alpha} transcripts, 6.5, 5, and 3 kb, were increased. The 6.5-kb transcript is the full-length form. As is suggested to occur in humans, the 5-kb transcript may be derived by use of an alternate polyadenylation site (30). The 3-kb transcript may be an alternatively spliced form of the PGC-1{alpha} transcript and lacks exon 8 and possibly exons 5, 6, and 7 (9). At a concentration of less than 0.01 mg/kg body weight, clenbuterol did not affect PGC-1{alpha} mRNA expression. At concentrations of more than 0.1 mg/kg body weight, clenbuterol increased PGC-1{alpha} mRNA levels 21- to 26-fold in skeletal muscle. This dose of clenbuterol is much lower than the 1 mg/kg body weight used previously to examine insulin resistance (31). However, the increase in PGC-1{alpha} mRNA in response to clenbuterol was not dose dependent; the increase in PGC-1{alpha} mRNA in response to 5 mg/kg body weight of clenbuterol was less than that of 1 mg/kg body weight of clenbuterol.


Figure 4
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  		FIG. 4. Effect of clenbuterol dose on PGC-1{alpha} expression in skeletal muscle and the inhibitory effects of propranolol. C57BL/6J mice were injected sc with saline (–) or propranolol (+) (10 mg/kg body weight) and then injected sc with saline (0 mg/kg body weight of clenbuterol) or selective ß2-AR agonist clenbuterol at a concentration of 0.01, 0.1, 1, or 5 mg/kg body weight. At 4 h after injection of saline or clenbuterol, skeletal muscle (gastrocnemius) was removed. A, Northern blot analysis of PGC-1{alpha} expression. Each lane represents an individual mouse. 18S rRNA is shown as a loading control. B, Graph of the percent in PGC-1{alpha} mRNA levels relative to those of 0 mg/kg body weight clenbuterol (saline)-injected mice. Open columns show saline preinjected mice, and closed columns show propranolol preinjected mice. Each value is the mean ± SEM of three mice. {dagger}, P < 0.05, {dagger}{dagger}{dagger}, P < 0.001 vs. saline preinjected mice; ***, P < 0.001 vs. 0 mg/kg body weight clenbuterol (saline)-injected mice.

 
The potency of the ß-AR antagonist propranolol was examined. Pretreatment of mice with propranolol at a dose of 10 mg/kg body weight effectively prevented the clenbuterol-induced increase in PGC-1{alpha}; however, this dose was not enough to inhibit substantially the increase in PGC-1{alpha} mRNA by clenbuterol at concentrations of 5 mg/kg body weight.

An increase in PGC-1{alpha} mRNA in response to clenbuterol is not observed in ß-less mice
To verify directly that the increase in PGC-1{alpha} mRNA in response to clenbuterol was mediated by ARs, the effect of clenbuterol (5 mg/kg body weight) on PGC-1{alpha} mRNA was investigated in ß-less mice (24, 25). As expected, the increase in PGC-1{alpha} mRNA in response to clenbuterol was not observed in ß-less mice, indicating that the increase in PGC-1{alpha} mRNA in response to clenbuterol was mediated by the ß1-, ß2-, and/or ß3-ARs (Fig. 5Go). However, in response to clenbuterol treatment, control WT mice with the FVB/C57BL6/DBA/2/129SvJ genetic background (24, 25) showed only a 3.6-fold increase in PGC-1{alpha} mRNA. This increase was much less than the 21- to 32-fold increases observed in C57BL/6J mice, indicating that the response to clenbuterol varied among mouse strains.


Figure 5
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  		FIG. 5. Effects of clenbuterol on PGC-1{alpha} expression in ß-less mice. ß-Less or WT mice were injected sc with saline or selective ß2-AR agonist clenbuterol at a concentration of 5 mg/kg body weight. At 4 h after injection of saline or clenbuterol, skeletal muscle (gastrocnemius) was removed. The level of PGC-1{alpha} mRNA was analyzed by Northern blotting and normalized to that of 18S rRNA. The graph shows the percent in PGC-1{alpha} mRNA levels relative to those of saline-injected WT mice. Open columns show WT mice, and closed columns show ß-less mice. Each value is the mean ± SEM of three mice. {dagger}{dagger}{dagger}, P < 0.001 vs. WT mice; ***, P < 0.001 vs. saline-injected mice.

 
Exercise-induced increase in PGC-1{alpha} expression is inhibited approximately 70% by pretreatment with ß-AR antagonists
Because the ß2-AR agonist clenbuterol stimulates PGC-1{alpha} expression in skeletal muscle and exercise stimulates SNS activity, an increase in PGC-1{alpha} mRNA in response to exercise might be due to stimulation of the ARs. To investigate this possibility, we first analyzed the time course of PGC-1{alpha} expression after 45 min of running on a treadmill. In control (propranolol untreated) mice, PGC-1{alpha} transcript levels were higher at 3, 6, and 9 h after exercise than in sedentary animals (Fig. 6Go). PGC-1{alpha} expression was not increased just after exercise (0 h), and PGC-1{alpha} expression was highest (3.5-fold) at 3 h after an exercise bout. Because a large increase in PGC-1{alpha} mRNA levels was observed just after 6 h of exercise (0 h) (data not shown), the increase in PGC-1{alpha} mRNA may occur during muscle contraction but requires at least 45 min of muscle contraction for the increase to be substantial. Similar to the response to clenbuterol stimulation, levels of all three endogenous PGC-1{alpha} transcripts (6.5, 5, and 3 kb) were increased after exercise. Pretreatment of mice with a ß-AR antagonist, propranolol, at a dose of 10 mg/kg body weight, inhibited partially the exercise-mediated increase in all three PGC-1{alpha} transcripts at 3, 6, and 9 h after exercise (Fig. 6Go). When the increase in PGC-1{alpha} expression at 3 h after exercise was measured, propranolol had inhibited the exercise-mediated increase in PGC-1{alpha} mRNA by 69% (Fig. 7AGo). Propranolol pretreated and nontreated mice received the same intensity and duration of exercise because the two groups of mice ran simultaneously side by side on the same treadmill machine. Pretreatment with the ß2-AR-specific inhibitor ICI 118,551 at a dose of 10 mg/kg body weight also inhibited the exercise-mediated increase in PGC-1{alpha} mRNA by 71% (data not shown), suggesting that exercise-mediated increases in PGC-1{alpha} mRNA may mediated by ß2-AR activation. Pretreatment of mice with the {alpha}-AR antagonist phentolamine at a dose of 10 mg/kg body weight did not inhibit the exercise-mediated increase in PGC-1{alpha} (data not shown).


Figure 6
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  		FIG. 6. Exercise-induced increase in PGC-1{alpha} expression with and without propranolol pretreatment. Skeletal muscles (gastrocnemius) at 0 (just after exercise), 3, 6, and 9 h after 45 min of exercise on a treadmill were taken from saline- (–) and 10 mg/kg body weight propranolol-preinjected C57BL/6J mice (+). A, Northern blot analysis of PGC-1{alpha} expression. Each lane represents an individual mouse. 18S rRNA is shown as a loading control. B, Graph showing the percent change in PGC-1{alpha} mRNA levels relative to that of sedentary mice. Open columns show saline-preinjected mice, and closed columns show propranolol-preinjected mice. Each value represents the mean ± SEM of three mice. Similar results were obtained in two additional independent experiments. {dagger}{dagger}, P < 0.01, {dagger}{dagger}{dagger}, P < 0.001 vs. saline-preinjected mice; *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs. sedentary mice.

 

Figure 7
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  		FIG. 7. Exercise-induced increase in PGC-1{alpha} expression was inhibited by propranolol pretreatment and by ablation of ß-ARs. A, Saline- (–) and 10 mg/kg body weight propranolol-preinjected C57BL/6J mice (+) were run for 45 min on a treadmill side by side. At 3 h after exercise, skeletal muscles (gastrocnemius) were taken, and the level of PGC-1{alpha} mRNA was analyzed by Northern blotting and normalized to that of 18S rRNA. The graph shows the percent change in PGC-1{alpha} mRNA levels relative to that of sedentary saline-preinjected mice. Open columns show saline-preinjected mice, and closed columns show propranolol-preinjected mice. Each value is the mean ± SEM of three mice. {dagger}{dagger}, P < 0.01 vs. saline-preinjected mice; *, P < 0.05 and ***, P < 0.001 vs. sedentary mice. B, ß-Less and WT mice were run for 45 min on a treadmill side by side. At 3 h after exercise, skeletal muscles (gastrocnemius) were taken and the level of PGC-1{alpha} mRNA was analyzed by Northern blotting and normalized to that of 18S rRNA. The graph shows the percent change in PGC-1{alpha} mRNA levels relative to those of sedentary WT mice. Open columns show WT mice, and closed columns show ß-less mice. Each value is the mean ± SEM of eight mice. {dagger}{dagger}, P < 0.01 vs. WT mice; ***, P < 0.001 vs. sedentary mice.

 
Exercise-induced increase in PGC-1{alpha} expression is inhibited by 36% in ß-less mice
We did not know what dose of propranolol was sufficient to inhibit the effects of exercise on skeletal muscles. Indeed, 10 mg/kg body weight of propranolol was not sufficient to inhibit substantially the increase in PGC-1{alpha} mRNA triggered by 5 mg/kg body weight of clenbuterol (Fig. 4Go). To examine to what extent ß-ARs activation was involved in the increase in PGC-1{alpha} mRNA in response to exercise more precisely, we measured PGC-1{alpha} mRNA levels in response to exercise in ß-less mice. The ß-less and control WT mice received the same intensity and duration of exercise. The exercise-mediated increase in PGC-1{alpha} mRNA at 3 h after exercise in ß-less mice was 36% lower than that in controls (Fig. 7BGo). However, control WT mice showed only 2.5-fold higher PGC-1{alpha} mRNA levels after exercise (Fig. 7BGo). This increase in response to exercise was less than the increase observed in C57BL/6J mice (~5-fold in Fig. 7AGo), indicating that the response to exercise as well as that to clenbuterol was decreased in this strain.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we found that a ß2-AR agonist, clenbuterol, but not {alpha}-, ß1-, or ß3-AR agonist, increased PGC-1{alpha} mRNA expression both in vivo and ex vivo and that an increase in PGC-1{alpha} mRNA expression in skeletal muscles in response to exercise was due, at least in part, to ß-ARs activation. Among ARs, ß2-AR may mediate an increase in PGC-1{alpha} by exercise.

In a previous study, a ß3-AR agonist, CL 316,243, injected ip increased the skeletal muscle PGC-1{alpha} mRNA level 2-fold in WT mice but not ß3-AR knockout mice (32). However, effects of other AR agonists on skeletal muscle PGC-1{alpha} expression have not been examined. In C57BL/6J mice, a marked 32-fold increase in PGC-1{alpha} mRNA was observed in ß2-AR agonist-injected mice. In ex vivo experiments, an increase in PGC-1{alpha} mRNA was observed only after ß2-AR stimulation. Physiologically, approximately 90% of the ß-AR in skeletal muscle is of the ß2-subtype (33). Therefore, in skeletal muscles, ß2-AR is a major AR subtype that regulates PGC-1{alpha} mRNA expression. In addition, it was recently reported that intracerebroventricular administration of an inhibitor of fatty acid synthase, C57, increased expression of PGC-1{alpha} and its target genes in skeletal muscle through the SNS (34). These data suggest that the SNS can transmit a signal to skeletal muscles to increase PGC-1{alpha} expression.

Promoter analyses of the PGC-1{alpha} gene revealed that catecholamine can stimulate PGC-1{alpha} expression in skeletal muscles. PGC-1{alpha} regulates the activity of its own promoter in a positive loop through MEF2 (19). A dominant-negative form of the CREB or a specific mutation of the CRE site (–222) reduced activation of the PGC-1{alpha} promoter by activated Ca2+/calmodulin-dependent protein kinase (19). In an in vivo study, MEF2- and CRE-binding sites were necessary for increased expression of a PGC-1{alpha} reporter construct in response to muscle contraction (21). Thus, it is possible that phosphorylated CREB binds CRE and increases PGC-1{alpha} transcription. Recently a new mechanism by which CREB activity regulates PGC-1{alpha} transcription was reported (22, 23). In muscle cells, TORCs, coactivators of CREB, markedly activate PGC-1{alpha} transcription and mitochondrial biogenesis (22). Phosphorylated TORCs are sequestered in the cytoplasm, whereas dephosphorylated TORCs by calcium or cAMP-dependent activation moves to the nucleus and coactivates CREB (23). Both cAMP-dependent pathway activation in response to SNS activation and Ca2+ signaling pathway activation in response to muscle contraction may activate TORCs, which would lead to PGC-1{alpha} expression. It is also conceivable that adrenergic activation increases PGC-1{alpha} mRNA by increasing PGC-1{alpha} mRNA stability. It is known that PGC-1{alpha} protein has an RNA recognition motif (35) and that PGC-1{alpha} expression is regulated by PGC-1{alpha} itself (19). Therefore, PGC-1{alpha} mRNA stability may be altered PGC-1{alpha} protein itself.

An exercise bout increases the norepinephrine concentration locally at the nerve endings that innervate contracting muscle and plasma levels of epinephrine from the adrenal medullas (4). Therefore, all tissues are exposed to higher concentrations of catecholamines during exercise than in the sedentary state, but contracting muscles are exposed to much higher concentrations of catecholamines from the nerve endings than are noncontracting muscles. It is expected that during exercise, contracting muscle increases PGC-1{alpha} expression more than noncontracting muscle. The increase in PGC-1{alpha} mRNA levels in clenbuterol injected mice (~30-fold) was much larger than that in exercised mice (3.5-fold), suggesting that the concentration of catecholamine at the nerve endings of skeletal muscles during contraction might be lower than that in the clenbuterol-injected mice in the present study.

Our study also indicated that a part of the increase in PGC-1{alpha} mRNA in response to exercise was not mediated by ß1-, ß2-, and/or ß3-ARs activation. ß4-AR may exist in skeletal muscles and adipocytes, as suggested previously (32, 36) and may be inhibited by propranolol. The exercise-mediated increase in PGC-1{alpha} mRNA was inhibited by 69% in propranolol-treated mice (Fig. 7AGo), whereas the inhibition was 36% in ß-less mice (Fig. 7BGo). This 33% difference might be due to ß4-AR activation by exercise. However, because the exercise-mediated increase in PGC-1{alpha} mRNA was inhibited 69% in propranolol-treated mice, 31% of the increase in PGC-1{alpha} in response to exercise was mediated by propranolol-insensitive pathway(s). The Ca2+-signaling pathway is another candidate for the propranolol-insensitive pathway in response to exercise. Because SNS activation may not occur in isolated rat epitrochlearis, the direct electrical stimulation of a muscle strip might be a good model to examine the Ca2+-signaling pathway specifically. In a previous study, a 1.5-fold increase in PGC-1{alpha} mRNA expression was observed in electrically stimulated, isolated rat epitrochlearis (16). This 1.5-fold increase might be mediated by the Ca2+-signaling pathway.

Increases in PGC-1{alpha} mRNA in response to exercise and clenbuterol treatment in control ß-less mice of the FVB/C57BL6/DBA/2/129SvJ genetic background were much less than those in C57BL6 mice. The reason is not clear at present; however, basal catecholamine concentrations in FVB/C57BL6/DBA/2/129SvJ mice were twice those in C57BL6 mice (Miura, S., and O. Ezaki, unpublished observation), suggesting that the ß-AR receptor sensing system may be blunted in FVB/C57BL6/DBA/2/129SvJ mice.

From our observations, the exercise-induced increases PGC-1{alpha} mRNA levels in skeletal muscle appear to be due, at least in part, to ß-ARs activation. An increase in PGC-1{alpha} mRNA was observed in a muscle strip incubated with a ß2-AR agonist, indicating that ß2-AR agonist can directly stimulate skeletal muscles. However, in vivo, it is possible that ß-AR antagonists and agonists affect the relaxation state of vascular smooth muscle in skeletal muscle tissues and alter blood flow, which could affect PGC-1{alpha} mRNA levels. In addition, they may affect ß-ARs in the brain and alter hormonal and SNS activities, which may also affect PGC-1{alpha} mRNA levels. Therefore, to identify tissues responsible for the increase in skeletal muscle PGC-1{alpha}, studies of PGC-1{alpha} expression in myocyte-specific ß2-AR knockout mice are needed.


   Acknowledgments
 
We thank Dr. Bradford B. Lowell (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA) for supply of ß-less mice.


   Footnotes
 
This work was supported in part by a grant-in-aid for scientific research KAKENHI from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Tokyo, Japan), research grants from the Japanese Ministry of Health, Labor, and Welfare, and a grant for the Promotion of Fundamental Studies in Health Sciences from the Organization for Pharmaceutical Safety and Research and National Institute of Biomedical Innovation.

First Published Online April 19, 2007

Abbreviations: AMPK, AMP-activated protein kinase; AR, adrenergic receptor; BAT, brown adipose tissue; ß-less mice, ß1-, ß2-, and ß3-AR knockout mice; COX, cytochrome c oxidase; CRE, cAMP response element; CREB, cAMP response element-binding protein; ERR, estrogen-related receptor; FFA, free fatty acids; MCAD, medium chain acetyl-coenzyme A dehydrogenase; MEF, myocyte enhancing factor; mtTFA, mitochondrial transcription factor A; PDK4, pyruvate dehydrogenase kinase 4; PGC, peroxisome proliferator-activated receptor-{gamma} coactivator; SNS, sympathetic nervous system; TORC, transducer of regulated CREB; WT, wild type.

Received December 7, 2006.

Accepted for publication April 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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