Endocrinology, doi:10.1210/en.2008-0466
Endocrinology Vol. 149, No. 9 4527-4533
Copyright © 2008 by The Endocrine Society
Isoform-Specific Increases in Murine Skeletal Muscle Peroxisome Proliferator-Activated Receptor-
Coactivator-1
(PGC-1) mRNA in Response to β2-Adrenergic Receptor Activation and Exercise
Shinji Miura,
Yuko Kai,
Yasutomi Kamei and
Osamu Ezaki
National Institute of Health and Nutrition (S.M., Y.K., O.E.), Shinjuku-ku, Tokyo 162-8636, Japan; and Tokyo Medical and Dental University (Y.K.), Bunkyo-ku, Tokyo 113-8510, Japan
Address all correspondence and requests for reprints to: Shinji Miura, Ph.D., or Osamu Ezaki, M.D., Ph.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.
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Abstract
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Adrenergic receptor (AR) activation increases expression of peroxisome proliferator-activated receptor (PPAR)-
coactivator 1
(PGC-1) mRNA, which may promote mitochondrial biogenesis in skeletal muscles. An AR-activated increase in PGC-1 mRNA was observed in exercise. PGC-1 mRNA is considered a single transcript (PGC-1-a); however, a transcript search of PGC-1 in expressed sequence tag libraries revealed that two novel isoforms of PGC-1 mRNA, named PGC-1-b and PGC-1-c, were expressed in mice tissues. Compared with PGC-1-a mRNA (a previously described isoform), PGC-1-b or PGC-1-c mRNA was transcribed by a different exon 1 of the PGC-1 gene and produced slightly smaller-sized proteins. PGC-1-b or PGC-1-c protein was functional; both isoforms possessed transcriptional activity and could coactivate PPARs, similar to those in PGC-1-a in vitro. Transgenic mice overexpressing PGC-1-b or PGC-1-c in skeletal muscles showed increased gene expression related to mitochondrial biogenesis and fatty acid oxidation. In C57BL/6J mice, injection of the β2-AR agonist clenbuterol increased PGC-1-b and PGC-1-c mRNA expression more than 350-fold, but not PGC-1-a, in skeletal muscle. A single bout of exercise also increased PGC-1-b and PGC-1-c mRNAs, but not PGC-1-a, in skeletal muscles. The increases in skeletal muscles in response to exercise were inhibited by pretreatment with the β2-AR-specific inhibitor ICI 118,551. However, in liver, fasting increased PGC-1-a mRNA, but not PGC-1-b and PGC-1-c mRNAs. These data indicate that AR activation is a major mechanism of an increase in PGC-1 expression in skeletal muscles, and the increase in PGC-1 mRNAs was isoform specific.
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Introduction
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PEROXISOME PROLIFERATOR-activated receptor (PPAR)- coactivator 1 (PGC-1), which was originally identified as a nuclear receptor coactivator, is expressed in brown adipose tissue (BAT), skeletal muscle, heart, kidney, liver, and brain; is markedly up-regulated in BAT and skeletal muscle after acute exposure to cold stress; and promotes mitochondrial biogenesis (1, 2). Cold is sensed in the central nervous system and results in increased sympathetic output to peripheral tissues, including skeletal muscle and BAT (3). Catecholamine production in response to cold triggers the activation of β-adrenergic receptors (β-ARs), resulting in the elevation of intracellular cAMP and stimulation of adaptive thermogenesis. We recently observed that exercise induced PGC-1 mRNA expression in skeletal muscles via the activation of β2-ARs (4). This might be one of the causes of mitochondrial biogenesis observed after regular exercise (5). PGC-1 plays another role in liver; fasting increased PGC-1 mRNA expression in liver to increase glucose production via increased gluconeogenesis (6).
During the study to elucidate the mechanism of AR-induced PGC-1 mRNA in skeletal muscles, we found two new transcripts of the PGC-1 gene, namely PGC-1-b and PGC-1-c, that were expressed in skeletal muscles. In vitro and in vivo experiments revealed that both isoforms were functional. We examined which isoforms of PGC-1 were increased in response to AR activation or exercise in skeletal muscles.
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Materials and Methods
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Experimental animals
Eight-week-old C57BL/6J mice were obtained from Japan SLC (Hamamatsu, Japan). 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. 0707).
Construct
Mouse PGC-1-b cDNA and PGC-1-c cDNA were obtained by PCR from first-strand cDNA using skeletal muscle total RNA that was prepared from mice after exercise. First-strand cDNA was prepared using an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA) after deoxyribonuclease I digestion. The coding region of mouse PGC-1-b cDNA and PGC-1-c cDNA were subcloned into a mammalian expression plasmid, pM (Clontech). PPAR cDNAs were also subcloned into pM. We used pM to generate a fusion of the GAL4 DNA binding protein (amino acids 1–147) and a protein of interest.
Transfection and luciferase assays
HEK293 cells were plated at a density of 1 x 105 cells/12-well plate in DMEM containing 10% fetal bovine serum. The luciferase reporter gene plasmid containing four copies of a GAL4 binding site followed by a minimum thymidine kinase promoter [(UAS)4-TK-Luc] was prepared as described previously (7). To measure the transcription activity of PGC-1 isoforms, luciferase reporter plasmid (0.8 µg), expression plasmids (pM-PGC-1-a, pM-PGC-1-b, pM-PGC-1-c, and empty pM, total 0.8 µg), and a phRL-TK vector (64 ng; Promega, Madison, WI) as an internal control for transfection efficiency were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). To measure the cotranscription activity of PGC-1 isoforms to PPARs, luciferase reporter plasmid (0.8 µg), expression plasmids of PPARs (pM-PPAR, pM-PPAR
, pM-PPAR, and empty pM, total 0.4 µg), expression plasmids of PGC-1 isoforms (pCMX flag-PGC-1-a, pCMX flag-PGC-1-b, pCMX flag-PGC-1-c, and empty pCMX flag, total 0.4 µg), and a phRL-TK vector (64 ng) as an internal control for transfection efficiency were transfected into cells. After an overnight transfection period, cells were lysed and assayed for luciferase activity using the dual luciferase assay kit (Promega). The activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity (internal control) and represented as the average of triplicate experiments.
Transgenic mice
The methods for the generation of transgenic mice overexpressing PGC-1-b or PGC-1-c in skeletal muscle were the same as those to generate PGC-1-a transgenic mice as described previously (8). The human skeletal muscle -actin promoter provided by Drs. E. D. Hardeman and K. Guven (Children’s Medical Research Institute, Australia) was used to express PGC-1 isoforms in skeletal muscle. The transgenic mice (heterozygotes, BDF 1 background) and wild-type C57BL6 mice were crossed, and the offspring (heterozygote and wild-type, born at the same period) were used for the experiments. Copy number of PGC-1 transgene in transgenic mice was estimated as described previously (8).
Experimental protocols
For β-AR agonist experiments, mice were injected sc with clenbuterol (1 mg/kg body weight) dissolved in saline. At 4 h after the injection, skeletal muscles (gastrocnemius) were isolated. For exercise experiments, mice were subjected to treadmill running at 15 m/min for 45 min. At 3 h after exercise, skeletal muscles (gastrocnemius) were isolated. For β-AR antagonist experiments, mice were injected sc with 10 mg/kg body weight ICI 118,551 (β2-AR-specific inhibitor) or the same volume of saline 1 h before exercise. All AR agonists and antagonist were purchased from Sigma Chemical Co. (St. Louis, MO). For fasting experiments, mice were fasted overnight (21 h), and liver was isolated. For cold exposure experiments, mice were placed in a room at 4 C for 24 h, and BAT was isolated. In all experiments, appropriate control mice were prepared. Skeletal muscle, liver, and BAT were rapidly (30–60 sec) removed from mice killed by decapitation. The samples were immediately frozen in liquid nitrogen and kept at –80 C.
Quantitative real-time RT-PCR
Methods of RNA preparation and quantitative real-time RT-PCR were as described previously (4). The mouse-specific primer pairs used were as follows: PGC-1-a forward, 5'-GCTTGACTGGCGTCATTCG-3'; PGC-1-a reverse, 5'-ACAGAGTCTTGGCTGCACATGT-3'; PGC-1-b forward, 5'-GACATGGATGTTGGGATTGTCA-3'; PGC-1-b reverse, 5'-ACCAACCAGAGCAGCACATTT-3'; PGC-1-c forward, 5'-AGTGACATGGATGTTGGGATTG-3'; PGC-1-c reverse, 5'-GAATGCCTCCGGTTACTCACTT-3'; glucose transporter 4 (GLUT4) forward, 5'-ATGGCTGTCGCTGGTTTCTC-3'; GLUT4 reverse, 5'-ACCCATGCCGACAATGAAGT-3'; cytochrome c oxidase (COX) 2 forward, 5'-CCGACTAAATCAAGCAACAGTAACA-3'; COX2 reverse, 5'-AAATTTCAGAGCATTGGCCATAG-3', COX4 forward, 5'-CTATGTGTATGGCCCCATCC-3'; and COX4 reverse, 5'-AGCGGGCTCTCACTTCTTC-3'. Other primers used for quantitative real-time RT-PCR were listed previously (4). PCR primers to detect all isoforms of PGC-1 (total PGC-1) were selected to correspond to sequences in the second and third exons of the PGC-1 gene (4).
For the comparison of the amount of mRNA in each isoform and total PGC-1, cDNA was amplified by the same primer sets in quantitative real-time RT-PCR. After purification and quantification of the amount, using cDNA from each isoform or total PGC-1 as a standard, we made a standard titration curve of each isoform or total PGC-1 mRNA by quantitative real-time RT-PCR. We then quantified the amount of mRNA in each isoform and total PGC-1, relative to amount of each cDNA. By these means, we were able to compare the amounts of mRNAs derived from each isoform PGC-1 gene (or total PGC-1). For comparison, relative values of the mean of PGC-1-a mRNA from the control mice are shown in Figs. 3–5
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Statistical analysis
Comparisons of data from multiple groups were made by two-way ANOVA. When differences were significant, data were compared by Fisher’s protected least significant difference test (Statview 5.0; Abacus Concepts, Berkeley, CA). Statistical significance was defined as P < 0.05. All values are shown as mean ± SEM.
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Results
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Isoforms of PGC-1 transcripts
In addition to the previously reported transcript (PGC-1-a, GenBank accession no. NM_008904) (1), we found two novel transcripts, PGC-1-b (GenBank accession no. BB853729) and PGC-1-c (GenBank accession no. AW012094) in the murine expressed sequence tag databases at NCBI. The predicted amino acid sequence is shown in Fig. 1A. Of 795 amino acids in total, only the N-terminal 16 amino acids in PGC-1-a differed from those in PGC-1-b or PGC-1-c. PGC-1-b and PGC-1-c was shorter by four and 13 amino acids, respectively, than PGC-1-a. A comparison of the sequence of these two transcripts with murine genomic sequence identified a novel exon; both PGC-1-b and PGC-1-c were transcripted by a novel exon 1 (exon 1b), which was located 13.7 kb upstream to the previously reported exon 1 (exon 1a) of the PGC-1-a gene (Fig. 1B). Other nucleotide sequences downstream of exon 2 were the same among these three transcripts.
Nucleotide sequences between the PGC-1-b and PGC-1-c transcripts derived from exon 1b were different; the nucleotide sequence of PGC-1-b lacked 50 bp of sequences of PGC-1-c in exon 1b (between the two arrowheads in Fig. 1C
). This suggested that alternative splicing had occurred within exon 1b in the PGC-1-b transcript. The predicted two spliced sites that possess the consensus sequence of murine 5' spliced sites (C/A AGGT A/G AGT) were found in exon 1b (shown underlined in Fig. 1C). The upstream-splicing site was used for PGC-1-b, whereas the downstream-splicing site was used for PGC-1-c. There were two ATG translation initiation codons and two translation stop codons (shown by an asterisk) near to the 3' end of exon 1b. For PGC-1-b, the upstream ATG codon was used for the initiation of protein synthesis. Its transcript derived from exon 1b was deleted by splicing located within exon 1b (shown by an upstream arrowhead in Fig. 1C). For PGC-1-c, the downstream ATG codon was used for the initiation of protein synthesis, and its transcript derived from exon 1b was deleted by splicing located at the end of exon 1b (shown by a downstream arrowhead in Fig. 1C).
PGC-1-b and PGC-1-c were functional in vitro
To examine whether PGC-1-b or PGC-1-c protein was functional, we first cloned their cDNAs. We obtained PCR products in a murine skeletal muscle cDNA library by appropriate primers. The sequence of PCR products confirmed that PGC-1-b and PGC-1-c cDNAs contained their unique exon 1b as shown in Fig. 1 and other exons (exon 2 to exon 13) that were identical to those of PGC-1-a. This suggested that the full length of PGC-1-b and PGC-1-c existed in the skeletal muscles of mice. The in vitro synthesized each isoform PGC-1 protein (
90-kDa protein) using reticulocyte lysate system was identified by Western blotting with antibody to PGC-1 (reacted to a common epitope of PGC-1 isoform) (data not shown). This suggested that PGC-1 isoform cDNA could produce PGC-1 protein.
Next, we made fusion constructs of PGC-1 isoform cDNA and Gal4 DNA-binding domain (Gal4 DB), transfected them into HEK293 cells, and examined whether these Gal4 DB-fused PGC-1 isoform proteins possessed the transcription activities by (UAS)4-luciferase reporter gene assay. Three PGC-1 isoforms showed similar transcription activity at different doses (Fig. 2A). Furthermore, when expression plasmids containing PGC-1 isoform cDNA and expression plasmids containing transcription factors (PPARs) fused with Gal4 DB were cotransfected to the HEK293 cell, (UAS)4-luciferase reporter gene activity was markedly increased to a similar extent in PGC-1 isoforms (Fig. 2B). This suggested that each PGC-1 isoform could bind PPARs and recruited the transcriptional machinery similarly. These data indicated that PGC-1-b or PGC-1-c was functional in vitro.
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FIG. 2. In vitro functional analysis of PGC-1 isoform. A, Dose-dependent transcriptional activation of Gal4 DB-fused PGC-1 isoforms. Expression plasmids harboring PGC-1 isoforms fused with Gal4 DB (0, 0.2, 0.4, and 0.8 µg) were transfected with luciferase (Luc) reporter plasmid containing 4x UAS, into HEK293 cells. B, Transcriptional activation of Gal4 DB-fused PPARs in the absence or presence of PGC-1 isoforms. Expression plasmids harboring PPARs fused with Gal4 DB domain, luciferase (Luc) reporter plasmid containing 4x UAS, and expression plasmids harboring each PGC-1 isoform were transfected into HEK293 cells. In both experiments, after an overnight transfection period, cells were lysed and assayed for luciferase (Luc) activity. The relative values compared with those obtained for empty vector transfection (set as 1) are shown. The activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity (internal control) and represented as the average of triplicate experiments. Each value represents means ± SEM (n = 3).
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Transgenic mice overexpressing PGC-1-b or PGC-1-c showed increased gene expression related to mitochondrial biosynthesis and fatty acid oxidation
To examine whether PGC-1-b or PGC-1-c protein was functional in vivo, we made transgenic mice to overexpress PGC-1-b or PGC-1-c in skeletal muscles. We obtained six and 10 independent mice lines of PGC-1-b and PGC-1-c transgenic mice, respectively, and examined their phenotype at 8–12 wk of age (Table 1). Compared with the control littermates, skeletal muscles (gastrocnemius) in PGC-1-b and PGC-1-c transgenic mice were reddish (data not shown) and showed 2- to 4-fold increases in mtTFA (mitochondrial transcription factor A), COX2 (mitochondrially encoded COX subunit), COX4 (nuclear encoded COX subunit), ERR (estrogen-related receptor ) and MCAD (a marker of mitochondrial fatty acid oxidation and PPAR target gene) mRNAs in most of lines. A mouse line PGC-1-c (line 03-8) that expressed PGC-1 only by 1.2-fold did not increase the expression in these genes. In our previous study (8), increases in COX2, COX4, and MCAD mRNAs were also increased in PGC-1-a transgenic mice. There were some differences in gene expression between PGC-1-b (or PGC-1-c) transgenic mice and PGC-1-a transgenic mice. GLUT4 mRNA was decreased in PGC-1-a transgenic mice (8). However, GLUT4 mRNA was not decreased in PGC-1-b and PGC-1-c transgenic mice; rather, they increased by 1.3- to 1.7-fold in some PGC-1-b transgenic mice lines (line 03-2, 04-5, and 05-5). PGC-1-a activates the expression of the gene encoding pyruvate dehydrogenase 4 (PDK4), a negative regulator of glucose oxidation (9). However, PDK4 mRNA was rather decreased in PGC-1-b mice and remained unchanged in PGC-1-c mice.
Clenbuterol injection increased PGC-1-b and PGC-1-c mRNAs in skeletal muscles
An injection of clenbuterol (β2-AR agonist) increases expression of total PGC-1 mRNA by more than 48-fold in skeletal muscles (4). We examined which isoform of PGC-1 was increased in response to β2-AR stimulation. Isoform-specific primers were used to measure each isoform mRNA by quantitative real-time RT-PCR. Total PGC-1 mRNA was measured using primers that could amplify all isoforms. Relative values to the mean of PGC-1-a mRNA from control, saline-injected mice are shown (Fig. 3). In control, the amounts of PGC-1-b and PGC-1-c mRNAs were less than 10% of PGC-1-a mRNA. The amount of PGC-1-a mRNA was similar to that of total PGC-1 mRNA. In response to clenbuterol, PGC-1-b and PGC-1-c mRNAs were increased by 350- and 1000-fold, respectively, relative to each of the control values, whereas PGC-1-a mRNA was not (Fig. 3). The increase in total PGC-1 mRNA in response to clenbuterol was well explained by increases in PGC-1-b and PGC-1-c mRNAs.
Exercise increased PGC-1-b and PGC-1-c mRNAs in skeletal muscles
A single bout of exercise increases PGC-1 mRNA transiently, and its increase was mediated by β2-AR stimulation (4). We examined which isoform of PGC-1 could be increased in response to exercise. In control, sedentary mice, the amounts of PGC-1-b and PGC-1-c mRNAs were very low, 7 and 1% of PGC-1-a mRNA, respectively, whereas the amount of PGC-1-a mRNA was similar to that of total PGC-1 mRNA (Fig. 4). Three hours after an exercise bout, PGC-1-b, PGC-1-c, and total PGC-1 mRNAs had increased by 28-, 41-, and 8-fold, respectively, relative to each of the control values, whereas PGC-1-a mRNA had not. Approximately 40% of the increase in total PGC-1 mRNA in response to exercise was explained by increases in PGC-1-b and PGC-1-c mRNAs, suggesting that other isoforms might have existed. It is also conceivable, however, that this imbalance was due to the variations in estimating mRNA levels by RT-PCR methods.
Pretreatment with the β2-AR-specific inhibitor ICI 118,551 at a dose of 10 mg/kg body weight inhibited the exercise-mediated increase in PGC-1-b and PGC-1-c mRNA by 87 and 66%, respectively (data not shown). Treatment with ICI 118,551, with or without exercise, did not affect PGC-1-a mRNA levels.
Regulation of PGC-1 isoform mRNAs in liver
Next, we examined the roles of PGC-1 isoform in liver. It is well known that PGC-1 mRNA is increased in liver from fasted mice (6). In the control mice, the amount of PGC-1-a mRNA was comparable to that of total PGC-1 mRNA. The amounts of PGC-1-b and PGC-1-c mRNAs were undetectable (Fig. 5A). Fasting for 21 h increased PGC-1-a and total PGC-1 mRNAs by 4.4- and 3.4-fold, respectively, relative to each of the control values but did not increase PGC-1-b and PGC-1-c mRNAs (they were also undetectable). These data suggested that in contrast to skeletal muscles, PGC-1-b and PGC-1-c isoforms might not play important roles in liver under fasting conditions.
Regulation of PGC-1 isoform mRNAs in BAT
Lastly, we examined the roles of PGC-1 isoform in BAT. It is well known that PGC-1 mRNA is increased in BAT from cold-exposed mice (1). In the control mice, the amounts of PGC-1-b and PGC-1-c mRNAs were less than 1 and 16% of PGC-1-a mRNA, respectively, and the amount of PGC-1-a mRNA was similar to that of total PGC-1 mRNA (Fig. 5B). Cold exposure for 24 h increased PGC-1-b and total PGC-1 mRNA by 300-and 7-fold, respectively, relative to each of the control values but did not increase PGC-1-a or PGC-1-c mRNAs. Half of the increase in total PGC-1 mRNA was explained by the sum of the increases in PGC-1 isoform mRNAs. These data suggested that other isoforms might exist or that this imbalance was due to the variations in mRNAs estimated by RT-PCR methods as observed in the exercise experiment (Fig. 4).
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Discussion
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In the present study, we found that two novel isoforms of PGC-1 transcripts were expressed in skeletal muscles and that they are functional. These two isoforms, named PGC-1-b and PGC-1-c, were not pseudogenes, because full-length cDNAs of PGC-1-b and PGC-1-c were obtained from the mouse skeletal muscle cDNA library by PCR. When full-length cDNAs of PGC-1-b and PGC-1-c were forced to express in cells and skeletal muscles, they were functional; they showed coactivation activity that increased in cotranscription with PPARs by reporter gene assay in cultured cells and increased gene expression related to mitochondrial biogenesis and fatty acid oxidation in transgenic mice. In addition, these two isoforms were physiologically functional. Although the basal expression levels in PGC-1-b and PGC-1-c mRNAs were less than 10% of PGC-1-a mRNA in skeletal muscles, PGC-1-b and PGC-1-c mRNAs, but not PGC-1-a, were increased in response to β2-AR activation, a mechanism by which exercise increased PGC-1 mRNA.
In skeletal muscles, the functions of PGC-1-b and PGC-1-c were similar to PGC-1-a, in respect to mRNA alterations related to mitochondrial biogenesis and fatty acid oxidation, whereas they were different in respect to mRNA alterations related to carbohydrate metabolism. GLUT4 mRNA was decreased in PGC-1-a transgenic mice (8), whereas it was rather increased in PGC-1-b mice and remained unchanged in PGC-1-c mice. PDK4 mRNA was increased in PGC-1-a transgenic mice (Miura, S., and O. Ezaki, unpublished observation), whereas it was rather decreased in PGC-1-b and remained unchanged in PGC-1-c mice. PGC-1-a encodes a protein of 795 amino acids with a predicted molecular mass of 92 kDa (1). Only the N-terminal 16 amino acids in PGC-1-a differed from those in PGC-1-b or PGC-1-c; this small difference may affect the binding to putative transcription factors, and this may lead to different responses in metabolism. The transcriptional activation domain in PGC-1 resides in the N-terminal 200 amino acids; this corresponds to the location of cAMP response element binding protein-binding protein (CBP)/p300 and SRC-1 binding sites, in which CBP/p300 and SRC-1 showed histone acetyltransferase activity (10). Functional studies of PGC-1 protein, including mutational analysis of amino acids near the N terminal, are required to prove this hypothesis.
Expression levels of isoforms PGC-1-b and PGC-1-c were markedly altered under physiological conditions in skeletal muscles and BAT, but not in liver. A single bout of exercise increased both PGC-1-b and PGC-1-c mRNAs in skeletal muscles, and cold exposure increased only PGC-1-b mRNA in BAT. Fasting, however, increased PGC-1-a mRNA, but not PGC-1-b and PGC-1-c mRNA in liver. The increases in PGC-1-b and PGC-1-c mRNAs in skeletal muscles in response to exercise were mainly mediated by β2-AR activation, because β2-AR-specific activation markedly increased expression in PGC-1-b and PGC-1-c mRNAs, and pretreatment of β2-AR-specific inhibitor prevented the increases in PGC-1-b and PGC-1-c mRNAs in response to exercise.
The increase in PGC-1 in skeletal muscles (or BAT) might be favorable in obese patients because it causes an increase in fatty acid oxidation, whereas that increase in liver was unfavorable because it resulted in an increase in blood glucose concentration. Because the regulatory mechanisms for PGC-1 expression are apparently different in skeletal muscle (or BAT) and liver (different isoforms were expressed in different tissues), we could increase PGC-1 mRNA in skeletal muscles (or BAT) without increasing PGC-1 mRNA in liver via promoting transcription in only the PGC-1-b and PGC-1-c mRNA. Promoter analyses of the PGC-1 gene in previous studies were based on the promoter of PGC-1-a that located upstream of exon 1a (Fig. 1) (11, 12). To clarify the regulation of PGC-1 expression in skeletal muscles, the promoters of PGC-1-b and PGC-1-c are required to be determined.
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Acknowledgments
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We thank Dr. Yasushi Ogawa (Tokyo Medical and Dental University, Japan) for discussion.
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Footnotes
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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 (MEXT, Tokyo, Japan); by research grants from the Japanese Ministry of Health, Labor, and Welfare; and by a grant for the Promotion of Fundamental Studies in Health Sciences from the Organization for Pharmaceutical Safety and Research (OPSR) and National Institute of Biomedical Innovation (NIBIO).
Disclosure Statement: The authors have nothing to disclose.
First Published Online May 29, 2008
Abbreviations: AR, Adrenergic receptor; BAT, brown adipose tissue; Gal4 DB, Gal4 DNA-binding domain; PGC-1, PPAR - coactivator 1; PPAR, peroxisome proliferator-activated receptor.
Received April 2, 2008.
Accepted for publication May 22, 2008.
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Abstract
Introduction
