PGC-1{alpha} Coactivates PDK4 Gene Expression via the Orphan Nuclear Receptor ERR{alpha}: a Mechanism for Transcriptional Control of Muscle Glucose Metabolism

The transcriptional coactivator PGC-1 ${alpha}$ is a key regulator ofenergy metabolism, yet little is known about its role in controlof substrate selection. We found that physiological stimuliknown to induce PGC-1 ${alpha}$ expression in skeletal muscle coordinatelyupregulate the expression of pyruvate dehydrogenase kinase 4(PDK4), a negative regulator of glucose oxidation. Forced expressionof PGC-1 ${alpha}$ in C₂C₁₂ myotubes induced PDK4 mRNA and protein expression.PGC-1 ${alpha}$ -mediated activation of PDK4 expression was shown to occurat the transcriptional level and was mapped to a putative nuclearreceptor binding site. Gel shift assays demonstrated that thePGC-1 ${alpha}$ -responsive element bound the estrogen-related receptor ${alpha}$ (ERR ${alpha}$ ), a recently identified component of the PGC-1 ${alpha}$ signalingpathway. In addition, PGC-1 ${alpha}$ was shown to activate ERR ${alpha}$ expression.Chromatin immunoprecipitation assays confirmed that PGC-1 ${alpha}$ andERR ${alpha}$ occupied the mPDK4 promoter in C₂C₁₂ myotubes. Additionally,transfection studies using ERR ${alpha}$ -null primary fibroblasts demonstratedthat ERR ${alpha}$ is required for PGC-1 ${alpha}$ -mediated activation of the mPDK4promoter. As predicted by the effects of PGC-1 ${alpha}$ on PDK4 genetranscription, overexpression of PGC-1 ${alpha}$ in C₂C₁₂ myotubes decreasedglucose oxidation rates. These results identify the PDK4 geneas a new PGC-1 ${alpha}$ /ERR ${alpha}$ target and suggest a mechanism whereby PGC-1 ${alpha}$ exerts reciprocal inhibitory influences on glucose catabolismwhile increasing alternate mitochondrial oxidative pathwaysin skeletal muscle.

INTRODUCTION

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Introduction

The transcriptional coactivator peroxisome proliferator-activatedreceptor ${gamma}$ (PPAR ${gamma}$ ) coactivator 1 ${alpha}$ (PGC-1 ${alpha}$ ) is a key regulator ofcellular energy metabolism (40), having known roles in thermogenesis(41), mitochondrial biogenesis (28, 56), fatty acid oxidation(51), and hepatic gluconeogenesis (14, 57). PGC-1 ${alpha}$ is enrichedin metabolically active tissues including brown adipose tissue,the heart, and slow-twitch skeletal muscles (30, 41). In contrastto most transcriptional coactivators, expression of the PGC-1 ${alpha}$ gene is highly inducible in accordance with tissue-specificenergy demands. For example, PGC-1 ${alpha}$ expression is rapidly increasedin brown adipose tissue following cold exposure (41), in theliver and heart following short-term starvation (28, 57), inskeletal muscle with exercise (2, 12, 38), and in the postnatalheart coincident with an increase in mitochondrial fatty acidoxidation (28). Gain-of-function and loss-of-function studieshave demonstrated that PGC-1 ${alpha}$ is necessary and sufficient toincrease mitochondrial content and respiratory capacity in responseto physiological stimuli in adipocytes (31, 41), skeletal muscle(29, 56), and the heart (1, 29, 44, 54). Skeletal-muscle-specificoverexpression of PGC-1 ${alpha}$ results in a fiber type switch fromfast-twitch (type II) to slow-twitch (type I) oxidative fibers(30). Slow-twitch fibers are characterized by increased insulinsensitivity, mitochondrial mass, and oxidative capacity. Recentstudies have also demonstrated altered expression of PGC-1 ${alpha}$ indiabetic skeletal muscles (36) and hearts (8).

A variety of transcription factors are known to be coactivatedby PGC-1 ${alpha}$ , including PPAR ${gamma}$ (41), PPAR ${alpha}$ (51), FOXO1 (39), and estrogen-relatedreceptor ${alpha}$ (ERR ${alpha}$ ) (18, 46). In brown adipose tissue, PGC-1 ${alpha}$ interactswith PPAR ${gamma}$ and other transcription factors to regulate the expressionof genes involved in adaptive thermogenesis (41). The PGC-1 ${alpha}$ /PPAR ${alpha}$ pathway is involved in the regulation of mitochondrial fattyacid oxidation genes in the heart (28, 51). The PGC-1 ${alpha}$ /FOXO1pathway activates expression of gluconeogenic genes in the liver(39). More recently, the PGC-1 ${alpha}$ /ERR ${alpha}$ pathway has been shown toplay a role in regulating both fatty acid oxidation and mitochondrialgenes in heart and skeletal muscle (20, 35, 45, 53).

In contrast to the role of PGC-1 ${alpha}$ in the regulation of fattyacid oxidation, mitochondrial respiratory function, and hepaticgluconeogenesis, its function as a potential regulator of glucoseutilization pathways has not been well defined. Indeed, thefew reports relating PGC-1 ${alpha}$ to glucose uptake are conflicting.PGC-1 ${alpha}$ has been reported to induce (33) or repress (34) expressionof the glucose transporter GLUT4. Given that regulatory mechanismsexist for reciprocal control of fatty acid and glucose oxidation,it is likely that the PGC-1 ${alpha}$ regulatory circuit directly or indirectlyinfluences both pathways. Cellular glucose utilization is tightlyregulated at multiple levels, including uptake by the glucosetransporters (e.g., GLUT4), glycolytic flux, and entry of pyruvateinto the citric acid cycle via the pyruvate dehydrogenase complex(PDC). In muscle, the PDC serves a critical, rate-limiting stepin the regulation of the glucose oxidation pathway by catalyzingthe irreversible decarboxylation of pyruvate to acetyl coenzymeA. A significant body of evidence indicates that multiple regulatorypathways converge on the PDC, including allosteric regulationintermediates of fatty acid oxidation and posttranslationalcontrol by a family of pyruvate dehydrogenase kinases (PDK1to -4) and corresponding phosphatases (reviewed in references13 and 16). The reversible phosphorylation of the PDC inactivatesthis complex, sparing glucose and favoring fatty acid oxidation.PDK4 has proven to be particularly important in the muscle andliver response to fasting and exercise (15, 37, 54). PDK4 activityand expression are increased in diabetes concurrent with reducedcapacity for muscle and hepatic glucose oxidation. Interestingly,PGC-1 ${alpha}$ is also activated by fasting and exercise. In addition,recent studies have shown that activators of PPAR ${alpha}$ , a known PGC-1 ${alpha}$ target, induce the expression of PDK4 (17, 55). However, themechanism whereby PPAR ${alpha}$ regulates PDK4 gene expression has notbeen delineated. Recent studies have also shown that the forkheadtranscription factor FOXO1 and the glucocorticoid receptor (GR),two additional potential PGC-1 ${alpha}$ partners, directly regulate PDK4expression through consensus binding sites in the PDK4 genepromoter (9, 26).

The present study was designed to investigate the potentialregulatory influence of the inducible transcriptional coactivatorPGC-1 ${alpha}$ on glucose oxidation through effects on PDK4 gene expression.We found that PGC-1 ${alpha}$ directly activates PDK4 gene transcriptionin skeletal myotubes, leading to a reduction in glucose oxidationrates. Surprisingly, the PGC-1 ${alpha}$ -mediated activation of PDK4 transcriptionwas shown to be independent of PPAR ${alpha}$ and FOXO1; rather, it isdependent on the muscle-enriched orphan nuclear receptor ERR ${alpha}$ .Collectively, these results identify a new PGC-1 ${alpha}$ gene targetand suggest a mechanism whereby PGC-1 ${alpha}$ reciprocally downregulatesglucose oxidation while activating mitochondrial fatty acidoxidation in skeletal muscle. Given the importance of PDK4-mediatedregulation of muscle glucose utilization, these results provideadditional insight into the proposed link between PGC-1 ${alpha}$ andmetabolic disorders such as diabetes.

MATERIALS AND METHODS

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Materials and Methods

Animal studies. All animal experiments and euthanasia protocols were conductedin strict accordance with the National Institutes of Healthguidelines for humane treatment of animals and were reviewedand approved by the Animal Care Committee of Washington University.Unless stated otherwise, 6- to 8-week-old male C57BL/6J micewere used for all animal experiments. A modified treadmill protocolwas used for acute exercise experiments (29). In brief, animalswere acclimated to the treadmill for 10 min, and on each ofthe following 2 days, animals exercised for 1 h total, alternating2 min of running (20 m/min at an 18° incline) with 1 minof rest. Twelve hours following the final bout of exercise,animals were sacrificed, and gastrocnemius muscles were dissectedand snap-frozen in liquid nitrogen for RNA extraction. For timecourse studies, animals underwent a single bout of running following2 days of 10-min acclimation to the treadmill. Runs were staggeredso that all samples would be collected at the same time of day(0900 h). For cold exposure experiments, animals were maintainedat 4°C for 6 h as previously described (29); immediatelyafterwards, they were sacrificed and their tissues dissectedfor RNA extraction.

Plasmid constructs. (i) Mammalian expression vectors. pcDNA3.1-myc/ his.PGC-1 ${alpha}$ , pcDNA3.1-ERR ${alpha}$ , CDM-PPAR ${alpha}$ , and CDM-RXR ${alpha}$ have been described previously (18, 51). The expression vectorpcDNA3-Flag-FKHR (FOXO1) was provided by W. R. Sellers (Dana-FaberCancer Institute) and has been described previously (49).

(ii) Reporter constructs. The mouse PDK4 (mPDK4) gene promoter deletion series was generatedby PCR amplification from C57BL/6J genomic DNA followed by cloninginto the pGL3 Basic firefly luciferase reporter plasmid usingMluI and BglII sites (Promega, Madison, WI). The following 5'primers were used: 5'-ACGCGTTCTAGATAAGAATATCATTCTTTGT-3' (mPDK4.Luc.2281),5'-ACGCGTACATGATCATTTCCTACATGCTTG-3' (mPDK4.Luc.2121), 5'-ACGCGTACACAAGTGGACACAACATTAGGC-3'(mPDK4.Luc.1377), 5'-ACGCGTACTGAGCCTCTGGGAAATGAAAGA-3' (mPDK4.Luc.874),5'-ACGCGTGGCTAGGAATGCGTGACATTGAGAT-3' (mPDK4.Luc.371), 5'-ACGCGTCTGAAGTCCTAGCGACCTGGGATCT-3'(mPDK4.Luc.308), and 5'-ACGCGTAGGCCATGGAAACCGTGTCGGGATCACTGT-3'(mPDK4.Luc.190). The same 3' primer was used with all constructs:5'-AGATCTGAGCCTGGGTGAAGGGTTGACACTT-3'. Site-directed mutagenesiswas performed using the QuikChange kit (Stratagene, La Jolla,CA) according to the manufacturer's protocol using complementaryoligonucleotides as follows (with mutated nucleotides shownin lowercase): 5'-GATGGCTCTGGAGTTGTAggggAGGACAAGTCTGGG-3' (IRSmut),5'-CTGGAGTTGTAAACAgatctAAGTCTGGGCGG-3' (NRmut), 5'-GGAGTTGTAAACAAGGACAAGTCTaaGCttGCCTGAAGTCCTAGCG-3'(Sp1mut), and 5'-CTGGAGTTGTAAACAAcGACAAGTCTGGGCGG-3' (NRptmut).Primers were designed, and clones were analyzed, using VectorNTI Suite 9 (InforMax, Rockville, MD) and the reported mPDK4gene sequence (GenBank accession number NT_039340). All cloneswere verified by ABI automated sequencing through the Proteinand Nucleic Acid Chemistry Laboratory (PNACL) at WashingtonUniversity. Numbering was determined by estimating the transcriptionstart site (+1) by homology to the human PDK4 (hPDK4) gene (43)(GenBank accession number NM_002612). Candidate transcriptionfactor response elements were identified using the Match program(http://www.gene-regulation.com).

(iii) Adenovirus expression vectors. The adenovirus (Ad)-green fluorescent protein (GFP), Ad-PGC-1 ${alpha}$ ,Ad-ERR ${alpha}$ , and Ad-PPAR ${alpha}$ expression vectors have been described previously(18, 19, 28).

Cell culture, transient transfection, and luciferase reporter studies. C₂C₁₂ myoblasts and CV-1 cells were maintained at 37°C under5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplementedwith 10% fetal calf serum. For experiments requiring myotubeformation, the medium of C₂C₁₂ myoblasts at >80% confluencewas changed to DMEM supplemented with 2% horse serum, and thecells were maintained for an additional 72 h. CV-1 cells andC₂C₁₂ myotubes were infected at a multiplicity of infectionsufficient to infect >95% of the cells based on GFP fluorescencewith minimal cell death. ERR ${alpha}$ ^–/– mice for primarycell culture isolation have been described elsewhere (32). Primarymouse fibroblasts from tail tissue of ERR ${alpha}$ ^+/+ and ERR ${alpha}$ ^–/–mice were isolated and cultured as described elsewhere (20).Transient transfections were performed by the calcium phosphatecoprecipitation method (4, 11). Briefly, reporter plasmids (4µg/ml) were cotransfected with simian virus 40 promoter-drivenRenilla luciferase (500 ng/ml), to control for transfectionefficiency, and the appropriate mammalian expression vector(500 ng/ml) or its corresponding empty vector. For luciferasereporter assays, CV-1 cells and C₂C₁₂ myotubes were harvestedand analyzed at 48 and 72 h, respectively, using Dual-Glo (Promega)and were read in a Chameleon plate reader (Hidex, Washington,D.C.) according to the manufacturer's protocols. All transfectiondata are presented as means ± standard errors (SE) forat least three separate transfection experiments done in triplicate.

RNA and protein analyses. Total cellular RNA was isolated from cells in culture (48 hfollowing adenovirus infection) or muscle tissue using the RNAzolmethod (Tel-Test, Friendswood, TX). Northern blot analysis wasperformed with QuikHyb (Stratagene) using random-primed ³²P-labeledcDNA clones for human ß-actin, mouse PGC-1 ${alpha}$ , and PDK4.Band intensities were quantified by phosphorimaging using aGS 525 Molecular Imager system (Bio-Rad) and normalized to theexpression of ß-actin. Real-time quantitative reversetranscription-PCR (RT-PCR) was performed as previously described(20) and results normalized to the expression of 36B4. The followingmouse-specific primer pairs were used: PGC-1 ${alpha}$ forward, 5'-CGGAAATCATATCCAACCAG-3';PGC-1 ${alpha}$ reverse, 5'-TGAGGACCGCTAGCAAGTTTG-3'; PDK4 forward, 5'-CCGCTGTCCATGAAGCA-3';PDK4 reverse, 5'-GCAGAAAAGCAAAGGACGTT-3'; 36B4 forward, 5'-GCAGACAACGTGGGCTCCAAGCAGAT-3';36B4 reverse, 5'-GGTCCTCCTTGGTGAACACGAAGCCC-3'.

Protein extracts were resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) (4 to 15% Criterion Precast;Bio-Rad) and transferred to nitrocellulose membranes (MidwestScientific, Valley Park, MO). Western blotting was performedusing antibodies against PGC-1 ${alpha}$ (28), medium-chain acyl coenzymeA dehydrogenase (MCAD) (24), PDK4 (a gift from R. A. Harris,Indiana University School of Medicine), ${alpha}$ -tubulin (MolecularProbes, Eugene, OR), PPAR ${alpha}$ , FKHR/FOXO1, and Sp1 (Santa Cruz Biotechnology,Santa Cruz, CA). An antibody against ERR ${alpha}$ was raised in rabbitsagainst an N-terminal peptide corresponding to amino acids 16to 33 of the mouse ERR ${alpha}$ protein conjugated to keyhole limpethemocyanin (KLH) or bovine serum albumin (BSA). This domainis conserved among species; therefore, the antibody recognizesrodent and human ERR ${alpha}$ isoforms. The KLH-conjugated peptide inFreund's adjuvant was used for the initial and second boosterimmunizations. Subsequent boosts were performed with the BSA-conjugatedpeptide. Peptide synthesis, conjugation, and purification byhigh-performance liquid chromatography were performed throughPNACL (Washington University). Peptide immunization and serumcollection were performed through Biosource (Hopkinton, MA).The specificity of the anti-ERR ${alpha}$ antibody was determined by theability of this antibody to detect recombinant ERR ${alpha}$ but not ERR ${gamma}$ and by comparison to the previously described anti-ERR ${alpha}$ antibody(47). Detection was performed by measuring the chemiluminescentsignal as assayed by SuperSignal Ultra (Pierce, Rockford, IL).

EMSA. Electrophoretic mobility shift assays (EMSA) were performedas described previously (6). Double-stranded complementary oligonucleotidescorresponding to the PGC-1 ${alpha}$ -responsive element (5'-gatccGGCTAGGAATGCGTGACATTGAGATGGCTCTGGAGTTGTAAACAAGGACAAGTCTGGGCGGGCg-3') or NRmut (5'-gatccGGCTAGGAATGCGTGACATTGAGATGGCTCTGGAGTTGTAAACAGATCTAAGTCTGGGCGGGCg-3')(overhangs are shown in lowercase; mutation is underlined) ofthe mPDK4 promoter were used to generate probes to assay recombinantand endogenous protein binding in vitro. Probes were radiolabeledby a Klenow fill-in reaction. Recombinant proteins for ERR ${alpha}$ ,PPAR ${alpha}$ , and FOXO1 were synthesized with the TNT Quick T7-coupledtranslation kit (Promega). Synthesis of appropriately sizedproteins was verified in reactions incorporating [³⁵S]methioninefollowed by SDS-PAGE analysis and autoradiographic detection.Nuclear extract was isolated as previously described (6). Briefly,C₂C₁₂ myotubes were infected with Ad-GFP or Ad-PGC-1 ${alpha}$ , nuclearextract was isolated 48 h following infection, and samples werealiquoted and stored at –80°C until use.

ChIP assays. Chromatin immunoprecipitation (ChIP) assays were performed aspreviously published (21, 52). In brief, C₂C₁₂ myotubes wereinfected with Ad-GFP or Ad-PGC-1 ${alpha}$ . Cross-linking was performed36 to 48 h postinfection byincubating cells with 0.4% formaldehyde(10 min) followed by addition of 0.125M glycine to halt cross-linking.Following cell and nuclear lysis, DNA was sheared by sonication;enrichment for 500-bp fragments was determined by agarose gelelectrophoresis. Sheared chromatin complexes were preclearedwith preimmune rabbit serum, followed by dilution of an aliquotin immunoprecipitation dilution buffer and incubation overnightat 4°C with immunoglobulin G (IgG) or a polyclonal antibodyagainst PGC-1 ${alpha}$ or ERR ${alpha}$ (described above). Antibody-chromatin complexeswere bound with ImmunoPure immobilized protein A (Pierce), followedby isolation and purification as described previously (20).Primers were designed to amplify a 164-bp amplicon correspondingto the region of the mPDK4 promoter flanking the nuclear receptor(NR) response element (nucleotides –449 to –285).The following mouse-specific primer pairs were used: PDK4proforward (5'-TGATTGGCTACTGTAAAAGTCCCGC-3') and PDK4pro reverse(5'-ATCCCAGGTCGCTAGGACTTCAGG-3'). Primers amplifying the 36B4gene (see above) were used to control for nonspecific enrichmentof DNA in the immunoprecipitation. Samples were analyzed by2% agarose gel electrophoresis and quantified relative to inputusing SYBR green RT-PCR (Applied Biosystems, Foster City, CA).

Glucose oxidation assay. Cellular glucose oxidation rates were determined using modificationsof previously published protocols (5, 22). C₂C₁₂ myoblasts weregrown in T25 flasks and differentiated into myotubes as describedabove. Forty-eight hours following infection with Ad-GFP orAd-PGC-1 ${alpha}$ , the medium was replaced with MEM supplemented with0.5% horse serum. Following 18 h of reduced serum incubation,new medium was added containing 0.2 µCi/ml [U-¹⁴C]glucose(10 mCi/mmol), 100 nM insulin, and 250 µM oleate. Theflasks were immediately capped with rubber stoppers fitted withplastic wells containing a piece of filter paper saturated with1 M NaOH. The culture flasks were incubated for 30 min at 37°C.Reactions were terminated with perchloric acid, followed byovernight storage at 4°C to trap CO₂. The release of ¹⁴CO₂from glucose was measured by scintillation counting of the filterpaper. Duplicate flasks were plated for each condition. Theresults were normalized to total protein (determined by theBradford method).

RESULTS

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Results

PGC-1 ${alpha}$ induces expression of the PDK4 gene in skeletal muscle. Physiological conditions that increase skeletal muscle energydemands induce the expression of the transcriptional coactivatorPGC-1 ${alpha}$ . As an initial step to investigate a potential role forPGC-1 ${alpha}$ in the regulation of muscle glucose metabolism via PDK4,we examined levels of mRNAs encoding PGC-1 ${alpha}$ and PDK4 followingacute exercise. For these experiments, a running protocol ona motorized treadmill was employed (29). PGC-1 ${alpha}$ mRNA levels weremarkedly induced in mouse gastrocnemius 12 h after two boutsof treadmill running (Fig. 1A). The expression of PDK4 was robustlyinduced in parallel with PGC-1 ${alpha}$ expression (Fig. 1A). Similarexperiments were conducted following cold exposure, which triggersshivering thermogenesis in skeletal muscle and has been shownto induce PGC-1 ${alpha}$ expression (41). The expression of PDK4 andPGC-1 ${alpha}$ genes was coordinately induced (two- to fourfold) in musclefollowing 6 h of cold exposure (Fig. 1B). To assess the temporalpatterns of the physiological induction of PGC-1 ${alpha}$ and PDK4 geneexpression in muscle, a time course experiment was performedfollowing a single bout of exercise. As shown in Fig. 1C, PGC-1 ${alpha}$ gene expression was markedly induced (at 1 h postexercise) priorto the activation of PDK4 gene expression.

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FIG. 1. Activation of PDK4 gene expression by physiological stimuli and by overexpression of PGC-1 ${alpha}$ . (A) Northern blot studies performed with 15 µg of total RNA isolated from the gastrocnemii of animals following two bouts of exercise as described in Materials and Methods (Ex) or from sedentary controls (C) (n = 4). Blots were sequentially hybridized with probes specific for the genes encoding PGC-1 ${alpha}$ and PDK4. Representative autoradiographs are shown at the top. Phosphorimage-based quantification of Northern blot signal intensities is shown in the graph. Data represent mean arbitrary units (±SE) normalized to sedentary control values (taken as 1.0). (B) Results of Northern blot analysis performed as described above on gastrocnemius samples immediately following 6 h of cold exposure (Cold) and from room temperature controls (C) (n = 6). Graph represents mean arbitrary units (±SE) as determined by real-time PCR quantification corrected to the 36B4 transcript and normalized to the values for control littermates (taken as 1.0). (C) RNA analysis of PGC-1 ${alpha}$ and PDK4 gastrocnemius gene expression 1 h and 3 h after a single bout of exercise (n = 4). The graph represents mean arbitrary units (±SE) as determined by RT-PCR corrected to the 36B4 transcript and normalized to the value for sedentary controls (taken as 1.0). (D) RNA analyses and Western blot analysis were performed with samples isolated from Ad-GFP- or Ad-PGC-1 ${alpha}$ -infected C₂C₁₂ myotubes. (Top) Results of Northern blotting performed as described above. Data represent mean arbitrary units (±SE) as determined by RT-PCR corrected to the 36B4 transcript and normalized to value for control Ad-GFP-infected samples (taken as 1.0). (Bottom) Western blot analysis of whole-cell extracts (25 µg) prepared from C₂C₁₂ myotubes infected with adenovirus vectors expressing GFP or PGC-1 ${alpha}$ . Asterisks indicate significant differences (P < 0.05) from controls.

To determine if PGC-1 ${alpha}$ plays a direct role in the regulationof PDK4 gene expression, we examined the effect of adenovirus-mediatedoverexpression of PGC-1 ${alpha}$ (Ad-PGC-1 ${alpha}$ ) compared to a control vector(Ad-GFP) in C₂C₁₂ myotubes. As expected, we observed an increasein levels of MCAD protein, a known PGC-1 ${alpha}$ target (51), followingAd-PGC-1 ${alpha}$ infection (Fig. 1D). Overexpression of PGC-1 ${alpha}$ markedlyactivated PDK4 mRNA and protein levels (Fig. 1D). Taken together,these results indicate that PGC-1 ${alpha}$ functions as an upstream activatorof PDK4 gene expression.

PGC-1 ${alpha}$ activates PDK4 gene transcription through a nuclear receptor binding site. To determine whether PGC-1 ${alpha}$ induces PDK4 gene expression at thetranscriptional level, we performed promoter-reporter transfectionsin C₂C₁₂ myotubes in culture. For these experiments, approximately2.3 kb of the mPDK4 gene promoter region was cloned into a promoterlessluciferase reporter vector (mPDK4.Luc.2281). Cotransfectionof mPDK4.Luc.2281 with an expression vector for PGC-1 ${alpha}$ resultedin robust (14-fold) activation of the reporter (Fig. 2A). Tomap the cis-acting region conferring the PGC-1 ${alpha}$ activation, cotransfectionexperiments were repeated with reporter constructs containingserial 5' deletions of the mPDK4 promoter (Fig. 2B). The PGC-1 ${alpha}$ -mediatedactivation was lost upon deletion of a region of the mPDK4 promoterfrom –371 to –308 bp upstream of the transcriptionstart site. In addition, the basal promoter activity increasedupon deletion of the region between –874 and –371bp.

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FIG. 2. PGC-1 ${alpha}$ activates the mPDK4 gene promoter through a cis-acting element within the proximal region. (A) A schematic of the reporter construct mPDK4.Luc.2281, containing the –2281-to-+31 region of the mouse PDK4 gene promoter, is shown at the top. The graph contains results of cotransfection studies with mPDK4.Luc.2281 in the presence (PGC-1 ${alpha}$ ) or absence [pcDNA(–)] of pcDNA-PGC-1 ${alpha}$ in C₂C₁₂ myotubes. The graph represents mean (±SE) relative light units (RLU) corrected for Renilla luciferase activity and normalized to the activity of mPDK4.Luc.2281 (taken as 1.0). (B) Transient transfections performed with a 5' deletion series of mPDK4.Luc.2281. All values represent at least three independent transfections conducted in triplicate. Asterisks indicate significant differences compared to individual reporters cotransfected with empty vector (P < 0.05). Daggers indicate significant differences in activity compared to mPDK4.Luc.2281 cotransfected with empty vector (P < 0.05).

Inspection of the nucleotide sequence of the PGC-1 ${alpha}$ -responsiveregion revealed a high degree of nucleotide identity acrossspecies (Fig. 3A). A screen of the 63-bp DNA sequence identifiedseveral putative transcription factor binding sites that couldsupport PGC-1 ${alpha}$ coactivation, including an NR half-site, an Sp1site, and a recently identified FOXO1 binding site (DBE [9],or IRS3 [26]) (Fig. 3A). Interestingly, coactivation of FOXO1by PGC-1 ${alpha}$ has been implicated in the regulation of hepatic gluconeogenicgene expression (39). Therefore, we first sought to determinewhether the FOXO1 response element was necessary for the PGC-1 ${alpha}$ -mediatedactivation. The FOXO1 binding site within the mPDK4.Luc.2281reporter was abolished using a mutation that has been shownpreviously to inactivate this element (9). Although the FOXO1site mutant was incapable of responding to FOXO1, PGC-1 ${alpha}$ -mediatedactivation of the mPDK4 promoter was unchanged (Fig. 3B). Itis noteworthy that the FOXO1 site mutant resulted in a 50% reductionin basal promoter activity, indicating a PGC-1 ${alpha}$ -independent rolefor FOXO1.

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FIG. 3. PGC-1 ${alpha}$ activates PDK4 gene transcription through a nuclear receptor binding site. (A) Species comparison of the nucleotide sequence (sense strand is shown) of the PGC-1 ${alpha}$ -activated region of the PDK4 promoter, demonstrating a high level of sequence identity between mouse, rat, and human genes. The numbers are relative to the transcription start site (+1). Shaded nucleotides indicate sequence that is not conserved. Computer analysis revealed numerous candidate response elements, including a nuclear receptor half-site (NR1/2), an Sp1 site, and the previously identified FOXO1 binding site (IRS) (9, 27), indicated by labels, boxes, and symbols. (B) (Top) Site-directed mutagenesis was used to abolish the FOXO1 response element. Mutated nucleotides are lowercased. (Bottom) The mPDK4.Luc.2281 (wild type [WT]) or IRSmut.mPDK4.Luc.2281 promoter reporter was used in cotransfections in C₂C₁₂ myotubes in the presence or absence of FOXO1 (left graph) or PGC-1 ${alpha}$ (right graph). (C) (Left) Schematic showing the mutations generated by site-directed mutagenesis of the minimal PGC-1 ${alpha}$ -responsive mPDK4.Luc.371 reporter in FOXO1 (IRSmut), NR (NRmut), or Sp1 (Sp1mut) candidate response elements (mutated nucleotides are lowercased). (Right) Cotransfection studies using WT or mutant mPDK4 promoter reporters were performed with C₂C₁₂ myotubes. Data represent mean (±SE) relative light units (RLU) corrected for rLuc and normalized to values for the WT reporter (taken as 1.0). All transient transfection studies represent at least three independent transfections conducted in triplicate. Asterisks indicate significant differences from individual reporters cotransfected with empty vector (P < 0.05). Daggers indicate significant differences from the wild-type reporter cotransfected with empty vector (P < 0.05).

Mutational studies were performed to examine the importanceof the putative nuclear receptor and Sp1 binding sites for PGC-1 ${alpha}$ coactivation. The nuclear receptor site was of particular interestgiven that several studies have shown that PPAR ${alpha}$ and its ligandsregulate PDK4 gene expression (7, 17, 55). Although mutationof the Sp1 response element significantly reduced the basalpromoter activity of the mPDK4.Luc.371 reporter, PGC-1 ${alpha}$ -mediatedactivation was maintained (Fig. 3C). In contrast, mutation ofthe putative NR response element abolished activation by PGC-1 ${alpha}$ .Together, these results demonstrate that the candidate NR responseelement is required for the observed PGC-1 ${alpha}$ -mediated activationof the PDK4 gene promoter.

The orphan nuclear receptor ERR ${alpha}$ , but not PPAR ${alpha}$ , directly confers PGC-1 ${alpha}$ -mediated coactivation of the mPDK4 promoter. To determine if PPAR ${alpha}$ was capable of binding to the PGC-1 ${alpha}$ -responsiveNR site identified within the mPDK4 gene promoter, EMSA wereperformed using recombinant PPAR ${alpha}$ and RXR ${alpha}$ , its obligate DNA bindingpartner. Surprisingly, PPAR ${alpha}$ /RXR ${alpha}$ did not bind to this regionbut did bind to a known PPAR response element from the musclecarnitine palmitoyltransferase I gene promoter (3) (data notshown).

The orphan nuclear receptor ERR ${alpha}$ is a recently identified mediatorof PGC-1 ${alpha}$ action (18, 46). ERR ${alpha}$ was an attractive candidate forbinding the nuclear receptor half-site within the PGC-1 ${alpha}$ -responsiveregion of the mPDK4 promoter, because it is capable of bindingas a monomer (23) and is enriched in slow-twitch muscle (10,20, 47). Therefore, EMSA were repeated using recombinant ERR ${alpha}$ and the 63-bp region as a probe. As shown in Fig. 4A, ERR ${alpha}$ boundthe 63-bp PGC-1 ${alpha}$ -responsive region of the mPDK4 promoter (lane3). Competition studies with unlabeled wild-type and mutatedfragments (Fig. 4A, lanes 6 to 10), as well as ERR ${alpha}$ antibodyrecognition experiments (lanes 3 to 5), confirmed the specificityof the ERR ${alpha}$ binding to the NR site. In addition, relative tothe wild-type probe, a ³²P-labeled mutant probe failed to supportERR ${alpha}$ binding (Fig. 4A, lanes 11 to 13).

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FIG. 4. The orphan nuclear receptor ERR ${alpha}$ binds to the PGC-1 ${alpha}$ -responsive region of the mPDK4 promoter. (A) Representative autoradiograph of EMSA conducted with ³²P-labeled probes of the 63-bp region of the mPDK4 promoter identified in Fig. 2B. Mutant probe corresponds to the same nucleotide substitutions for NRmut as in Fig. 3C. Probes were incubated with recombinant in vitro-translated ERR ${alpha}$ (rERR ${alpha}$ ) or unprogrammed reticulocyte lysate (Unprog.) as indicated at the top. Antibody (Ab) supershift studies were performed by incubation with an anti-ERR ${alpha}$ (lane 5) or nonspecific (n.s.) (lane 4) Ab. Competition analysis was performed by adding a molar excess of the unlabeled wild-type (lanes 7 and 8) or NRmut (lanes 9 and 10) probe in increasing amounts as indicated. For lanes 11 to 13, the ³²P-labeled NRmut probe was used. n.s. bands represent protein complexes identified in unprogrammed reticulocyte lysate. (B) Autoradiograph of an EMSA using nuclear (Nuc.) extracts isolated from C₂C₁₂ myotubes incubated with the ³²P-labeled wild-type probe. Four prominent complexes, labeled Ia, Ib, II, and III, were observed. Complexes were identified by antibody supershift analysis using antibodies specific for the transcription factors indicated at the top.

To determine whether endogenous ERR ${alpha}$ bound to the mPDK4 genepromoter, EMSA were repeated with nuclear extracts preparedfrom C₂C₁₂ myotubes. Three complexes formed with the 63-bp mPDK4probe (Fig. 4B, complexes I to III). Complex I, the slowest-migratingband, exhibited a doublet (Ia and Ib). To identify proteinswithin the complexes, antibody recognition experiments wereperformed using antibodies to ERR ${alpha}$ , Sp1, FOXO1, and PPAR ${alpha}$ . ComplexIb was abolished by anti-Sp1 (Fig. 4B, lane 5). Complex II wassupershifted with anti-ERR ${alpha}$ (Fig. 4B, lane 4). Additionally,the anti-ERR ${alpha}$ antibody disrupted complexes Ia and Ib, suggestinga potential requirement for ERR ${alpha}$ binding in the formation ofthese two complexes (Fig. 4B, lane 4). Alternatively, the latterresult could be due to the "mass action" of the supershiftedcomplex II. Surprisingly, FOXO1 was not identified within anyof the complexes, though the antibody was shown to supershiftrecombinant FOXO1 (Fig. 4B and data not shown). As expected,none of the complexes were recognized by anti-PPAR ${alpha}$ . The identitiesof complexes Ia and III are under investigation. Taken together,the EMSA studies demonstrate that the PGC-1 ${alpha}$ -mediated regionof the mPDK4 gene promoter is capable of binding both exogenousand endogenous ERR ${alpha}$ .

Cotransfection studies were performed to examine the functionalcorrelate of the binding studies. For these studies, CV-1 cellswere used because they are relatively deficient in ERR ${alpha}$ and manyother nuclear receptors, presenting the opportunity to reconstitutethe system with factors required for maximal PGC-1 ${alpha}$ activation.Cotransfection of a PGC-1 ${alpha}$ expression vector resulted in fourfoldactivation of mPDK4.Luc.2281, while ERR ${alpha}$ alone resulted in amodest twofold activation (Fig. 5). However, cotransfectionof both PGC-1 ${alpha}$ and ERR ${alpha}$ increased reporter activity nearly 10-fold,comparable to that observed in C₂C₁₂ myotubes (compare Fig.5 and 2A). The PGC-1 ${alpha}$ /ERR ${alpha}$ response was dependent on the NR responseelement, because the mutant construct did not exhibit activationby either factor alone, showing only a very modest increasewith the combination (Fig. 5). These data indicate that ERR ${alpha}$ is sufficient to confer the PGC-1 ${alpha}$ -mediated transcriptional activation.

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FIG. 5. ERR ${alpha}$ and PGC-1 ${alpha}$ regulate the mPDK4 promoter through the NR response element. (Top) Schematic showing the mutation (lowercase) of the NR response element in mPDK4.Luc.2281 (NRptmut.mPDK4.Luc.2281). (Bottom) Graph depicting the results of transient cotransfections in CV-1 cells performed with either the mPDK4.Luc.2281 reporter (wild type [WT]) or NRptmut.mPDK4.Luc.2281 (NRptmut). Asterisks indicate significant differences from WT mPDK4.Luc.2281 cotransfected with empty vector (P < 0.05). Dagger indicates a significant difference from mPDK4.Luc.2281 with ERR ${alpha}$ (P < 0.05). Double dagger indicates a significant difference from the value obtained using NRptmut.mPDK4.Luc.2281 with empty vector (P < 0.05).

PGC-1 ${alpha}$ increases ERR ${alpha}$ expression and PDK4 promoter binding in skeletal myotubes. PGC-1 ${alpha}$ has been shown to directly coactivate ERR ${alpha}$ (18). In addition,in certain cell contexts, PGC-1 ${alpha}$ activates ERR ${alpha}$ gene expression(27, 46). To determine whether the activation of PDK4 expressionby PGC-1 ${alpha}$ involved simultaneous activation of ERR ${alpha}$ expression,endogenous ERR ${alpha}$ protein expression levels were assessed followingadenovirus-mediated overexpression of PGC-1 ${alpha}$ in C₂C₁₂ myotubes.ERR ${alpha}$ protein levels were markedly increased in Ad-PGC-1 ${alpha}$ -infectedC₂C₁₂ myotubes, while the expression of PPAR ${alpha}$ , FOXO1, and Sp1was unchanged (Fig. 6A). Consistent with upregulation of ERR ${alpha}$ expression, EMSA performed with nuclear extracts prepared fromC₂C₁₂ myotubes following Ad-PGC-1 ${alpha}$ infection revealed that theintensity of complex II on the PGC-1 ${alpha}$ -responsive region of thePDK4 promoter was markedly increased, accompanied by a modestincrease in complex III (Fig. 6B, lanes 2 and 3). Addition ofan ERR ${alpha}$ -specific antibody again confirmed the identity of complexII (Fig. 6B, lane 5). As a separate control, EMSA using extractsfrom Ad-PPAR ${alpha}$ -infected cells did not show any changes in complexintensity compared to the control (data not shown).

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FIG. 6. PGC-1 ${alpha}$ induces expression of the endogenous ERR ${alpha}$ gene in skeletal muscle cells.(A) Representative autoradiograph of Western blot analysis performed on nuclear extracts isolated from C₂C₁₂ myotubes following infection with Ad-GFP or Ad-PGC-1 ${alpha}$ as indicated at the top. The antibodies used are given on the left. Arrows on the right indicate specific (PGC-1 ${alpha}$ and ERR ${alpha}$ ) bands. (B) EMSA study performed with nuclear extracts isolated from C₂C₁₂ myotubes following infection with Ad-GFP (lane 2) or Ad-PGC-1 ${alpha}$ (lanes 3 to 8) and the wild-type probe described in the legend to Fig. 4. Complexes II and Ib were identified by antibody (Ab) supershift analysis using Abs specific for ERR ${alpha}$ and Sp1, respectively (lanes 5 and 6). n.s., nonspecific. (C) Chromatin immunoprecipitation assays performed on C₂C₁₂ myotubes following infection by Ad-PGC-1 ${alpha}$ . Formaldehyde cross-linked protein-DNA complexes were isolated. Sheared protein-DNA complexes were immunoprecipitated with a nonspecific antibody (IgG), anti-ERR ${alpha}$ , or anti-PGC-1 ${alpha}$ . Isolated fragments were amplified by PCR to detect the enrichment of amplicons corresponding to a 190-bp region of the 36B4 gene (negative control) or a 164-bp region encompassing the NR response element of the mPDK4 gene promoter. (Top) Results of agarose gel analysis of a representative trial showing relative band intensities. Input represents 2% of the total chromatin used in the immunoprecipitation reactions. (Bottom) Graphs contain mean values as determined by SYBR green quantification of three independent chromatin isolations and immunoprecipitations (arbitrary units ± SE) normalized to the value for the IgG control (taken as 1.0). Asterisks indicate significant differences from the IgG control (P < 0.05).

The transfection and gel shift studies strongly suggested thata cooperative interaction between ERR ${alpha}$ and PGC-1 ${alpha}$ at the NR half-siteleads to activation of PDK4 gene transcription. To determinewhether these factors simultaneously occupy the endogenous promotersite in cells, ChIP assays were performed on chromatin isolatedfrom Ad-PGC-1 ${alpha}$ -infected C₂C₁₂ myotubes. Neither the anti-ERR ${alpha}$ nor the anti-PGC-1 ${alpha}$ antibody enriched for the promoter regionover a nonspecific IgG control following infection with theAd-GFP backbone (data not shown). However, following Ad-PGC-1 ${alpha}$ infection, antibodies specific to ERR ${alpha}$ or PGC-1 ${alpha}$ significantlyenriched for the endogenous PDK4 promoter region, four- to fivefoldover the IgG control (Fig. 6C). These results confirm that bothPGC-1 ${alpha}$ and ERR ${alpha}$ occupy the PGC-1 ${alpha}$ -responsive region of the mPDK4promoter in skeletal myotubes and that PGC-1 ${alpha}$ enriches the levelof binding, likely via increasing ERR ${alpha}$ gene expression.

ERR ${alpha}$ is required for PGC-1 ${alpha}$ -mediated activation of the PDK4 gene promoter. The data shown above support the conclusion that both ERR ${alpha}$ andPGC-1 ${alpha}$ are capable of activating PDK4 gene transcription. Todetermine if ERR ${alpha}$ is actually required for PGC-1 ${alpha}$ -mediated activation,we assessed the ability of PGC-1 ${alpha}$ to activate the PDK4 promoterin ERR ${alpha}$ -null cells. For these experiments, fibroblasts were isolatedfrom ERR ${alpha}$ -null (ERR ${alpha}$ ^–/–) (32) and wild-type (ERR ${alpha}$ ^+/+)mice. Cotransfection of a PGC-1 ${alpha}$ or ERR ${alpha}$ expression vector withthe mPDK.Luc.2281 reporter in wild-type cultured primary fibroblastsled to 3.5-fold and 2.5-fold increases in reporter activity,respectively; while addition of both PGC-1 ${alpha}$ and ERR ${alpha}$ increasedreporter activity 6-fold (Fig. 7). In ERR ${alpha}$ ^–/– fibroblasts,cotransfection of either a PGC-1 ${alpha}$ or an ERR ${alpha}$ expression vectoralone failed to induce mPDK4.Luc.2281 reporter activity (Fig.7). However, cotransfection of both ERR ${alpha}$ and PGC-1 ${alpha}$ rescued PGC-1 ${alpha}$ -mediatedactivation in ERR ${alpha}$ ^–/–fibroblasts, resulting in anearly fivefold induction (Fig. 7). These results demonstratethat ERR ${alpha}$ is required for PGC-1 ${alpha}$ -mediated activation of the mPDK4gene promoter.

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FIG. 7. ERR ${alpha}$ is required for PGC-1 ${alpha}$ -mediated activation of PDK4 gene transcription. Primary fibroblasts isolated from wild-type or ERR^–/– mice were cotransfected with mPDK4.Luc.2281 and an expression vector for PGC-1 ${alpha}$ or ERR ${alpha}$ , or both. Asterisks indicate significant differences from wild-type mPDK4.Luc.2281 cotransfected with empty vector (P < 0.05). Dagger indicates a significant difference from mPDK4.Luc.2281 cotransfected with ERR ${alpha}$ (P < 0.05).

PGC-1 ${alpha}$ decreases glucose oxidation in C₂C₁₂ myotubes. To determine the functional relevance of PGC-1 ${alpha}$ -mediated activationof PDK4 expression, cellular glucose oxidation rates were determinedin the presence and absence of overexpressed PGC-1 ${alpha}$ . For theseexperiments, glucose oxidation was measured as the release of¹⁴CO₂ from [U-¹⁴C]glucose in C₂C₁₂ myotubes. Mean glucose oxidationrates were markedly reduced (by approximately 75%) in C₂C₁₂myotubes following infection with Ad-PGC-1 ${alpha}$ compared to Ad-GFP-infectedcontrols (Fig. 8).

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FIG. 8. PGC-1 ${alpha}$ decreases glucose oxidation in C₂C₁₂ myotubes. The graph represents mean (±SE) [U-¹⁴C]glucose oxidation rates in C₂C₁₂ myotubes infected with Ad-PGC-1 ${alpha}$ or Ad-GFP (control). Results are based on three independent experiments performed in triplicate. Asterisk indicates a significant difference from the GFP-infected control (P < 0.001).

DISCUSSION

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Discussion

Skeletal muscle is capable of rapid adjustments in ATP productionand fuel preference in accordance with diverse physiologicalconditions. In order to match energy requirements with activitydemands, muscle substrate utilization pathways must be tightlycontrolled. During intense bouts of exercise, the majority ofthe muscles' energy demands are met by glucose. Following exercise,muscle glucose is spared through a shift toward mitochondrialfatty acid oxidation so that glycogen levels may be quicklyreplenished. The molecular basis for the metabolic plasticityof muscle involves rapid changes in the activity of enzymesvia allosteric control and posttranslational modifications aswell as chronic changes through the regulation of numerous genescontrolling mitochondrial content and substrate preference.Delineation of the mechanisms involved in the gene expressionchanges is critical for the understanding of the molecular pathogenesisof disease states where substrate utilization is chronicallyderanged, such as heart failure, obesity, and diabetes. In thisreport, we identify PDK4, an important negative regulator ofmuscle glucose oxidation, as a new target of PGC-1 ${alpha}$ -mediatedregulation.

The inducible transcriptional coactivator PGC-1 ${alpha}$ has been identifiedas a key regulator of multiple ATP-generating pathways in striatedmuscle through its regulatory influence on genes involved inmitochondrial biogenesis, respiration, oxidative phosphorylation,and fatty acid oxidation (reviewed in references 25 and 40).However, little is known about the potential role of PGC-1 ${alpha}$ inthe control of muscle glucose utilization pathways. The pioneeringstudies of Randle et al. demonstrated that glucose oxidationis decreased coincident with increased fatty acid oxidationin heart muscle (42). This inhibition of glucose oxidation occursat the level of the pyruvate dehydrogenase complex, among otherenzymes, via allosteric effects of the products of fatty acidoxidation and through changes in the cellular redox state. Ourresults indicate that PGC-1 ${alpha}$ may also contribute to this counter-regulatoryeffect at the level of gene expression in skeletal muscle throughmodulation of PDK4, a negative regulator of the PDC complex.Recent reports have shown a role for PGC-1 ${alpha}$ in glucose replenishmentin the liver through the regulation of gluconeogenic enzymegenes (14, 39, 57). The regulatory mechanism described by thecurrent study, in which PGC-1 ${alpha}$ increases muscle PDK4 expression,may serve to replenish glycogen levels in muscle through a differentmechanism, by reducing glucose oxidation. Interestingly, PGC-1 ${alpha}$ has also been shown to activate the expression of the insulin-sensitiveglucose transporter GLUT4 in muscle (33), which would also serveto increase glycogen stores in the context of increased PDK4levels.

Our results identify the orphan nuclear receptor ERR ${alpha}$ as themediator of PGC-1 ${alpha}$ -induced activation of mPDK4 gene expression.Recently, ERR ${alpha}$ was identified as a critical transcriptional regulatorof cellular energy metabolism. The activity of ERR ${alpha}$ appears tobe highly dependent on PGC-1 ${alpha}$ (18,45). PGC-1 ${alpha}$ -mediated coactivationof some ERR ${alpha}$ targets, such as MCAD (47), ATP synthase ß,and cytochrome c (45), is conferred directly by ERR ${alpha}$ bindingto elements within the promoter regions of these genes. In addition,we recently demonstrated that ERR ${alpha}$ is capable of regulating geneexpression indirectly by activating the transcription of PPAR ${alpha}$ (20). ERR ${alpha}$ has also been shown to cooperate with the transcriptionfactors NRF-1 and NRF-2 to activate the expression of genesinvolved in mitochondrial respiratory function (45). The "PGC-1 ${alpha}$ /ERR ${alpha}$ circuit" was further defined when it was shown that PGC-1 ${alpha}$ couldinduce ERR ${alpha}$ expression through ERR ${alpha}$ by an autoregulatory feed-forwardmechanism (27). Thus, ERR ${alpha}$ serves a central function in the PGC-1 ${alpha}$ gene regulatory cascade. The current study expands the listof ERR ${alpha}$ target genes to include PDK4. Our results show that PGC-1 ${alpha}$ not only directly coactivates ERR ${alpha}$ but also activates its expressionthrough the autoregulatory loop described previously.

Previous work has implicated the nuclear receptor PPAR ${alpha}$ , a knownPGC-1 ${alpha}$ target factor, in the regulation of PDK4 gene expression.Specifically, the PPAR ${alpha}$ selective ligand compound WY-14,643 inducesPDK4 gene expression (17), and this response is lost in thehearts and kidneys of PPAR ${alpha}$ -null mice (55). Somewhat surprisingly,our results demonstrate that PGC-1 ${alpha}$ -mediated activation of PDK4gene expression occurs independently of PPAR ${alpha}$ . It should be notedthat a direct mechanism for PPAR ${alpha}$ -mediated activation of PDK4gene expression has not been shown. However, the current studydoes not rule out an indirect role for PPAR ${alpha}$ in regulating PDK4gene expression or direct PPAR ${alpha}$ -mediated activation through elementsoutside of the promoter region examined in our study.

The transcription factor FOXO1 has been shown to function withPGC-1 ${alpha}$ in the regulation of liver gluconeogenesis (39). Recentstudies have shown that the GR in cooperation with FOXO1 activatestranscription of the human PDK4 gene. This transcriptional regulatoryeffect is mediated by a complex mechanism involving the interactionof a single GR response element and three FOXO1 response elementswithin the hPDK4 gene promoter region (26). This region is notconserved in the mouse PDK4 promoter. As with PPAR ${alpha}$ , we werealso surprised to find that the FOXO1 response element in theproximal mPDK4 promoter was dispensable for the observed PGC-1 ${alpha}$ effect. To our knowledge, the influence of PGC-1 ${alpha}$ on the humanPDK4 gene promoter has not been studied. However, inspectionof the human PDK4 promoter sequence indicates that a PGC-1 ${alpha}$ /ERR ${alpha}$ -responsiveregion may be conserved in the human promoter. Although theERR response element we identified in the mPDK4 gene is notspatially conserved in the hPDK4 promoter, a candidate monomericERR response element (AAGGACA; bp –823 to –817)does exist, overlapping the previously identified GR responseelement in the hPDK4 gene (26). Interestingly, a previous reportdescribes the modulation of GR activity by ERR isoforms (50).It is tempting to speculate that ERR ${alpha}$ and GR regulatory pathwaysconverge on the human PDK4 gene.

In summary, our findings, together with the known regulatoryeffects of PGC-1 ${alpha}$ on cellular energy metabolism, suggest a mechanismby which PGC-1 ${alpha}$ is able to suppress muscle glucose oxidationwhile increasing the expression of genes involved in mitochondrialfatty acid oxidation (Fig. 9). Energy demands placed on skeletalmuscle result in increased utilization of fatty acids, mediatedin part through the activating effects of PGC-1 ${alpha}$ on the expressionof genes involved in cellular fatty acid uptake and oxidation.Induction of PGC-1 ${alpha}$ activates ERR ${alpha}$ expression, leading to occupationof the PGC-1 ${alpha}$ /ERR ${alpha}$ complex on the PDK4 promoter. The resultantincreased expression of PDK4 leads to phosphorylation and inactivationof the PDC, which catalyzes the rate-limiting step of glucoseoxidation (48). This model is consistent with the response toendurance training, a known stimulus for increasing fatty acidoxidative capacity and mitochondrial biogenesis. Collectively,these findings expand the role of the PGC-1 ${alpha}$ /ERR ${alpha}$ complex to aregulator of muscle substrate selection, leading to a betterunderstanding of transcriptional mechanisms involved in themaintenance of energy homeostasis.

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FIG. 9. Proposed mechanism for the regulation of muscle glucose metabolism by PGC-1 ${alpha}$ /ERR ${alpha}$ . Increased energy demands related to physiological stress (e.g., exercise) result in the induction of muscle PGC-1 ${alpha}$ gene expression, which in turn induces ERR ${alpha}$ gene expression. ERR ${alpha}$ then binds to the PDK4 gene promoter, where PGC-1 ${alpha}$ directly coactivates ERR ${alpha}$ to induce PDK4 gene expression. PDK4 has known roles in the negative regulation of the PDC, resulting in decreased glucose oxidation coincident with increased mitochondrial fatty acid oxidation driven by PGC-1 ${alpha}$ .

ACKNOWLEDGMENTS

Special thanks to Robert A. Harris for providing the PDK4 antibodyand to William R. Sellers for providing the FKHR expressionvector. We thank Mary Wingate and Teresa Leone for assistancein preparing the manuscript.

This work is supported by NIH grant RO1 DK45416, the DigestiveDiseases Research Core Center grant (P30 DK52574), and the ClinicalNutrition Research Unit Core Center (P30 DK56341). A.R.W. issupported by the Washington University School of Medicine CardiovascularTraining grant T32 HL007275.

FOOTNOTES

* Corresponding author. Mailing address: Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, MO 63110. Phone: (314) 362-8908. Fax: (314) 362-0186. E-mail: dkelly{at}im.wustl.edu.

REFERENCES

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