Fatty Acids Activate Transcription of the Muscle Carnitine Palmitoyltransferase I Gene in Cardiac Myocytes via the Peroxisome Proliferator-activated Receptor α

To explore the gene regulatory mechanisms involved in the metabolic control of cardiac fatty acid oxidative flux, the expression of muscle-type carnitine palmitoyltransferase I (M-CPT I) was characterized in primary cardiac myocytes in culture following exposure to the long-chain mono-unsaturated fatty acid, oleate. Oleate induced steady-state levels of M-CPT I mRNA 4.5-fold. The transcription of a plasmid construct containing the human M-CPT I gene promoter region fused to a luciferase gene reporter transfected into cardiac myocytes, was induced over 20-fold by long-chain fatty acid in a concentration-dependent and fatty acyl-chain length-specific manner. The M-CPT I gene promoter fatty acid response element (FARE-1) was localized to a hexameric repeat sequence located between 775 and 763 base pairs upstream of the initiator codon. Cotransfection experiments with expression vectors for the peroxisome proliferator-activated receptor α (PPARα) demonstrated that FARE-1 is a PPARα response element capable of conferring oleate-mediated transcriptional activation to homologous or heterologous promoters. Electrophoretic mobility shift assays demonstrated that PPARα bound FARE-1 with the retinoid X receptor α. The expression of M-CPT I in hearts of mice null for PPARα was approximately 50% lower than levels in wild-type controls. Moreover, a PPARα activator did not induce cardiac expression of the M-CPT I gene in the PPARα null mice. These results demonstrate that long-chain fatty acids regulate the transcription of a gene encoding a pivotal enzyme in the mitochondrial fatty acid uptake pathway in cardiac myocytes and define a role for PPARα in the control of myocardial lipid metabolism. To explore the gene regulatory mechanisms involved in the metabolic control of cardiac fatty acid oxidative flux, the expression of muscle-type carnitine palmitoyltransferase I (M-CPT I) was characterized in primary cardiac myocytes in culture following exposure to the long-chain mono-unsaturated fatty acid, oleate. Oleate induced steady-state levels of M-CPT I mRNA 4.5-fold. The transcription of a plasmid construct containing the human M-CPT I gene promoter region fused to a luciferase gene reporter transfected into cardiac myocytes, was induced over 20-fold by long-chain fatty acid in a concentration-dependent and fatty acyl-chain length-specific manner. The M-CPT I gene promoter fatty acid response element (FARE-1) was localized to a hexameric repeat sequence located between 775 and 763 base pairs upstream of the initiator codon. Cotransfection experiments with expression vectors for the peroxisome proliferator-activated receptor α (PPARα) demonstrated that FARE-1 is a PPARα response element capable of conferring oleate-mediated transcriptional activation to homologous or heterologous promoters. Electrophoretic mobility shift assays demonstrated that PPARα bound FARE-1 with the retinoid X receptor α. The expression of M-CPT I in hearts of mice null for PPARα was approximately 50% lower than levels in wild-type controls. Moreover, a PPARα activator did not induce cardiac expression of the M-CPT I gene in the PPARα null mice. These results demonstrate that long-chain fatty acids regulate the transcription of a gene encoding a pivotal enzyme in the mitochondrial fatty acid uptake pathway in cardiac myocytes and define a role for PPARα in the control of myocardial lipid metabolism. fatty acid oxidation carnitine palmitoyltransferase muscle type liver type fatty acid response element-1 peroxisome proliferator-activated receptor bovine serum albumin polymerase chain reaction base pair(s) retinoid X receptor thymidine kinase electrophoretic mobility shift assay. Mammalian cardiac energy substrate utilization rates are regulated during development and in response to physiologic and pathophysiologic stimuli. During the fetal period, glucose serves as the chief myocardial substrate (1Neely J.R. Rovetto M.J. Oram J.F. Prog. Cardiovasc. Dis. 1972; 15: 289-329Crossref PubMed Scopus (380) Google Scholar). 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Chem. 1995; 270: 16308-16314Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Carter M.E. Gulick T. Moore D.D. Kelly D.P. Mol. Cell. Biol. 1994; 14: 4360-4372Crossref PubMed Google Scholar). Carnitine palmitoyltransferase I (CPT I; palmitoyl-CoA:l-carnitine O-palmitoyltransferase; EC2.3.1.21) catalyzes the initial reaction in the mitochondrial import of long-chain fatty acids, a tightly regulated step in the cellular fatty acid utilization pathway (17McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (292) Google Scholar, 18McGarry J.D. Biochem. Soc. Trans. 1995; 23: 321-324Crossref PubMed Scopus (101) Google Scholar). The activity of CPT I is an important determinant of cellular fatty acid oxidative flux. CPT I catalyzes the transfer of a long-chain fatty acyl group from coenzyme A to carnitine. A specific translocase (carnitine-acylcarnitine carrier) located in the inner mitochondrial membrane delivers long-chain acylcarnitines into the mitochondrial matrix where they are re-esterified to acyl-thioesters by carnitine palmitoyltransferase II (CPT II). Acyl-thioesters in the mitochondria undergo β-oxidation generating reducing equivalents used to produce ATP via oxidative phosphorylation. Recent studies have demonstrated that CPT I exists as two isoforms encoded by separate genes: liver-type (L-CPT I or CPT IA), a hepatic-enriched, ubiquitously expressed protein (19Esser V. Britton C.H. Weis B.C. Foster D.W. McGarry J.D. J. Biol. Chem. 1993; 268: 5817-5822Abstract Full Text PDF PubMed Google Scholar, 20Britton C.H. Schultz R.A. Zhang B. Esser V. Foster D.W. McGarry J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1984-1988Crossref PubMed Scopus (127) Google Scholar) and muscle-type (M-CPT I or CPT IB), which is expressed abundantly in heart, skeletal muscle, and brown adipose tissue (21Yamazaki N. Shinohara Y. Shima A. Terada H. FEBS Lett. 1995; 363: 41-45Crossref PubMed Scopus (112) Google Scholar, 22Yamazaki N. Shinohara Y. Shima A. Yamanaka Y. Terada H. Biochim. Biophys. Acta. 1996; 1307: 157-161Crossref PubMed Scopus (102) Google Scholar, 23Esser V. Brown N.F. Cowan A.T. Foster D.W. McGarry J.D. J. Biol. Chem. 1996; 271: 6972-6977Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). CPT I activity is inhibited by the reversible binding of malonyl-CoA, the first committed intermediate in the pathway of fatty acid synthesis (17McGarry J.D. Woeltje K.F. Kuwajima M. Foster D.W. Diabetes Metab. Rev. 1989; 5: 271-284Crossref PubMed Scopus (292) Google Scholar, 18McGarry J.D. Biochem. Soc. Trans. 1995; 23: 321-324Crossref PubMed Scopus (101) Google Scholar). Malonyl-CoA is proposed to inhibit hepatic fatty acid oxidation during periods of fatty acid synthesis. Much less is known about the regulation of CPT I activity in heart. The IC50of M-CPT I for malonyl-CoA is approximately 100-fold lower than that of L-CPT I (24McGarry J.D. Mills S.E. Long C.S. Forter D.W. Biochem. J. 1983; 214: 21-28Crossref PubMed Scopus (467) Google Scholar), yet the malonyl-CoA concentration in liver and heart is similar (25Singh B. Stakkestad J.A. Bremer J. Borrebaek B. Anal. Biochem. 1983; 138: 107-111Crossref Scopus (18) Google Scholar). These observations have led to speculation that control of M-CPT I activity occurs, at least in part, via malonyl-CoA independent mechanisms. We hypothesized that long-chain fatty acids regulate M-CPT I gene expression. In this study we demonstrate that expression of the M-CPT I gene is regulated in cardiac myocytes, at the transcriptional level, by long-chain fatty acids via the peroxisome proliferator-activated receptor α (PPARα). Our results suggest a mechanism for the control of myocardial fatty acid utilization at the mitochondrial import step, by long-chain acyl substrate levels. Cardiac myocytes were prepared from 1-day-old Sprague-Dawley rats as described (13Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Crossref PubMed Scopus (67) Google Scholar). In brief, ventricle was removed and digested in 0.2% collagenase (Wako Chemicals, Richmond, VA), the cells were pooled in Dulbecco's modified Eagle's medium containing 10% horse serum, 5% fetal calf serum (Sigma) and subjected to differential plating (1 h) to reduce fibroblast contamination. Nonadherent cells (enhanced cardiocyte fraction) were plated to 50% confluence on 60-mm diameter dishes pretreated with collagen (Sigma). After 24 h the cell medium was switched to serum-free Dulbecco's modified Eagle's medium containing 0.10 mm 5-bromo-2′-deoxyuridine (Sigma), 10 μg/ml insulin (Sigma), 10 μg/ml transferrin (Sigma), and 1 mg/ml fatty acid-free BSA (Sigma). Oleate was diluted to 20 mm in water preheated to 70 °C. NaOH (1n) was added dropwise until oleate was solubilized. The oleate was then complexed to BSA (molar ratio 7:1) to yield a final oleate concentration of 4.7 mm. The sodium salts of n-decanoic acid and n-caproic acid were each diluted in water to a concentration of 100 mm. The sodium salt of etomoxir (Research Biochemicals International) was diluted in water to a concentration of 4 mm. Each of these stock solutions were diluted in cell culture medium to the concentrations outlined under “Results.” RNA blotting was performed as described (26Kelly D.P. Gordon J.I. Alpers R. Strauss A.W. J. Biol. Chem. 1989; 264: 18921-18925Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from rat neonatal cardiac myocytes in cell culture or mouse ventricle using the RNAzol (Tel-Test, Inc.) method. Probes used included a rat M-CPT I cDNA, a mouse ATPase subunit e cDNA (27Levy F.H. Kelly D.P. Am. J. Physiol. 1997; 41: C457-C465Crossref Google Scholar), a cDNA probe encoding 18 S ribosomal RNA, and a human β-actin cDNA. Band intensities were quantified by laser densitometry using an LKB Ultrascan XL (Matsushita Electric Industrial Co., Ltd.) or by phosphorimaging using a Bio-Rad GS 525 Molecular Imager System. For Northern blot analyses with mouse tissues, total RNA was isolated from ventricles of adult (2–3-month-old) male mice null for PPARα or age-matched wild-type controls. The production and initial characterization of mice lacking PPARα have been described (28Lee S.S.T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar). The 5′-flanking region of the human M-CPT I gene was cloned by PCR amplification of a BAC subclone template known to contain the M-CPT I gene (GenBank data base accession number U62317; a gift from the Institute for Genomic Research, Rockville, MD). The region from 1025 to 12 bp 5′ of the initiation codon adenine (1Neely J.R. Rovetto M.J. Oram J.F. Prog. Cardiovasc. Dis. 1972; 15: 289-329Crossref PubMed Scopus (380) Google Scholar) was amplified using the proofreading polymerase Pwo (Boehringer Mannheim). Automated DNA sequencing confirmed that the nucleotide sequence of the PCR product was 100% identical to the template sequence. MCPT.Luc.1025 was constructed by cloning the human M-CPT I gene 5′-flanking region from 12 to 1025 bp upstream of the start codon adenine into the promoterless pGL2-Basic plasmid (Promega). The deletion constructs MCPT.Luc.915, MCPT.Luc.781, and MCPT.Luc.724 were generated by PCR amplification of the BAC subclone template using the same 3′ primer used to construct MCPT.Luc.1025 and 5′ primers beginning 915, 781, and 724 base pairs 5′ of the start codon adenine, respectively. The M-CPT I promoter point mutant construct, MCPT.Luc.781m1, was generated by PCR amplification of the M-CPT I gene 5′-flanking genomic DNA extending from 781 to 12 base pairs 5′ of the start codon adenine using a 5′ PCR primer containing a cytidine for guanine mismatch substitution at position −771 (as illustrated in Fig. 3 B). All PCR products were ligated into the XhoI (5′) and HindIII (3′) sites in pGL2-Basic. The heterologous promoter-reporter constructs MCPT(FARE)2TKLuc and MCPT(FAREm1)2TKLuc were constructed by ligation of two copies of a double-stranded oligonucleotide (single-copy sense-strand sequences: MCPT(FARE)2TKLuc: 5′-GATCCGGTGACCTTTTCCCTACAG-3′ and MCPT(FAREm1)2TKLuc: 5′-GATCCGGTGACgTTTTCCCTACAG-3′ (lowercase bold letter denotes mutation)) containing BamHI overhangs into aBamHI site upstream of the herpes simplex virus thymidine kinase (TK) promoter in a plasmid described previously (16Carter M.E. Gulick T. Moore D.D. Kelly D.P. Mol. Cell. Biol. 1994; 14: 4360-4372Crossref PubMed Google Scholar). PCR-generated mutations were excluded by automated sequence analysis of each promoter-reporter plasmid. Transient transfection of rat neonatal cardiac myocytes was performed as described (13Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Crossref PubMed Scopus (67) Google Scholar) using N-[1-(2, 3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Boehringer Mannheim). For each transfection, 1–2 μg of reporter plasmid was cotransfected with 100 ng of pRSVβ-Gal, a plasmid containing a β-galactosidase gene downstream of the Rous sarcoma virus promoter, to control for transfection efficiency. For the cotransfection experiments with the PPARα expression vector, human HepG2 cells were plated in 12-well dishes at 2 × 105cells/well and maintained in an atmosphere containing 5% CO2 in minimal essential medium supplemented with 10% fetal calf serum. Transient cell transfections were performed using a modified calcium-phosphate precipitation method (29Gorman C. Glover D.M. DNA Cloning: A Practical Approach. II. IRL Press, Oxford1985: 143-190Google Scholar). Four micrograms of reporter plasmid and 100 ng of pRSVβ-Gal were added to each well of transfected cells. In some wells, 1 μg of pCDM.PPAR, a mammalian expression vector containing a mouse PPARα cDNA (30Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Crossref PubMed Scopus (491) Google Scholar), or 1 μg of pCDM(−), the expression vector backbone lacking the PPARα cDNA, was added. Oleate, etomoxir, and vehicle were added to the cell medium 16 h after transfection. The cells were harvested 24 h later. Luciferase activities were determined by the standard luciferin-ATP assay, and β-galactosidase activity was measured by the Galacto-Light chemiluminescence assay (Tropix, Bedford, MA) in an Analytical Luminescence Monolight 2010 luminometer. EMSAs were performed as described (16Carter M.E. Gulick T. Moore D.D. Kelly D.P. Mol. Cell. Biol. 1994; 14: 4360-4372Crossref PubMed Google Scholar). The pT7lac-RXRα bacterial expression vector was generously provided by Dr. Tod Gulick (Harvard University). Nuclear receptor was overproduced in bacterial cells and partially purified as described (16Carter M.E. Gulick T. Moore D.D. Kelly D.P. Mol. Cell. Biol. 1994; 14: 4360-4372Crossref PubMed Google Scholar). pCal-n (m.PPAR) was generated by PCR cloning of mouse PPARα cDNA into the pCAL-n vector (Stratagene) and used for in vitro transcription/translation in the TnT coupled reticulocyte lysate system (Promega) per standard protocol. The M-CPT I FARE-1 probe sense strand sequence is 5′-GATCCGGTGACCTTTTCCCTACAG-3′. Double-stranded oligonucleotide probe was 32P-labeled by Klenow “fill-in” of a 5′-GATC overhang. Antibody “supershift” experiments were performed with polyclonal antibodies directed against human PPARα (a gift of Dr. Michael Arand, University of Mainz), or a monoclonal antibody directed against RXRα (a gift from Dr. Pierre Chambon, INSERM, Strasbourg, France). Specific antibody or preimmune sera was added to a mixture containing labeled probe and protein, and incubated for 10 min at room temperature, followed by resolution on a 5% nondenaturing polyacrylamide gel followed by autoradiography. Differences between mean mRNA levels were determined by unpaired Student's t test analysis. A statistically significant difference was defined as P < 0.05. All values shown represent the mean ± the standard error of the mean (S.E.). To determine whether M-CPT I gene expression is regulated by long-chain fatty acids in cardiac myocytes, M-CPT I mRNA levels were delineated in primary ventricular myocytes in culture following exposure to oleate (C18:1). For these experiments, myocytes isolated from 1-day-old rat ventricle were exposed to 0.5 mm oleate complexed to bovine serum albumin (BSA) or vehicle (BSA alone) for 90 h in serum-free medium. Mean M-CPT I mRNA levels were 4.5-fold higher in cardiac myocytes exposed to oleate compared with vehicle-treated cells (p < 0.01; Fig. 1). Expression of the genes encoding several mitochondrial β-oxidation cycle enzymes including very-long-chain and medium-chain acyl-CoA dehydrogenase also increased in the presence of oleate (data not shown). The level of β-actin mRNA, a control for loading, was not different in vehicle compared with oleate-treated cells. Similarly, the expression of the mRNA encoding ATPase subunit e, a nuclear encoded mitochondrial protein, was not affected by exposure to oleate indicating that the regulatory effect does not involve all nuclear genes encoding mitochondrial proteins. To determine whether the oleate-induced increase in M-CPT I gene expression occurs at the transcriptional level, the oleate experiments were repeated with cardiac myocytes transiently transfected with a plasmid containing the human M-CPT I gene promoter region fused to a luciferase reporter gene. A 1013-bp fragment of M-CPT I gene 5′-flanking DNA was cloned by PCR amplification of a BAC subclone template containing the human M-CPT I gene (see “Materials and Methods”). The human M-CPT I gene 5′-flanking DNA contains two untranslated exons, 1A and 1B, that extend from 746 to 633 and 523 to 470 base pairs upstream of the start codon, respectively (31van der Leij F.R. Takens J. van der Veen A.Y. Terpstra P. Kuipers J.R.G. Biochim. Biophys. Acta. 1997; 1352: 123-128Crossref PubMed Scopus (29) Google Scholar, 32Yamazaki N. Yamanaka Y. Hashimoto Y. Shinohara Y. Shima A. Terada H. FEBS Lett. 1997; 409: 401-406Crossref PubMed Scopus (56) Google Scholar). The 5′ ends of exons 1A and 1B function as independent transcription start sites (32Yamazaki N. Yamanaka Y. Hashimoto Y. Shinohara Y. Shima A. Terada H. FEBS Lett. 1997; 409: 401-406Crossref PubMed Scopus (56) Google Scholar). The M-CPT I gene 5′-flanking region (from 1025 to 12 base pairs 5′ of the start codon adenine) was cloned upstream of the luciferase gene in a promoterless reporter plasmid to generate MCPT.Luc.1025 (Fig. 2 A). The basal transcriptional activity of MCPT.Luc.1025 in cardiac myocytes was significantly higher (over 15-fold) than that of the promoterless vector backbone (data not shown). Oleate markedly induced the transcriptional activity of MCPT.Luc.1025 (approximately 25-fold; Fig. 2 A). The dose dependence of the oleate response was delineated by repeating the cardiac myocyte transfection experiments in serum-deprived medium containing 50, 250, or 500 μm oleate versusvehicle alone. As shown in Fig. 2 B, the transcriptional activity of MCPT.Luc.1025 increases with increasing oleate concentration. To determine whether the fatty acid-induced transcription of the M-CPT I gene is acyl chain length-specific, dose-response experiments were repeated using decanoate (C10:0) and hexanoate (C6:0). Compared with the oleate response, the activity of MCPT.Luc.1025 was induced only modestly by decanoate and was not affected by hexanoate at any of the concentrations tested (Fig. 2 B). Taken together, these data indicate that M-CPT I gene transcription is activated by long-chain fatty acids in a concentration-dependent and acyl chain length-specific manner. These results are consistent with the role of M-CPT I in the mitochondrial import of long-chain fatty acids. Cardiac myocyte transfections were repeated with a MCPT.Luc deletion series to localize the oleate-responsive region within the human M-CPT I gene promoter region. Luciferase reporter plasmids containing M-CPT I 5′-flanking DNA extending from 1025 (MCPT.Luc.1025), 915 (MCPT.Luc.915), 781 (MCPT.Luc.781), and 724 (MCPT.Luc.724) bp upstream of the M-CPT I start codon adenine were used for these experiments. The oleate response was preserved with the MCPT.Luc.915 and MCPT.Luc.781 constructs but was absent with MCPT.Luc.724 (Fig. 3 A). The basal transcriptional activity of MCPT.Luc.724 was approximately 50% lower than that of MCPT.Luc.781 but still significantly greater than that of the promoterless, reporter vector backbone indicating that the lack of an oleate response is not due to the loss of promoter function. Thus, the M-CPT I gene 5′-flanking region between −781 and −724 contains a fatty acid response element. DNA sequence analysis of the M-CPT I gene promoter oleate-responsive region revealed the presence of an imperfect hexameric repeat separated by a single nucleotide (DR-1;−775-TGACCTTTTCCCT-−763). The antisense sequence of each half-site of the putative fatty acid-responsive element (Fig. 3 B) conforms to the consensus ((A/G)GG(T/G)NA) for binding class II members of the nuclear receptor superfamily. The transfections were repeated with a mutated MCPT.Luc.781 construct containing a cytidine substitution for the invariant consensus second position guanine within the 5′-most hexameric half-site of the DR-1 sequence (MCPT.Luc.781m1; Fig. 3 B). The basal transcriptional activity of MCPT.Luc.781m1 was not significantly different than that of MCPT.Luc.781 (data not shown). However, the oleate response was abolished by this single bp substitution (Fig. 3 B). These results indicate that the DR-1 located between −775 and −763 is required for the M-CPT I gene fatty acid response in cardiac myocytes. This element will be referred to as thefatty acid responseelement-1 or FARE-1. Previous studies have shown that DR-1 elements serve as binding sites for PPAR·RXR heterodimers (33Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (640) Google Scholar). Given the results of previous studies demonstrating that PPARα regulates the transcription of genes encoding other mitochondrial and peroxisomal fatty acid oxidation enzymes (33Lemberger T. Desvergne B. Wahli W. Annu. Rev. Cell Dev. Biol. 1996; 12: 335-363Crossref PubMed Scopus (640) Google Scholar), we speculated that this receptor was a candidate for the fatty acid-mediated control of M-CPT I gene expression. Indeed, PPARα has been shown to be activated by fatty acids (34Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1898) Google Scholar, 35Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1870) Google Scholar). To determine whether FARE-1 is activated by PPARα, hepatoma G2 (HepG2) cells were cotransfected with a PPARα mammalian expression vector (pCDM.PPAR) and MCPT.Luc.781 or MCPT.Luc.781m1 in the presence or absence of oleate or etomoxir, known PPARα activators (30Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Crossref PubMed Scopus (491) Google Scholar, 34Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1898) Google Scholar, 35Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1870) Google Scholar). The HepG2 cell line was chosen because, in contrast to cardiac myocytes, the MCPT.Luc.781 oleate response is minimal allowing for a “null” background. The transcriptional activity of MCPT.Luc.781 increased less than 1.5-fold in the presence of oleate or etomoxir alone (Fig. 4 A). Cotransfection of pCDM.PPAR in the absence of an exogenous activator induced MCPT.Luc.781 transcription nearly 7-fold. The addition of oleate or the CPT I inhibitor etomoxir to cells cotransfected with pCDM.PPAR induced MCPT.Luc.781 transcription 2–3.5-fold higher than pCDM.PPAR cotransfection alone (total activation 16–24-fold; Fig. 4 A). In contrast, transcription of the point mutant construct MCPT.Luc.781m1 was not induced by pCDM.PPAR cotransfection in the absence or presence of activators (Fig. 4 A). To test whether FARE-1 could confer PPARα responsiveness to a heterologous promoter, two copies of FARE-1 were cloned upstream of the herpes simplex virus TK promoter fused to a luciferase reporter gene (MCPT(FARE)2TKLuc). Cotransfection studies were performed with MCPT(FARE)2TKLuc and pCDM.PPAR in the presence and absence of oleate or etomoxir (Fig. 4 B). Neither oleate nor etomoxir alone activated MCPT(FARE)2TKLuc. Overexpression of PPARα resulted in a 5-fold activation of MCPT(FARE)2TKLuc activity with an additional 1.6–2-fold induction with addition of oleate or etomoxir (total activation 8–10-fold; Fig. 4 B). When a point mutation identical to that present in MCPT.Luc.781m1 was introduced into both copies of FARE-1 in the context of TKLuc (MCPT(FAREm1)2TKLuc), PPARα responsiveness was abolished (Fig. 4 B). Taken together, these results define FARE-1 as a PPARα response element. PPARα binds cognate DNA elements as a heterodimer with RXR (36Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1525) Google Scholar). EMSAs were performed to characterize the interaction of PPARα·RXRα heterodimers with the PPARα-responsive element, FARE-1. EMSA was performed with a radiolabeled FARE-1 oligonucleotide probe, RXRα (produced by overexpression in bacteria), and PPARα produced byin vitro coupled reticulocyte lysate transcription/translation. The FARE-1 probe formed a light complex with PPARα alone and no complex with RXRα alone (Fig. 5, lanes 2 and 3). A prominent FARE·protein complex formed when both PPARα and RXRα were added to the incubation (Fig. 5, lane 4). Competition experiments performed with a molar excess of specific (FARE-1) or an unrelated, size-matched, double-stranded nonspecific oligonucleotide confirmed that the prominent complex of lowest mobility formed with PPARα and RXRα represented a specific DNA-protein interaction (Fig. 5, lanes 5–7). Antibody recognition studies confirmed that PPARα and RXRα were present in the FARE-1/protein complex. The FARE-1/protein complex was supershifted by either a polyclonal antibody that recognizes mouse PPARα (37Gebel T. Arand M. Oesch F. FEBS Lett. 1992; 309: 37-40Crossref PubMed Scopus (103) Google Scholar) or an anti-RXRα antibody, whereas addition of preimmune sera did not alter its mobility (Fig. 5,lanes 8–10). Taken together, these findings demonstrate that, as reported for other PPARα response elements, PPARα and RXRα bind FARE-1 as a heterodimer. To determine the importance of PPARα in the regulation of cardiac fatty acid oxidative enzyme gene expression in vivo, M-CPT I gene expression was characterized in mice null for PPARα (PPARα−/−; Ref. 28Lee S.S.T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar) and compared with age-matched controls (PPARα+/+). Previous studies have shown that mitochondrial and peroxisomal fatty acid β-oxidation cycle enzyme gene expression is reduced in the liver of PPARα−/− mice (28Lee S.S.T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar, 38Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Northern blot studies demonstrated that steady-state levels of M-CPT I mRNA are significantly lower (by 51 ± 9%) in the hearts of adult PPARα−/− mice compared with controls (Fig. 6 A). The studies were repeated following a 5-day administration of etomoxir, a known activator of PPARα. As expected, etomoxir induced myocardial expression of M-CPT I mRNA in PPARα+/+ mice (194 ± 15versus 100 ± 7). In contrast, the PPAR activator did not induce M-CPT I gene expression in the hearts of PPARα−/− mice (64 ± 10 versus 58 ± 10) (Fig. 6 B). These results confirm the role of PPARα as an activator of M-CPT I gene expression in heart in vivo. The energy substrate preference of the mammalian heart is tightly controlled during development and in response to diverse physiologic and pathophysiologic conditions (2Bing R.J. Harvey Lect. 1955; 50: 27-70Google Scholar, 3Taegtmeyer H. Curr. Prob. Cardiol. 1994; 19: 57-116Crossref Scopus (336) Google Scholar, 4Bishop S.P. Altschuld R.A. Am. J. Physiol. 1970; 218: 153-159Crossref PubMed Scopus (185) Google Scholar, 5Taegtmeyer H. Overturf M.L. Hypertension. 1988; 11: 416-426Crossref PubMed Scopus (201) Google Scholar, 6Scheuer J. Circulation. 1993; 87: VII54-VII57Google Scholar, 7Moalic J.-M. Charlemagne D. Mansier P. Chevalier B. Swynghedauw B. Circulation. 1993; 87: IV21-IV26Crossref PubMed Scopus (54) Google Scholar, 8Takeyama D. Kagaya Y. Yamane Y. Shiba N. Chida M. Takahashi T. Ido T. Ishide N. Takishima T. Cardiovasc. Res. 1995; 29: 763-767Crossref PubMed Scopus (39) Google Scholar, 9Massie B.M. Schaefer S. Garcia J. McKirnan D. Schwartz G.G. Wisneski J.A. Weiner M.W. 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During cardiac hypertrophy and in the failing heart, the myocardium reverts to the fetal energy substrate utilization pattern using glucose as the chief energy substrate (4Bishop S.P. Altschuld R.A. Am. J. Physiol. 1970; 218: 153-159Crossref PubMed Scopus (185) Google Scholar, 5Taegtmeyer H. Overturf M.L. Hypertension. 1988; 11: 416-426Crossref PubMed Scopus (201) Google Scholar, 6Scheuer J. Circulation. 1993; 87: VII54-VII57Google Scholar, 7Moalic J.-M. Charlemagne D. Mansier P. Chevalier B. Swynghedauw B. Circulation. 1993; 87: IV21-IV26Crossref PubMed Scopus (54) Google Scholar, 8Takeyama D. Kagaya Y. Yamane Y. Shiba N. Chida M. Takahashi T. Ido T. Ishide N. Takishima T. Cardiovasc. Res. 1995; 29: 763-767Crossref PubMed Scopus (39) Google Scholar, 9Massie B.M. Schaefer S. Garcia J. McKirnan D. Schwartz G.G. Wisneski J.A. Weiner M.W. White F.C. Circulation. 1995; 91: 1814-1823Crossref PubMed Scopus (80) Google Scholar, 10Alpert N.R. Mulieri L.A. Circ. Res. 1982; 50: 491-500Crossref PubMed Scopus (181) Google Scholar, 11Christe M.E. Rodgers R.L. J. Mol. Cell. Cardiol. 1994; 26: 1371-1375Abstract Full Text PDF PubMed Scopus (167) Google Scholar). Previous studies have shown that expression of nuclear genes encoding mitochondrial fatty acid β-oxidation cycle enzymes is regulated at the transcriptional level in parallel with fatty acid utilization rates during development and in the hypertrophied and failing heart (12Sack M.N. Rader T.A. Park S. McCune S.A. Kelly D.P. Circulation. 1996; 94: 2837-2842Crossref PubMed Scopus (547) Google Scholar, 13Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Crossref PubMed Scopus (67) Google Scholar, 14Sack M.N. Disch D. Rockman H. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6438-6443Crossref PubMed Scopus (177) Google Scholar, 39Nagao M. Parimoo B. Tanaka K. J. Biol. Chem. 1993; 268: 24114-24124Abstract Full Text PDF PubMed Google Scholar). Thus, the capacity for myocardial fatty acid oxidation is dictated, at least in part, via transcriptional regulatory mechanisms. In this report we describe a mechanism for the induction of M-CPT I gene expression by long-chain fatty acids in heart, namely transcriptional control by the fatty acid-activated nuclear receptor, PPARα. These results extend the gene regulatory paradigm established for the cardiac FAO cycle to mitochondrial long-chain fatty acid import, a highly regulated step in the myocardial lipid utilization pathway. Prior to this report, several lines of evidence suggested that fatty acids induce the expression of M-CPT I and other enzymes in the cellular fatty acid utilization pathway. First, the activity of CPT I and FAO cycle enzymes is up-regulated in parallel with the ingestion of a fatty acid-enriched diet during the suckling period (2Bing R.J. Harvey Lect. 1955; 50: 27-70Google Scholar, 3Taegtmeyer H. Curr. Prob. Cardiol. 1994; 19: 57-116Crossref Scopus (336) Google Scholar). The cardiac expression of these enzymes falls upon weaning in rats and mice (13Disch D.L. Rader T.A. Cresci S. Leone T.C. Barger P.M. Vega R. Wood P.A. Kelly D.P. Mol. Cell. Biol. 1996; 16: 4043-4051Crossref PubMed Scopus (67) Google Scholar, 26Kelly D.P. Gordon J.I. Alpers R. Strauss A.W. J. Biol. Chem. 1989; 264: 18921-18925Abstract Full Text PDF PubMed Google Scholar, 39Nagao M. Parimoo B. Tanaka K. J. Biol. Chem. 1993; 268: 24114-24124Abstract Full Text PDF PubMed Google Scholar). Second, long-chain fatty acids induce expression of CPT I in hepatocytes and pancreatic islet cells in culture (40Assimacopoulos-Jeannet F. Thumelin S. Roche E. Esser V. McGarry J.D. Prentki M. J. Biol. Chem. 1997; 272: 1659-1664Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 41Chatelain F. Kohl C. Esser V. McGarry J.D. Girard J. Pegorier J-P. Eur. J. Biochem. 1996; 235: 789-798Crossref PubMed Scopus (112) Google Scholar). Third, FAO enzyme gene expression is induced by fasting in liver and heart (39Nagao M. Parimoo B. Tanaka K. J. Biol. Chem. 1993; 268: 24114-24124Abstract Full Text PDF PubMed Google Scholar). The results shown here demonstrate that M-CPT I gene transcription is activated by long-chain fatty acids via FARE-1, a PPARα response element. The activation of FARE-1 is acyl-chain length-specific and can be conferred to a heterologous promoter. Moreover, the reduced expression of M-CPT I in the PPAR−/− mouse heart suggests that, in vivo, fatty acids influence the basal transcription of this gene which is consistent with the presence of circulating lipids and reliance on fatty acids as the chief energy substrate in the adult mammalian heart. These results strongly suggest that intracellular fatty acid derivatives, several of which are known to activate PPARα, comprise a metabolic signaling pathway in heart. The role of PPARα in the control of hepatic lipid metabolism is well established. PPARα was first identified as a transcription factor involved in the hepatic response to peroxisome proliferators (42Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3059) Google Scholar). The expression of PPARα target genes encoding enzymes involved in peroxisomal, cytochrome P450, and mitochondrial FAO is reduced in the liver of mice null for PPARα (28Lee S.S.T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar). Further, the expected induction of peroxisomal PPARα target enzymes in response to peroxisome proliferators is absent in PPARα−/− mice (28Lee S.S.T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1506) Google Scholar). In addition to liver, PPARα is expressed abundantly in tissues with high capacity for fatty acid oxidation such as heart, kidney, and brown adipose tissue. The role of PPARα in extrahepatic tissues has not been characterized. Our results demonstrate one important function for PPARα in heart: transcriptional control of the gene encoding M-CPT I, a pivotal enzyme in the uptake of long-chain fatty acids into mitochondria. Taken together with the previous observation that the promoter of the gene encoding the cardiac-enriched mitochondrial FAO cycle enzyme medium-chain acyl-CoA dehydrogenase contains a PPARα response element (30Gulick T. Cresci S. Caira T. Moore D.D. Kelly D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11012-11016Crossref PubMed Scopus (491) Google Scholar), our results indicate that PPARα regulates myocardial as well as hepatic lipid metabolism. In summary, we have demonstrated that long-chain fatty acids regulate M-CPT I gene expression in heart through PPARα. We speculate that this transcriptional regulatory mechanism is activated as a component of the coordinate control of myocardial fatty acid utilization pathways following birth and is altered in pathophysiologic settings such as cardiac hypertrophy and failure. We thank Dr. Frank Gonzalez (National Cancer Institute) for the use of the PPARα−/− mice, Dr. Tod Gulick (Harvard University) for helpful discussions, and Kelly Hall for expert secretarial assistance. Recently, Mascaró et al. (43Mascaró C. Acosta E. Ortiz J. Marrero P. Hegardt F. Haro D. J. Biol. Chem. 1998; 273: 8560-8563Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) described the regulation of M-CPT I gene expression by PPARα.