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.

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 (1). Following birth, myocardial energy is produced primarily via mitochondrial ␤-oxidation of long-chain fatty acids (2,3). During the develop-ment of cardiac hypertrophy in rodents and humans, fatty acid oxidation (FAO) 1 rates decrease and glucose utilization increases (4 -11): a reversion to the fetal energy metabolic program. Recent studies have demonstrated that the expression of genes encoding FAO enzymes is regulated, at the transcriptional level, in parallel with fatty acid utilization rates during development and in the hypertrophied and failing heart (12)(13)(14). The gene regulatory mechanisms governing cardiac fatty acid utilization have not been delineated; however, recent studies focused on the gene encoding medium-chain acyl-CoA dehydrogenase have implicated nuclear receptors and members of the Sp transcription factor family in the metabolic control of FAO enzyme gene expression (13)(14)(15)(16).
Carnitine palmitoyltransferase I (CPT I; palmitoyl-CoA:Lcarnitine O-palmitoyltransferase; EC 2.3.1.21) catalyzes the initial reaction in the mitochondrial import of long-chain fatty acids, a tightly regulated step in the cellular fatty acid utilization pathway (17,18). 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 ␤-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 (19,20) and muscle-type (M-CPT I or CPT IB), which is expressed abundantly in heart, skeletal muscle, and brown adipose tissue (21)(22)(23). CPT I activity is inhibited by the reversible binding of malonyl-CoA, the first committed intermediate in the pathway of fatty acid synthesis (17,18). 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 IC 50 of M-CPT I for malonyl-CoA is approximately 100-fold lower than that of L-CPT I (24), yet the malonyl-CoA concentration in liver and heart is similar (25). 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 ␣ (PPAR␣). Our results suggest a mechanism for the control of myocardial fatty acid utilization at the mitochondrial import step, by long-chain acyl substrate levels.
Preparation of Fatty Acid and Etomoxir Solutions-Oleate was diluted to 20 mM in water preheated to 70°C. NaOH (1 N) 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." Northern Blot Analysis-RNA blotting was performed as described (26). 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 (27), a cDNA probe encoding 18 S ribosomal RNA, and a human ␤-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 PPAR␣ or age-matched wild-type controls. The production and initial characterization of mice lacking PPAR␣ have been described (28).
Cloning of the Human M-CPT I Gene 5Ј-Flanking Region-The 5Јflanking 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 5Ј of the initiation codon adenine (1) 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.
Reporter Plasmids-MCPT.Luc.1025 was constructed by cloning the human M-CPT I gene 5Ј-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 3Ј primer used to construct MCPT.Luc.1025 and 5Ј primers beginning 915, 781, and 724 base pairs 5Ј 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 5Ј-flanking genomic DNA extending from 781 to 12 base pairs 5Ј of the start codon adenine using a 5Ј PCR primer containing a cytidine for guanine mismatch substitution at position Ϫ771 (as illustrated in Fig. 3B). All PCR products were ligated into the XhoI (5Ј) and HindIII (3Ј) sites in pGL2-Basic.
Mammalian Cell Transient Transfections-Transient transfection of rat neonatal cardiac myocytes was performed as described (13) using N- [1-(2, 3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Boehringer Mannheim). For each transfection, 1-2 g of reporter plasmid was cotransfected with 100 ng of pRSV␤-Gal, a plasmid containing a ␤-galactosidase gene downstream of the Rous sarcoma virus promoter, to control for transfection efficiency. For the cotransfection experiments with the PPAR␣ expression vector, human HepG2 cells were plated in 12-well dishes at 2 ϫ 10 5 cells/well and maintained in an atmosphere containing 5% CO 2 in minimal essential medium supplemented with 10% fetal calf serum. Transient cell transfections were performed using a modified calcium-phosphate precipitation method (29). Four micrograms of reporter plasmid and 100 ng of pRSV␤-Gal were added to each well of transfected cells. In some wells, 1 g of pCDM.PPAR, a mammalian expression vector containing a mouse PPAR␣ cDNA (30), or 1 g of pCDM(Ϫ), the expression vector backbone lacking the PPAR␣ 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 ␤-galactosidase activity was measured by the Galacto-Light chemiluminescence assay (Tropix, Bedford, MA) in an Analytical Luminescence Monolight 2010 luminometer.
Electrophoretic Mobility Shift Assays (EMSAs)-EMSAs were performed as described (16). The pT7lac-RXR␣ bacterial expression vector was generously provided by Dr. Tod Gulick (Harvard University). Nuclear receptor was overproduced in bacterial cells and partially purified as described (16). pCal-n (m.PPAR) was generated by PCR cloning of mouse PPAR␣ 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 5Ј-GATCCGGTGACCTTTTCCCTACAG-3Ј. Double-stranded oligonucleotide probe was 32 P-labeled by Klenow "fillin" of a 5Ј-GATC overhang. Antibody "supershift" experiments were performed with polyclonal antibodies directed against human PPAR␣ (a gift of Dr. Michael Arand, University of Mainz), or a monoclonal antibody directed against RXR␣ (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.
Statistical Analysis-Differences between mean mRNA levels were determined by unpaired Student's t test analysis. A statistically significant difference was defined as P Ͻ 0.05. All values shown represent the mean Ϯ the standard error of the mean (S.E.).

Long-chain Fatty Acids Induce Expression of the M-CPT I
Gene-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 Ͻ 0.01; Fig. 1). Expression of the genes encoding several mitochondrial ␤-oxidation cycle enzymes including very-longchain and medium-chain acyl-CoA dehydrogenase also increased in the presence of oleate (data not shown). The level of ␤-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.
Oleate  (31,32). The 5Ј ends of exons 1A and 1B function as independent transcription start sites (32). The M-CPT I gene 5Ј-flanking region (from 1025 to 12 base pairs 5Ј of the start codon adenine) was cloned upstream of the luciferase gene in a promoterless reporter plasmid to generate MCPT.Luc.1025 ( Fig. 2A). 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. 2A).
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 M oleate versus vehicle alone. As shown in Fig. 2B, 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. 2B). Taken together, these data indicate that M-CPT I gene transcription is activated by longchain 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.
The  .781m1; Fig. 3B). 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. 3B). These results indicate that the DR-1 located between Ϫ775 and Ϫ763 is required for the M-CPT I gene fatty acid response in cardiac myocytes. This element will be referred to as the fatty acid response element-1 or FARE-1.
The M-CPT I Gene Promoter Element, FARE-1, Is a PPAR␣ Response Element-Previous studies have shown that DR-1 elements serve as binding sites for PPAR⅐RXR heterodimers (33). Given the results of previous studies demonstrating that PPAR␣ regulates the transcription of genes encoding other mitochondrial and peroxisomal fatty acid oxidation enzymes (33), we speculated that this receptor was a candidate for the fatty acid-mediated control of M-CPT I gene expression. Indeed, PPAR␣ has been shown to be activated by fatty acids (34,35). To determine whether FARE-1 is activated by PPAR␣, hepatoma G2 (HepG2) cells were cotransfected with a PPAR␣ mammalian expression vector (pC-DM.PPAR) and MCPT.Luc.781 or MCPT.Luc.781m1 in the presence or absence of oleate or etomoxir, known PPAR␣ activators (30,34,35). 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. 4A). 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. 4A). 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. 4A).
To test whether FARE-1 could confer PPAR␣ 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) 2 TKLuc). Cotransfection studies were performed with MCPT(FARE) 2 TKLuc and pCDM.PPAR in the presence and absence of oleate or etomoxir (Fig. 4B). Neither oleate nor etomoxir alone activated MCPT(FARE) 2 TKLuc. Overexpression of PPAR␣ resulted in a 5-fold activation of MCPT(FARE) 2 TKLuc activity with an additional 1.6 -2-fold induction with addition of oleate or etomoxir (total activation 8 -10-fold; Fig. 4B). 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) 2 TKLuc), PPAR␣ responsiveness was abolished (Fig. 4B). Taken together, these results define FARE-1 as a PPAR␣ response element.
PPAR␣⅐RXR␣ Heterodimers Bind FARE-1-PPAR␣ binds cognate DNA elements as a heterodimer with RXR (36). EM-SAs were performed to characterize the interaction of PPAR␣⅐RXR␣ heterodimers with the PPAR␣-responsive element, FARE-1. EMSA was performed with a radiolabeled FARE-1 oligonucleotide probe, RXR␣ (produced by overexpression in bacteria), and PPAR␣ produced by in vitro coupled reticulocyte lysate transcription/translation. The FARE-1 probe formed a light complex with PPAR␣ alone and no complex with RXR␣ alone (Fig. 5, lanes 2 and 3). A prominent FARE⅐protein complex formed when both PPAR␣ and RXR␣ 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 PPAR␣ and RXR␣ represented a specific DNA-protein interaction (Fig. 5, lanes 5-7). Antibody recognition studies confirmed that PPAR␣ and RXR␣ were present in the FARE-1/protein complex. The FARE-1/protein complex was supershifted by either a polyclonal antibody that recognizes mouse PPAR␣ (37) or an anti-RXR␣ 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 PPAR␣ response elements, PPAR␣ and RXR␣ bind FARE-1 as a heterodimer.
PPAR␣ Regulates FAO Enzyme Gene Expression in Vivo-To determine the importance of PPAR␣ in the regulation of cardiac fatty acid oxidative enzyme gene expression in vivo, M-CPT I gene expression was characterized in mice null for PPAR␣ (PPAR␣Ϫ/Ϫ; Ref. 28) and compared with age-matched controls (PPAR␣ϩ/ϩ). Previous studies have shown that mitochondrial and peroxisomal fatty acid ␤-oxidation cycle enzyme gene expression is reduced in the liver of PPAR␣Ϫ/Ϫ mice (28,38). Northern blot studies demonstrated that steady-state levels of M-CPT I mRNA are significantly lower (by 51 Ϯ 9%) in the hearts of adult PPAR␣Ϫ/Ϫ mice compared with controls (Fig. 6A). The studies were repeated following a 5-day administration of etomoxir, a known activator of PPAR␣. As expected, etomoxir induced myocardial expression of M-CPT I mRNA in PPAR␣ϩ/ϩ mice (194 Ϯ 15 versus 100 Ϯ 7). In contrast, the PPAR activator did not induce M-CPT I gene expression in the hearts of PPAR␣Ϫ/Ϫ mice (64 Ϯ 10 versus 58 Ϯ 10) (Fig. 6B). These results confirm the role of PPAR␣ as an activator of M-CPT I gene expression in heart in vivo.

DISCUSSION
The energy substrate preference of the mammalian heart is tightly controlled during development and in response to diverse physiologic and pathophysiologic conditions (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The fetal heart produces energy primarily through catabolism of glucose and lactate (1). Following birth, the mammalian heart switches to fatty acids as the chief energy substrate. Compared with glucose, the oxidation of fatty acids provides more ATP per mole of substrate, albeit at the expense of increased oxygen consumption. Thus, fatty acid oxidation provides a greater capacity for energy production to meet the physiologic demands imposed on the postnatal mammalian heart. 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 (4 -11). Previous studies have shown that expression of nuclear genes encoding mitochondrial fatty acid ␤-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 (12)(13)(14)39). 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, PPAR␣. 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 (2,3). The cardiac expression of these enzymes falls upon weaning in rats and mice (13,26,39). Second, long-chain fatty acids induce expression of CPT I in hepatocytes and pancreatic islet cells in culture (40,41). Third, FAO enzyme gene expression is induced by fasting in liver and heart (39). The results shown here demonstrate that M-CPT I gene transcription is activated by long-chain fatty acids via FARE-1, a PPAR␣ 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 PPARϪ/Ϫ 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 PPAR␣, comprise a metabolic signaling pathway in heart.
The role of PPAR␣ in the control of hepatic lipid metabolism is well established. PPAR␣ was first identified as a transcription factor involved in the hepatic response to peroxisome pro-liferators (42). The expression of PPAR␣ target genes encoding enzymes involved in peroxisomal, cytochrome P450, and mitochondrial FAO is reduced in the liver of mice null for PPAR␣ (28). Further, the expected induction of peroxisomal PPAR␣ target enzymes in response to peroxisome proliferators is absent in PPAR␣Ϫ/Ϫ mice (28). In addition to liver, PPAR␣ is expressed abundantly in tissues with high capacity for fatty acid oxidation such as heart, kidney, and brown adipose tissue. The role of PPAR␣ in extrahepatic tissues has not been characterized. Our results demonstrate one important function for PPAR␣ 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 cardiacenriched mitochondrial FAO cycle enzyme medium-chain acyl-CoA dehydrogenase contains a PPAR␣ response element (30), our results indicate that PPAR␣ 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 PPAR␣. 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.
FIG. 6. Altered M-CPT I gene expression in heart of PPAR␣؊/؊ mice. A, top, autoradiogram of a representative Northern blot analysis performed with total RNA isolated from the hearts of PPAR␣Ϫ/Ϫ mice or age-matched control wild-type mice (ϩ/ϩ). The blot was hybridized with the radiolabeled cDNA probes denoted on the left. Bottom, bars represent mean (Ϯ S.E.) laser densitometric quantification of band intensities normalized first to 18 S rRNA levels and second to results from PPAR␣ϩ/ϩ mice (ϭ100%). B, top, an autoradiogram of a representative Northern blot analysis of total RNA isolated from the hearts of PPAR␣Ϫ/Ϫ mice (Ϫ/Ϫ) or age-matched controls (ϩ/ϩ) treated with the PPAR␣ activator/CPT I inhibitor etomoxir (Eto, 50 mg/kg body weight) or vehicle (Veh). The blot was hybridized with the radiolabeled cDNA probes denoted on the left. Bottom, bars represent mean (Ϯ S.E.) laser densitometric quantification of band intensities normalized first to 18 S rRNA levels and second to results in vehicle-treated PPAR␣ϩ/ϩ mice (ϭ100). The values in A and B represent the mean signal obtained from 15 g of total RNA isolated from at least three different animals.