Control of Human Muscle-type Carnitine Palmitoyltransferase I Gene Transcription by Peroxisome Proliferator-activated Receptor

The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial β-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5′-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy. The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial β-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5′-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy. The incorporation of activated long-chain fatty acids into the mitochondria to be catabolized through β-oxidation is produced by the mitochondrial carnitine palmitoyltransferase (CPT) 1The abbreviations used are: CPT, carnitine palmitoyltransferase; CAT, chloramphenicol acetyltransferase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-responsive element; RXR, retinoid X receptor; PCR, polymerase chain reaction; hRXRα, human 9-cis-retinoic acid receptor α; TK, thymidine kinase; NIDDM, non-insulin-dependent diabetes mellitus; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA. enzyme system. CPT I, the outer membrane component of this system, is the main control point in the β-oxidation pathway. CPT I is thus a suitable site for pharmacological control of fatty acid oxidation in conditions such as diabetes or heart diseases. Two isoforms of CPT I have been described, which have been designated LCPT I and MCPT I since these isoforms are mainly expressed in liver and muscle respectively. The MCPT I gene is expressed not only in skeletal muscle but also in heart and brown and white adipose tissue (1Weiss B.C. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 18712-18715Abstract Full Text PDF PubMed Google Scholar, 2Yamazaki N. Shinohara Y. Shima A. Terada H. FEBS Lett. 1995; 363: 41-45Crossref PubMed Scopus (112) Google Scholar, 3Yamazaki N. Shinohara Y. Shima A. Yamanaka Y. Terada H. Biochim. Biophys. Acta. 1996; 1307: 157-161Crossref PubMed Scopus (102) Google Scholar, 4Esser 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). This expression pattern may be of great significance since fatty acids are a major source of energy for heart, skeletal muscle, and brown adipose tissue. The CPT I gene expression is regulated by fatty acids and peroxisome proliferators (5Asins G. Serra D. Hegardt F.G. Biochem. Pharmacol. 1994; 47: 1373-1379Crossref PubMed Scopus (7) Google Scholar, 6Chatelain 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). To gain more insight into the control of CPT I gene expression by fatty acids, we have examined the transcriptional regulation of CPT I genes. The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial β-oxidation, is mediated by ligand-activated receptors collectively referred to as peroxisome proliferator-activated receptors (PPARs); these receptors are members of the nuclear receptor superfamily. PPARs are activated by a wide array of peroxisome proliferators and also by natural and synthetic fatty acids (7Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1870) Google Scholar, 8Kliewer S.A. Sundseth S.S. Jones S.A. Broen 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), antidiabetic drugs (9Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkinson W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar, 10Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2740) Google Scholar), prostaglandin J2 (10Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2740) Google Scholar), and leukotriene B4 (11Devchand P.R. Keller H. Peters J.M. Vázquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1214) Google Scholar). We have amplified by polymerase chain reaction (PCR) the 5′ region of the human heart and brown adipose tissue CPT I gene and demonstrate, first, the transcriptional activity of this fragment and, second, the presence of a PPRE in the 5′-flanking region of this gene. In CV1 cells, the activation of the CPT I gene by PPAR was dependent on the addition of exogenous ligands. pCPTCAT, containing an 882-base pair fragment of the human MCPT I gene, was constructed by the application of the PCR using a pair of oligonucleotide primers, CPTF (5′-CCTGGCTGCAGCTTAGAATAA) and CPTR (5′-GGAGTTGATCCCAGACAGG TAGAC), corresponding to coordinates −909 to −889 and +126 to +92, respectively, of the human MCPT I gene (12Yamazaki N. Yamanka Y. Hashimoto Y. Shinohara Y. Shima A. Terada H. FEBS Lett. 1997; 409: 401-406Crossref PubMed Scopus (56) Google Scholar) and human genomic DNA as a template. The PstI-AvrII-digested PCR product was cloned into the PstI-XbaI sites of chloramphenicol acetyltransferase (CAT) vector pCAT-BASIC reporter gene (Promega). To confirm the sequence, the PCR-amplified fragment was automatically sequenced using the fluorescent terminator kit (Perkin-Elmer). Heterologous promoter plasmids were constructed in the herpesvirus thymidine kinase gene promoter upstream of the CAT reporter gene pBLCAT2 (13Luckow B. Schütz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). pTKCATCPT contains a fragment corresponding to coordinates −774 to −755 of the mitochondrial HMG-CoA synthase gene. It was constructed by cloning the oligonucleotide 5′-agctTGACCTTTTCCCTACATTTG annealed to 5′-tcgaCAAATGTAGGGAAAAGGTCA into pBLCAT2 (nucleotides designated in lowercase were added to provide cohesive HindIII-SalI ends at the 5′ and 3′ termini, respectively). The insert in this plasmid had the same 5′ → 3′ orientation as found in the human MCPT I gene promoter. DNA sequence analysis, by the fluorescent terminator kit was performed to confirm insert orientation. CV1 cells were cultured in minimal essential media supplemented with 10% fetal calf serum. Cells were typically cotransfected with 10 μg of the reporter MCPT I-CAT gene construct and, when indicated, with 1 μg of effector plasmids expressing full-length cDNAs for mouse PPARα, PPARγ2, or PPARδ. 4 μg of plasmid pRSVβGAL (Rous sarcoma virus promoter β-galactosidase) was included as internal control in cotransfections. Transfection experiments were carried out by the calcium-phosphate method as described (14Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates/Wiley-Interscience, New York1987: 9.1.4-9.1.6Google Scholar, 15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.66-16.67Google Scholar). After removal of the calcium-phosphate-DNA precipitate, cells were re-fed with medium supplemented with 10% delipidated calf serum. Experiments with ligand included either vehicle (dimethyl sulfoxide or ethyl alcohol) or ligand (10 μm PGJ2 (15-deoxy-Δ12,14-prostaglandin J2), 30 μm LY-171883, or 30 μm linoleic acid). All ligands used were from Sigma. Cells were harvested 48 h after re-feeding. Extracts of harvested cells were prepared by liquid nitrogen freeze/thaw disruption (three times) after resuspension in 100 μl of 0.25 m Tris-HCl, pH 7.5. β-Galactosidase activity was determined (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.66-16.67Google Scholar) in a 10–20-μl volume of extract to normalize for transfection efficiency. All samples assayed for CAT activity were first incubated at 65 °C for 5 min. CAT assays were performed (14Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates/Wiley-Interscience, New York1987: 9.1.4-9.1.6Google Scholar) for 60 min. Radioactivity of samples was measured on an LKB-1217 liquid scintillation counter. cDNAs for the receptors (mouse PPARα, PPARγ2, PPARδ, and human RXRα) were transcribed and translated by using a commercially available kit according to the instructions of the manufacturer (Promega). 2 μl of mPPARα, mPPARγ, and mPPARδ with or without hRXRα (2 μl) synthesizedin vitro were preincubated on ice for 10 min in 10 mm Tris-HCl, pH 8.0, 40 mm KCl, 0.05% (v/v) Nonidet P-40, 6% glycerol, 1 mm dithiothreitol, and 2 μg of poly(dI-dC). The total amount of reticulocyte lysate was kept constant in each reaction (4 μl) by the addition of unprogrammed lysate. For competition experiments, a 25–100-fold molar excess of MCPT I PPRE or MCPT I MPPRE double-stranded probes, relative to the labeled probe, was included during preincubation. MCPT I PPRE is the fragment corresponding to coordinates −774 to −755 of the MCPT I gene, which was used to prepare pTKCATCPT. MCPT I MPPRE is the fragment corresponding to coordinates −782 to −748 of the MCPT I gene, but the nucleotides corresponding to the PPAR binding sequence have been mutated (CACATCGGTGACCctcgagggatccTTGGCTATTT, nucleotides described in lowercase correspond to those that have been changed from the wild type sequence). Next, 2 ng of MCPT I PPRE, 32P-labeled by fill-in with Klenow polymerase, was added, and the incubation was continued for 15 min at room temperature. The final volume for all reactions was 20 μl. Samples were electrophoresed at 4 °C on a 4.5% polyacrylamide gel in 0.5× TBE buffer (45 mm Tris, 45 mm boric acid, 1 mm EDTA, pH 8.0). PPAR α, γ, and δ bind to the MCPT I PPRE as heterodimers with RXR. To elucidate the control of CPT I gene expression by fatty acids, we have examined the transcriptional regulation of CPT I genes. A BLAST search performed using the NCBI BLAST WWW Server revealed that the sequence for the human muscle type CPT I gene was included in the sequence of a BAC clone containing a part of the q arm of chromosome 22 (GenBankTM accession number U62317). The analysis of the 5′-flanking region of this gene by the TFSEARCH routine, performed using the Kyoto Center's GenomeNet WWW Server, shows the presence of a putative PPAR binding sequence upstream of exon 1A. The comparison of this sequence with the consensus sequence required for the binding of the PPAR-RXR heterodimer, as proposed by Palmer et al. (16Palmer C.N.A. Hsu M-H. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), shows the coincidence of 17 out of 20 bases (Fig. 1). We performed gel mobility shift assays to analyze whether PPAR-RXR heterodimers bind to the putative PPAR binding sequence of the human muscle type CPT I gene. As can be seen in Fig. 2 neither PPARs nor RXR alone binds significantly to this sequence. However, incubation of this probe with a mixture of PPAR (α, β, or γ) and RXRα resulted in a prominent complex. An oligonucleotide containing a mutated PPRE was not able to compete with the wild-type probe for the formation of the complex. The binding of the three subtypes of PPAR to the MCPT I PPRE is as strong as the binding to the mitochondrial HMG-CoA synthase PPRE, which allows the formation of the strongest complexes for all PPAR subtypes (17Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) (data not shown).Figure 2Electrophoretic mobility shift assay of the muscle CPT I PPRE with PPAR-RXR heterodimers. PPAR α, γ, and δ and RXRα were translated in vitro, incubated with the proposed CPT I PPRE labeled probe, and analyzed by electrophoretic mobility shift assay. Additions were as indicated on the topof the figure. Shown in panel B is a competition of the complex PPARα-RXR-PPRE with a 25–100-fold molar excess of two different unlabeled oligonucleotides: MCPT I PPRE, containing the proposed PPRE, or MCPT I MPPRE, with the proposed PPRE mutated. All isoforms of PPAR are identically competed (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the effect of the observed binding of PPAR to the human MCPT I gene promoter on its transcriptional activity, we made constructs in which the 5′-flanking region of this gene was linked to a promoter-less bacterial CAT gene. These plasmids were introduced into cultured CV1 cells by the calcium-phosphate method, with or without an expression vector for PPARs, together with a plasmid that contains the β-galactosidase coding region driven by the SV40 promoter as a control of the efficiency of the transfection. Following transfection, cells were incubated in the presence or absence of a PPAR activator, and after 48 h, the cells were harvested and CAT activity measured. As can be seen in Fig. 3 cotransfection of PPAR expression vectors lead to a marked increase in CAT activity in the presence of the PPAR activators. Surprisingly, even though PPARδ is able to bind the MCPT I PPRE in vitro, it does not activate the expression of the chimeric gene even in the presence of linoleic acid as activator. Next a pair of oligonucleotides containing the human MCPT I PPRE were inserted into pBLCAT2, a plasmid containing the CAT gene under the control of the thymidine kinase gene promoter. As can be seen in Fig. 4, this sequence conferred PPAR responsiveness to the otherwise unresponsive thymidine kinase gene promoter. The results demonstrate that this human MCPT I element is able to confer PPARα and γ responsiveness both on its natural context and on a normally unresponsive promoter. Our data provide evidence that extends the influence of PPARs in the regulation of mitochondrial fatty acid metabolism. They influence not only activation, through the control of acyl-CoA synthetase (18Schoonjans K. Watanabe M. Suzuki H. Mahfoudi A. Krey G. Wahli W. Grimaldi P. Staels B. Yamamoto T. Auwerx J. J. Biol. Chem. 1995; 270: 19269-19276Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), β-oxidation, through medium-chain acyl-CoA dehydrogenase (19Gulick T. Cresce 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), and ketogenesis, through mitochondrial HMG-CoA synthase (20Rodrı́guez J.C. Gil-Gómez G. Hegardt F.G. Haro D. J. Biol. Chem. 1994; 269: 18767-18772Abstract Full Text PDF PubMed Google Scholar), but also, mitochondrial import through CPT I (Fig. 5). These results also support the suggestion that in higher organisms, as well as in bacteria and yeast, there is metabolic control of gene expression. Non-insulin-dependent diabetes mellitus (NIDDM) affects between 5 and 20% of the population in Western industrialized societies (21Harris M.I. Diabetes Care. 1989; 12: 464-474Crossref PubMed Scopus (189) Google Scholar), but despite decades of research, the pathogenesis of NIDDM remains incompletely understood. It has recently been suggested that NIDDM may have more to do with abnormalities in fat than in carbohydrate metabolism (22McGarry J.D. Science. 1992; 258: 766-770Crossref PubMed Scopus (578) Google Scholar). There is evidence that free fatty acids are an important link between obesity and insulin resistance and NIDDM (reviewed in Ref. 23Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar). There is also evidence that the antidiabetic action of the thiazolidinediones (insulin sensitizers that significantly reduce glucose, lipid, and insulin levels in animal models of NIDDM and obesity) are directly mediated through binding to PPARγ and the resulting active conformation of the receptor (24Berger J. Bailey P. Biswas C. Cullinan C.A. Doebber T.W. Hayes N.S. Saperstein R. Smith R.G. Leibowitz M.D. Endocrinology. 1996; 137: 4189-4195Crossref PubMed Scopus (346) Google Scholar), whose expression is high in the skeletal muscle of obese and type II diabetic subjects (25Park K.S. Ciaraldi T.P. Abrams-Carter L. Mudaliar S. Nikoulina S.E. Henry R.R. Diabetes. 1997; 46: 1230-1234Crossref PubMed Google Scholar). Our hypothesis is that the transcriptional control of the muscle type CPT I gene produced by thiazolidinedione-activated PPARγ may contribute to the antidiabetic effect of these agents by controlling glucose utilization in skeletal muscle through modulation of fatty acids catabolism in such cells, and studies to examine this hypothesis are now under way. We are indebted to Drs. Ronald M. Evans, Stephen Green, and Bruce M. Spiegelman for supplying the expression vectors for RXRα and PPARδ, PPARα, and PPARγ, respectively. We are also grateful to Robin Rycroft of the Language Service, University of Barcelona, for valuable assistance in the preparation of the English manuscript.