Co-regulation of tissue-specific alternative human carnitine palmitoyltransferase Iβ gene promoters by fatty acid enzyme substrate

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining step in mitochondrial fatty acid beta-oxidation. CPT-I has two structural genes (alpha and beta) that are differentially expressed among tissues. Our CPT-I beta isolates from a human cardiac cDNA library contained two different extreme 5'-sequences derived from short alternative first untranslated exons that utilize a common splice acceptor site in exon 2. Primer extension identified single dominant start sites for each transcript, and ribonuclease protection assays showed the presence of one 5'-exon in liver, muscle, and heart mRNAs, indicating that the cognate promoter U (upstream/ubiquitous) is active in each of these tissues. By contrast, mRNAs containing the alternative 5'-exon were present only in muscle and heart, indicating a muscle-specific promoter M (muscle). CPT-I beta mRNA levels increased markedly in tissues of fasted rats, when circulating free fatty acid concentrations are elevated. Using CPT-I beta promoter/reporter transient transfection of murine C2C12 myotubes and HepG2 hepatocyte, fatty acids were found to increase promoter activity in a peroxisome proliferator-activated receptor alpha (PPAR alpha)-dependent fashion. A promoter fatty acid response element (FARE) was mapped, mutation of which ablated fatty acid-mediated production of both transcripts. PPAR alpha/retinoid X receptor a formed specific complexes with oligonucleotides containing the FARE, and anti-PPAR alpha antibody shifted nuclear protein-DNA complexes, confirming the role of this factor in regulating the expression of this critical metabolic enzyme gene. The constitutive repressor chicken ovalbumin upstream promoter transcription factor competitively binds at the FARE and modulates fatty acid induction of the promoters.