Carnitine palmitoyltransferase I (CPT-1) catalyzes the rate-limiting step in mitochondrial FA oxidation. Catalytic activity of previously identified CPT-I enzymes (B 1 ['muscle'] & CPT-IA ['liver']) can be completely suppressed by malonyl-CoA, the concentration of which is governed by glucose availability, cell energy state, and pancreatic endocrine hormones. This mechanism effects reciprocal glucose vs FA utilization. The co-residence of CPT-I N- and C-termini in the cytosol necessary for regulation by malonyl-CoA is achieved by enzyme polytopy in the outer mito membrane (OMM): CPT-IA and B 1 have N-terminal hybrid mito targeting/stop transfer signals with 2 transmembrane domains (TMD). We hypothesized the existence of and found additional CPT-Is that may account for perpetually active cardiac FA oxidation. Up to 30 percent of cardiac CPT-I mRNA is the novel B2 variant, a product of alternative CPT-IB splicing. The encoded B2 isozyme has intact mito leader and catalytic domains, but only onecandidate TMD, and overexpressed isozyme is insensitive to mal-CoA. Cardiac expression of B2 is induced during the perinatal period. The objective of this project is to ascertain the role of CPT-I isozymes in cellular fuel metabolism. Kinetic features of rat heart mito CPT-I will be assessed before and after B2 expression. Observations will be compared with predictions based on isozyme abundance as judged by immunoblots using isoform-specific antibodies, and activities of each CPT-I isozyme when overexpressed using recombinant adenoviruses. The impact of CPT-IB isozyme expression on cell metabolism will be determined using cardiocytes pre- and post-B2 expression, and isozyme-complemented CPT-I-deficient fibroblasts. [14C]-FA oxidation rates will be assessed as a function of cellular mal-CoA content, to be modulated by providing medium glucose, FA, and insulin over a range of physiological concentrations. The basis of differential CPT-I isozyme sensitivity to mal-CoA will be assessed using radioligand binding assays with mitos from cells expressing each isoform. This will be correlated with isozyme submito loci and topology in parallel strategies: 1. Efficacy of Sepharose-coupled substrate and mal-CoA (which are cytosol-restricted) on isozyme activity; 2. Protease sensitivity of [35S]-CPT-I isozymes and derivative fusion proteins after in vitro mito import; and 3. N- and C-terminal epitope:antibody interactions. CPT-IB minigene reporters that specifically detect B2 splicing will be used to map intronic and exonic splicing enhancers as a first step in the analysis of alternative CPT-IB splcing. We hypothesize that the previously unrecognized B2 isozyme contributes to ceaseless brisk cardiac FA oxidation despite [mal-CoA] that vastly exceeds the Ki of the known enzymes, and to the partial uncoupling of FA oxidation from glucose availability in this tissue.
