Carnitine palmitoyltransferase (CPT) is a regulatory membrane enzyme which controls the entry of long-chain fatty acids into the mitochondria for beta-oxidation. The regulation of fatty acid metabolism is a focus of nutritional and pharmacological investigation since fatty acids represent a significant energy source in American diets. Further, CPT has generated clinical interest because of the potential to modify it's activity and produce clinical improvement in cardiac ischemia, diabetes, and trauma/ sepsis, as well as in CPT deficiency. Previous studies in vivo have shown that CPT activity and synthesis increased in starvation, diabetes, with administration of hypolipidemic drugs, in riboflavin deficiency, and with increased dietary fat. In vivo and in vitro studies have shown that increased CPT synthesis is correlated with increased transcription rate of CPT mRNA from DNA and increased CPT mRNA concentrations. In H4IIE cells and hepatocytes CPT mRNA is induced by peroxisomal proliferators, cAMP, dexamethasone, epinephrine, fatty acids and dicarboxylic acids, and repressed by insulin. The first goal of the present proposal is to identify the 5'regulatory response unit of the CPT genomic DNA and to compare it to response units of other enzymes that respond to cAMP and dexamethasone. Since CPT transcription is also induced by fatty acids, we will define the fatty acid responsive element(s) of the CPT genome. The second goal is to assess the impact of specific CPT regulation on fatty acid metabolism. This will be accomplished by infection of rat and human hepatoma cell lines, as well as CPT-deficient human fibroblast lines, with retroviral vector constructs containing the phosphoenolpyruvate carboxykinase (PEPCK) promoter, the mouse mammary tumor virus (MMTV) promoter or the metallothionein promoter with a CPT structural gene (sense constructs) or CPT antisense constructs. Selection of the cells for incorporation of the gene construct will be by resistance to G418 (neo), since the neo element can be included in all of the vectors. CPT over- or under-expression should have characteristic specific effects on lipid synthesis and lipid oxidation. These experiments should give us further insight into the specific function and regulation of carnitine palmitoyltransferase and provide a framework for the study of the regulation of the peroxisomal carnitine octanoyltransferase, and peroxisomal and mitochondrial carnitine acetyltransferase in the future. The proposed studies should also help to define specific conditions under which it would be metabolically fruitful to insert a CPT gene construct into liver.
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