The studies outlined in this proposal have as a long-term objective the elucidation of mechanisms whereby the liver cell adapts, on a molecular level, to increased fatty acid flux. The proposed studies will determine the mechanisms whereby the microsomal, peroxisomal and mitochondrial pathways of fatty acid metabolism interact in response to high fluxes of long-chain fatty acids caused by nutritional, hormonal and pharmacological factors. The peroxisomes may be the sole sites of metabolism of dicarboxylic fatty acids formed via the microsomal cytochrome P-450 IVA1- mediated omega-hydroxylation, and of very long-chain fatty acids. Elevated liver and blood levels of dicarboxylic and very long-chain fatty acids are seen in clinical disorders of peroxisomal and mitochondrial beta-oxidation such as Zellweger's and Reye's syndromes, as well as in diabetes and starvation, and may contribute significantly to the morbidity of these disorders. Induction of peroxisomal beta-oxidation occurs after exposure to peroxisome proliferators (PP). These agents inhibit mitochondrial beta- oxidation while they induce P-450 IVA1, the dicarboxylic fatty acid products of which may be mediators of the induction of peroxisomal beta- oxidation. The proposed studies are designed to further elucidate the mechanisms of induction of proxisomal beta-oxidation by PP and dicarboxylic fatty acids.
Specific Aims : 1) Utilizing primary hepatocyte cultures, to elucidate the role of fatty acid metabolites in the induction of peroxisomal fatty acyl-CoA oxidase (FAO). 2) Determine the role of inositol phospholipid signal transduction pathway in the PP- and dicarboxylic fatty acid-mediated induction of FAO. 3) Define the role of soluble proteins/receptors in mediating the effects of PP and dicarboxylic fatty acids. Experimental Design and Methods: Using primary hepatocyte cultures, the effects of selective inhibition of the enzymes of mitochondrial and microsomal fatty acid oxidation pathways on PP-mediated induction of FAO will be used to determine the role of fatty acid metabolites such as acyl-carnitines or dicarboxylic fatty acids in induction of peroxisomal beta-oxidation. The effects of fatty acids of varying chain-length and of dicarboxylic fatty acids on P450 IVA1 and on FAO will also be directly determined. The time course of gene activation of P450 IVA1 and FAO will also be established. The role of protein kinase C in mediating PP effects will be determined by establishing the effects of a) PP and fatty acids on protein kinase C activity in cell culture, b) inhibitors of protein kinase C on the dicarboxylic fatty acid and PP- mediated induction of FAO and P-450 IVA1. Since the effects of PP may be mediated by cytosolic receptor, proteins in high binding affinity for PP and for fatty acids will be isolated using affinity chromatography. Evidence for the occurrence of nuclear translocation of a radiolabeled PP will also be sought as an indirect evidence for the presence of a receptor.
