NADPH-Dependent Metabolism of Arachidonic Acid

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DESCRIPTION (provided by applicant): The cytochrome P450 (P450) arachidonic acid (AA) epoxygenases and omega-hydroxylases participate in the in vivo metabolism of AA, to epoxyeicosatrienoic (EET) and 20-hydroxyeicosatetraenoic (20HETE) acids. The EETs and 20-HETE, have been identified as mediators of vascular reactivity, ion transport, and transmembrane signaling, and alterations in CYP2C and 4A function associated with the pathophysiology of cardiovascular disease. Genetic models of P450 dysfunction show that P450 expression levels and product selectivity are important determinants of EET and 20-HETE functional role, site, and mechanism of action. As master transcriptional regulators of fatty acid and lipid metabolism, the peroxisome proliferator-activated nuclear receptors (PPARs) play important roles in the control of fatty acid metabolism and body lipolytic/lipogenic balance. Interest in the identification and functional characterization of the PPARs endogenous ligands stem from their documented roles in dyslipidemias, obesity, cancer, diabetes, and cardiovascular diseases. Hepatic fatty acid beta-oxidation and CYP4A omega/omega-1hydroxylations, are under regulatory control by the PPAR alpha-subtype, and the CYP4A omega-hydroxylated EETs have been identified as PPARalpha ligands and activators. Disruption of the Cyp4a14 gene impairs liver fatty acid metabolism, and causes plasma and liver dyslipidemia, and hepatic steatosis. Based on this, we propose that: a) the omega-hydroxylated EETs (HEETs,) are endogenous PPARalpha ligands, and b) the AA CYP2C epoxygenase and 4A omega-hydroxylases participate in their biosynthesis, and in the control of hepatic fatty acid oxidation and lipolysis. To test these hypotheses, we are proposing to: a) characterize the enzymatic mechanisms of HEET formation in vivo, and b) to study the role of Cyp4a isoforms in hepatic lipid metabolism and PPARalpha signaling. To accomplish these goals we propose to utilize a combination of analytical, chemical, biochemical, and molecular approaches develop by this project, in order to define the role of these reactions in endogenous lipid homeostasis, and the mechanisms of action their metabolites. The identification and characterization of endogenous regulators of PPAR activity should contribute to a better understanding of their physiological and pathophysiological significance, and facilitate the development of novel pharmacological targets for clinical intervention.