NADPH-Dependent Metabolism of Arachidonic Acid

描述（由申请人提供）：细胞色素P450 （P450）花生四烯酸（AA）环氧化酶和omega-羟化酶参与AA在体内代谢为环氧二十碳三烯酸（EET）和20-羟基二十碳四烯酸（20HETE）。eet和20-HETE已被确定为血管反应性、离子转运和跨膜信号传导的介质，以及与心血管疾病病理生理相关的CYP2C和4A功能改变的介质。P450功能障碍的遗传模型表明，P450表达水平和产物选择性是EET和20-HETE功能作用、位点和作用机制的重要决定因素。过氧化物酶体增殖体激活核受体（PPARs）是脂肪酸和脂质代谢的主要转录调控因子，在脂肪酸代谢和体内脂质代谢平衡中发挥重要作用。对ppar内源性配体的鉴定和功能表征的兴趣源于它们在血脂异常、肥胖、癌症、糖尿病和心血管疾病中的作用。肝脂肪酸β -氧化和CYP4A ω / ω -1羟基化受PPAR α亚型的调控，CYP4A ω -羟基化eet已被确定为PPAR α配体和激活剂。Cyp4a14基因的破坏会损害肝脏脂肪酸代谢，导致血浆和肝脏血脂异常，以及肝脂肪变性。基于此，我们提出：a) omega-羟基化EETs （HEETs）是内源性ppar - α配体，b) AA CYP2C环氧化酶和4A omega-羟化酶参与其生物合成，并控制肝脏脂肪酸氧化和脂肪分解。为了验证这些假设，我们建议：a)表征体内HEET形成的酶促机制，b)研究Cyp4a亚型在肝脏脂质代谢和ppar α信号传导中的作用。为了实现这些目标，我们建议利用本项目开发的分析、化学、生化和分子方法的结合，以确定这些反应在内源性脂质稳态中的作用，以及它们的代谢产物的作用机制。PPAR活性内源性调节因子的鉴定和表征将有助于更好地了解其生理和病理生理意义，并促进临床干预的新药理靶点的开发。

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.