NADPH-Dependent Metabolism of Arachidonic Acid

描述（由申请方提供）：细胞色素P450（P450）花生四烯酸（AA）环氧合酶和ω-羟化酶参与AA体内代谢为环氧二十碳三烯酸（EET）和20-羟基二十碳四烯酸（20 HETE）。已确定Ehrs和20-HETE是血管反应性、离子转运和跨膜信号传导以及与心血管疾病病理生理学相关的CYP 2C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。过氧化物酶体增殖物激活核受体（peroxisome proliferator-activated nuclear receptor，PPARs）作为脂肪酸和脂质代谢的主要转录调节因子，在脂肪酸代谢和体内脂肪分解/脂肪生成平衡的调控中发挥重要作用。对PPARs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记录作用。肝脂肪酸β-氧化和CYP 4A omega/omega-1羟基化受PPAR α亚型的监管控制，CYP 4A omega-羟基化雌二醇已被鉴定为PPAR α配体和激活剂。Cyp 4a 14基因的破坏损害肝脏脂肪酸代谢，并引起血浆和肝脏血脂异常和肝脂肪变性。基于此，我们提出：a）ω-羟基化的E3（HE 3）是内源性的PPARalpha配体，和B）AA CYP 2C环氧酶和4A ω-羟基化酶参与其生物合成，并参与肝脂肪酸氧化和脂解的控制。为了验证这些假设，我们建议：a）表征体内HEET形成的酶机制，和B）研究Cyp 4a亚型在肝脂质代谢和PPARalpha信号传导中的作用。为了实现这些目标，我们建议利用该项目开发的分析、化学、生物化学和分子方法的组合，以定义这些反应在内源性脂质稳态中的作用及其代谢物的作用机制。识别和表征的内源性调节因子的过氧化物酶体增殖物激活受体的活性，应有助于更好地了解其生理和病理生理意义，并促进新的药理学靶点的临床干预的发展。

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.