Role of Hepatic GDPD3 in Mechanisms of Lipid Metabolism

项目总结/摘要 非酒精性脂肪性肝病（NAFLD）影响约30%的美国人口，预计将取代 到2020年，丙型肝炎将成为肝移植的主要原因。开发有效的NAFLD治疗是 由于对其基本机制的理解不足，包括 遗传和环境因素。含甘油磷酸二酯磷酸二酯酶结构域蛋白3 GDPD 3是一种新发现的具有溶血磷脂酶D活性的酶， 溶血磷脂酸（lysoPA）在非肝细胞。哺乳动物GDPD3以前未涉及 肝脏脂质代谢。我们的初步数据表明，人类GDPD3 表达和甘油三酯（TG）积累在肝细胞和小鼠肝脏，表明一种新的基因，在肝细胞和小鼠肝脏中， 调节肝脏TG稳态。尽管如此，细胞内的位置，底物特异性， 人GDPD 3在肝细胞/肝脏中的生理功能和分子机制尚不清楚。 因此，在本研究中，在经验丰富的多学科指导小组的指导下，我们 我建议研究人GDPD3的酶特性，并探索人GDPD3是否是致病的 肝脂肪变性的基因更具体地说，我们提出了三个问题：1）人类GDPD 3是否是一种 内质网膜相关酶含有溶血磷脂酶D活性？2)并 人GDPD 3通过增加lysoPA产生，导致肝TG合成和积累增加 甘油磷酸途径3)人GDPD3产生的lysoPA是否激活过氧化物酶体增殖物- 活化受体γ（PPAR γ），通过增加脂肪酸（FA）摄取增强肝脂肪变性， TG合成？为了回答这些问题，我们将在肝癌细胞系中过表达人GDPD 3，并在肝癌细胞系中过表达人GDPD 3。 a）人GDPD 3过表达对油酸诱导的TG的影响 在小鼠中肝细胞瘤细胞中的积累和饮食诱导的肝脂肪变性; B） 肝癌细胞和小鼠原代肝细胞中的人GDPD 3;和c）量和分子种类 GDPD3脂质底物和产物在小鼠肝脏中的表达。肝脏特异性人GDPD3过表达小鼠 在PPAR γ功能丧失或功能获得的患者中，将喂食普通饲料或西式饮食以诱导肝细胞凋亡。 脂肪变性我们将对这些小鼠进行全面的肝脏和全身代谢表型分析。初级 肝细胞将用于研究从头脂肪生成、FA摄取和掺入TG、FA 氧化和使用放射性同位素的极低密度脂蛋白-TG分泌。当提出的目标 实现，我们将有一个更好的机制理解人类GDPD3和 肝脂肪变性，以解决关于NAFLD发病机制的知识的差距，并为其提供策略， 治疗最后，本建议书为申请人提供必要的培训和指导， 过渡到一个成功的职业生涯作为一个独立的研究者在脂质和葡萄糖代谢。

PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD), affecting ~30% of the U.S. population, is projected to replace hepatitis C as the leading cause of liver transplantation by 2020. Developing effective NAFLD treatments is hampered by a poor understanding of its underlying mechanisms, including complex interactions between genetic and environmental factors. Glycerophosphodiester phosphodiesterase domain-containing protein 3 (GDPD3) is a newly discovered enzyme containing lysophospholipase D activity that converts lysophospholipid to lysophosphatidic acid (lysoPA) in non-hepatic cells. Mammalian GDPD3 has not previously been implicated in hepatic lipid metabolism. Our preliminary data indicates a positive correlation between human GDPD3 expression and triglyceride (TG) accumulation in hepatocytes and mouse livers, suggesting a novel gene in the regulation of hepatic TG homeostasis. Nonetheless, the intracellular locations, substrate specificity, physiological function, and molecular mechanisms of human GDPD3 in hepatocytes/livers are unknown. Therefore, in this study, with the guidance of a highly experienced multi-disciplinary mentoring group, we propose to investigate enzymatic properties of human GDPD3 and explore whether human GDPD3 is a causal gene for hepatic steatosis. More specifically, we are asking three questions: 1) Is human GDPD3 an endoplasmic reticulum membrane-associated enzyme containing lysophospholipase D activity? 2) Does human GDPD3 increase lysoPA production resulting in increased hepatic TG synthesis and accumulation via the glycerol phosphate pathway? 3) Does human GDPD3-produced lysoPA activate peroxisome proliferator- activated receptor gamma (PPARγ) which enhances hepatic steatosis via increased fatty acid (FA) uptake and TG synthesis? To answer these questions, we will overexpress human GDPD3 in hepatoma cell lines and in mouse liver to determine: a) the effect of human GDPD3 overexpression on oleic acid-induced TG accumulation in hepatoma cells and diet-induced hepatic steatosis in mice; b) the subcellular localization of human GDPD3 in hepatoma cells and mouse primary hepatocytes; and c) the amount and molecular species of GDPD3 lipid substrates and products in mouse livers. Liver-specific human GDPD3 overexpressing mice with loss-of-function or gain-of-function in PPARγ will be fed chow or a Western-type diet to induce hepatic steatosis. We will perform comprehensive hepatic and systemic metabolic phenotyping on these mice. Primary hepatocytes will be used to investigate de novo lipogenesis, FA uptake and incorporation into TG, FA oxidation, and very low density lipoprotein-TG secretion using radioactive isotopes. When the proposed aims are achieved, we will have a better mechanistic understanding of the relationship between human GDPD3 and hepatic steatosis, to address the gap in knowledge regarding NAFLD pathogenesis and inform strategies for its treatment. Finally, this proposal provides the necessary training and mentored guidance for the applicant to transition to a successful career as an independent investigator in lipid and glucose metabolism.