Role of Hepatic GDPD3 in Mechanisms of Lipid Metabolism

项目摘要/摘要 非酒精性脂肪性肝病(NAFLD)，影响到大约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治疗方法是 由于对其基本机制缺乏了解而受到阻碍，包括 遗传和环境因素。甘油磷酸二酯磷酸二酯酶结构域含蛋白3 (GDPD3)是一种新发现的含有溶血磷脂酶D活性的酶，可以转化溶血磷脂 对非肝细胞中的溶血磷脂酸(LysoPA)。哺乳动物的GDPD3此前并未受到牵连 在肝脏的脂质代谢中。我们的初步数据表明，人类GDPD3与 在肝细胞和小鼠肝脏中的表达和甘油三酯(TG)的积累，表明一个新的基因在 调节肝脏甘油三酯动态平衡。尽管如此，细胞内的位置，底物的特异性， 人GDPD3在肝细胞/肝脏中的生理功能和分子机制尚不清楚。 因此，在这项研究中，在一个经验丰富的多学科辅导小组的指导下，我们 建议研究人类GDPD3的酶学性质，并探讨人类GDPD3是否是原因 肝脏脂肪变性的基因。更具体地说，我们问了三个问题：1)人类GDPD3和 含有溶血磷脂酶D活性的内质网膜相关酶？2) 人GDPD3通过以下途径增加LysoPA的产生，从而增加肝脏TG的合成和积聚 甘油磷酸途径？3)人GDPD3产生的溶菌素PA能激活过氧化物酶增殖物吗？ 活化受体γ(PPARγ)通过增加脂肪酸(FA)摄取和 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GDPD3，在 小鼠肝脏测定：a)人GDPD3过表达对油酸诱导的甘油三酯的影响 肝癌细胞蓄积与饮食诱导的小鼠肝脏脂肪变性；b)亚细胞定位 人GDPD3在肝癌细胞和小鼠原代肝细胞中的表达；以及c)数量和分子种类 GDPD3脂质底物和产物在小鼠肝脏中的含量。肝脏特异性人GDPD3过表达小鼠 在PPAR中功能丧失或功能获得时，γ将被喂养食物或西式饮食来诱导肝脏 脂肪变性。我们将对这些小鼠进行全面的肝脏和系统代谢表型鉴定。主要 肝细胞将被用于研究新生脂肪生成、FA摄取和掺入TG、FA 氧化，以及使用放射性同位素分泌极低密度脂蛋白-甘油三酯。当提议的目标是 我们将更好地从机制上理解人类GDPD3和GDPD3之间的关系 肝脏脂肪变性，以解决对NAFLD发病机制的认识差距，并为其提供策略 治疗。最后，该提案为申请人提供了必要的培训和指导，以 过渡到成功的职业生涯，成为一名脂肪和葡萄糖代谢的独立研究员。

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.