Role of Hepatic GDPD3 in Mechanisms of Lipid Metabolism

肝脏 GDPD3 在脂质代谢机制中的作用

基本信息

批准号：
10242757
负责人：
Chia-Chi Chuang Key
金额：
$ 11.64万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10242757
关键词：
Address Adenoviruses Adult Affect Albumins Anabolism Antibodies Automobile Driving Calnexin Cell Line Cell membrane Complex Confocal Microscopy Data Developed Countries Diabetes Mellitus Diet Disease Endoplasmic Reticulum Environmental Risk Factor Enzymes Etiology Fatty Acids Fatty Liver Fatty acid glycerol esters Gene Expression Genes Genetic Glycerol Glycerophospholipids Goals Golgi Apparatus Green Fluorescent Proteins HepG2 Hepatic Hepatitis C Hepatocyte High Prevalence Homeostasis Human Hyperlipidemia Incubated Knowledge Link Lipids Liver Location Lysophosphatidic Acid Receptors Lysophosphatidylcholines Lysophospholipase Lysophospholipids Mass Spectrum Analysis Membrane Membrane Proteins Mentors Molecular Mus Na(+)-K(+)-Exchanging ATPase Natural History Oleic Acids PPAR alpha PPAR gamma Pathogenesis Pathway interactions Pharmaceutical Preparations Physiological Population Prevalence Primary carcinoma of the liver cells Production Property Proteins Radioisotopes Rattus Regulation Reporting Research Personnel Role Small Interfering RNA Substrate Specificity Time Training Triglyceride Metabolism Triglycerides United States United States Food and Drug Administration adult obesity career causal variant enzyme pathway experience fatty acid oxidation gain of function glucose metabolism glycerophosphodiester phosphodiesterase hepatoma cell improved inorganic phosphate interdisciplinary approach knock-down lipid biosynthesis lipid metabolism liver transplantation loss of function lysophosphatidic acid macrogolgin male metabolic phenotype multidisciplinary non-alcoholic fatty liver disease novel overexpression promoter receptor small hairpin RNA standard care targeted treatment therapeutic target uptake vector control very low density lipoprotein triglyceride western diet

项目摘要

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.

项目概要/摘要非酒精性脂肪肝 (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 治疗方法是由于对其基本机制了解不足而受到阻碍，包括之间复杂的相互作用遗传和环境因素。含有甘油磷酸二酯磷酸二酯酶结构域的蛋白3。 (GDPD3)是一种新发现的含有溶血磷脂酶D活性的酶，可将溶血磷脂转化为此前并未涉及非肝细胞中的溶血磷脂酸 (lysoPA)。我们的初步数据表明人类 GDPD3 之间呈正相关。肝细胞和小鼠肝脏中的表达和甘油三酯（TG）积累，表明在肝细胞和小鼠肝脏中存在一个新基因然而，肝脏TG稳态的调节，细胞内位置、底物特异性、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，在经验丰富的多学科指导小组的指导下，我们研究提出人类 GDPD3 的酶学特性并探讨人类 GDPD3 是否是因果关系更具体地说，我们问三个问题：1) 人类 GDPD3 是一个吗？内质网膜相关酶含有溶血磷脂酶 D 活性吗？人 GDPD3 增加 lysoPA 的产生，导致肝脏 TG 合成和积累增加 3) 人 GDPD3 产生的 lysoPA 是否激活过氧化物酶体增殖物？激活的γ受体（PPARγ）通过增加脂肪酸（FA）的摄取和增强肝脂肪变性为了回答这些问题，我们将在肝癌细胞系和小鼠肝脏以确定：a) 人 GDPD3 过表达对油酸诱导的 TG 的影响小鼠肝癌细胞中的积累和饮食诱导的肝脂肪变性；b) 的亚细胞定位；人肝癌细胞和小鼠原代肝细胞中的GDPD3；c) 数量和分子种类；小鼠肝脏特异性人 GDPD3 过表达小鼠中 GDPD3 脂质底物和产物的研究。 PPARγ功能丧失或功能获得的患者将被喂食食物或西式饮食以诱导肝损伤我们将对这些小鼠进行全面的肝脏和全身代谢表型分析。肝细胞将用于研究从头脂肪生成、FA 摄取以及并入 TG、FA 当提出的目标时，使用放射性同位素进行氧化和极低密度脂蛋白-TG的分泌。如果实现了，我们将对人类 GDPD3 与肝脂肪变性，以解决有关 NAFLD 发病机制的知识差距并为其提供策略最后，该提案为申请人提供了必要的培训和指导。作为脂质和葡萄糖代谢的独立研究者，过渡到成功的职业生涯。