Determining the Function of PNPLA3 Utilizing Metabolomics and Stable Isotope Labe

利用代谢组学和稳定同位素标记确定 PNPLA3 的功能

基本信息

批准号：
8743229
负责人：
Matthew Alvin Mitsche
金额：
$ 10.32万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8743229
关键词：
Adipose tissue Affect Alleles Chronic Disease Cirrhosis Cultured Cells Data Deuterium Development Diet Epidemic Fatty Acids Fatty Liver Fatty acid glycerol esters Genes Genetic Genetic Variation Glycerol Health Hepatic Hepatocyte Homozygote Human Isoleucine Isotope Labeling Laboratories Life Lipids Liquid Chromatography Liver Liver Failure Liver diseases Mass Spectrum Analysis Measures Medical Metabolic Metabolism Methionine Methods Missense Mutation Modeling Mus Names Phospholipase Physiological Plasma Population Positioning Attribute Predisposition Primary carcinoma of the liver cells Proteins Research Proposals Risk Role Stable Isotope Labeling Technology Training Triglycerides Variant Water Work base design gene function high risk human disease insight metabolomics mouse model mutant non-alcoholic fatty liver nonalcoholic steatohepatitis novel therapeutics overexpression prevent protein function public health relevance research study risk variant stable isotope tandem mass spectrometry

项目摘要

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent medical problem that affects approximately a third of the US population. Patatin-like phospholipase domain containing 3 (PNPLA3) was first implicated in the metabolism of hepatic triacylglycerides (TAGs) when our lab found that a missense mutation that substitutes isoleucine at position 148 with methionine (I148M) was associated with non-alcoholic fatty liver disease in humans. Homozygotes with the variant allele (148M) have a 2-fold higher risk of hepatic TAG accumulation than homozygotes with the wild type allele (148I). The function of PNPLA3 and the cause of I148M induced hepatic TAG accumulation remains unclear. Since fatty liver represents an accumulation of fatty acids in the form of triglycerides, I will adapt and/or develop targeted liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods to measure the concentrations of different classes of fatty acid-containing lipids in liver adipose tissue, and plasma from mouse models of PNPLA3-associated steatosis. Mice will be studied on both a normal chow diet and on diets designed to exacerbate or alleviate PNPLA3-I148M associated accumulation of hepatic TAG. I will then extend these LC-MS/MS-based methods to determine the flux of each lipid class by measuring the rate of incorporation of deuterium from deuterated water (D2O) in cultured cells that overexpress PNPLA3, and in genetically manipulated mouse models. Alternative stable isotope labeling strategies, such as 13C-glycerol or 13C-fatty acid incorporation, will also be employed to compliment the D2O incorporation data. Execution of this research proposal will provide training in the application of metabolomics technology to study human disease and shed insight into the function of PNPLA3 and its role in the progression of fatty liver disease.

描述（由申请人提供）：非酒精性脂肪肝病 (NAFLD) 是一种日益普遍的医疗问题，影响着大约三分之一的美国人口。当我们的实验室发现用蛋氨酸 (I148M) 取代 148 位异亮氨酸的错义突变与非酒精性脂肪肝相关时，含有 3 (PNPLA3) 的 Patatin 样磷脂酶结构域首次与肝三酰甘油酯 (TAG) 的代谢有关。在人类中。具有变异等位基因 (148M) 的纯合子的肝脏 TAG 积累风险比具有野生型等位基因 (148I) 的纯合子高 2 倍。 PNPLA3 的功能和 I148M 诱导肝脏 TAG 积累的原因尚不清楚。由于脂肪肝代表脂肪酸以甘油三酯的形式积累，我将采用和/或开发有针对性的液相色谱-串联质谱 (LC-MS-MS) 方法来测量不同类别的含脂肪酸脂质的浓度肝脏脂肪组织和 PNPLA3 相关脂肪变性小鼠模型的血浆中。将对小鼠进行正常饮食和旨在加剧或减轻与PNPLA3-I148M相关的肝脏TAG积累的饮食的研究。然后，我将扩展这些基于 LC-MS/MS 的方法，通过测量过度表达 PNPLA3 的培养细胞和基因操纵的小鼠模型中氘水 (D2O) 中氘的掺入速率来确定每种脂质类别的通量。替代的稳定同位素标记策略，例如 13C-甘油或 13C-脂肪酸掺入，也将用于补充 D2O 掺入数据。该研究计划的执行将提供应用培训代谢组学技术用于研究人类疾病并深入了解 PNPLA3 的功能及其在脂肪肝疾病进展中的作用。