Determining the Function of PNPLA3 Utilizing Metabolomics and Stable Isotope Labe

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是一种日益普遍的医学问题，影响了大约三分之一的美国人口。Patatin-like phospholipase domain containing 3（PNPLA 3）首次与肝甘油三酯（TAG）代谢有关，我们的实验室发现在148位的异亮氨酸被甲硫氨酸（I148 M）取代的错义突变与人类非酒精性脂肪肝相关。具有变异等位基因（148 M）的纯合子的肝脏TAG蓄积风险比具有野生型等位基因（148 I）的纯合子高2倍。PNPLA 3的功能和I148 M诱导肝TAG蓄积的原因尚不清楚。由于脂肪肝代表了甘油三酯形式的脂肪酸积累，因此我将调整和/或开发靶向液相色谱-串联质谱法（LC-MS-MS）方法，以测量肝脏脂肪组织和PNPLA 3相关脂肪变性小鼠模型血浆中不同类别含脂肪酸脂质的浓度。将对正常食物饮食和设计用于加重或减轻PNPLA 3-I148 M相关的肝TAG积累的饮食研究小鼠。然后，我将扩展这些基于LC-MS/MS的方法，通过测量过表达PNPLA 3的培养细胞和遗传操作小鼠模型中氘水（D2 O）中氘的掺入率来确定每种脂质的通量。替代稳定同位素标记策略，如13 C-甘油或13 C-脂肪酸掺入，也将用于补充D2 O掺入数据。本研究计划的实施将提供以下方面的培训： 代谢组学技术来研究人类疾病，并深入了解PNPLA 3的功能及其在脂肪肝疾病进展中的作用。

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.