Role of PNPLA3 in Fatty Liver Disease

描述（由申请人提供）：非酒精性脂肪性肝病 (NAFLD) 是一种新兴的健康问题，影响美国三分之一的成年人和越来越多的儿童。该疾病过程始于肝脏中甘油三酯 (TG) 的积累（脂肪变性），这在某些个体中会引起炎症反应（脂肪性肝炎），可能发展为肝硬化，甚至可能发展为肝癌。尽管在横断面研究中多种因素（例如肥胖、胰岛素抵抗）与 NAFLD 相关，但 NAFLD 的发病机制仍然知之甚少，目前的治疗选择也非常有限。我们的团队采用遗传学方法来确定导致 NAFLD 的致病因素。最近，我们在含 patatin 样磷脂酶结构域的蛋白 PNPLA3 中发现了一个错义突变 (I148M)，该突变与肝脏 TG 含量和肝损伤密切相关。这种变异在西班牙裔美国人中最常见，该群体的肝脂肪变性患病率最高，而在脂肪变性发生率最低的非裔美国人中最不常见。随后的研究证实了我们的发现，并表明 PNPLA3-I148M 变异在活检证实的脂肪性肝炎和酒精相关性肝硬化受试者中富集。因此，PNPLA3 被认为是全谱 NAFLD 以及酒精性肝硬化的影响因素。 关于 PNPLA3 的生理作用以及该酶的遗传变异如何促进肝脏 TG 积累、炎症和纤维化的基本问题仍然存在。本申请的总体目标是阐明 PNPLA3 在脂肪肝疾病中的作用。为此，我们将结合经典生物化学和生理学以及最先进的质谱分析技术，对 PNPLA3 功能发生遗传性改变的小鼠进行鉴定，以确定该酶的底物和产物、PNPLA3 在脂质代谢中的作用以及其与 TG 积累和肝损伤相关的分子基础。将使用两种互补的方法来鉴定 PNPLA3 的生物底物：i) 使用纯化酶的候选底物方法（目标 1a）和转基因小鼠中的比较脂质组学方法（目标 1b），以鉴定因 PNPLA3 活性变化而改变的脂质。在目标 2 中，我们将使用小鼠模型来检查 PNPLA3-I148M 对肝脏脂质代谢的影响。目标 3 侧重于确定 PNPLA3-I148M 促进肝脏中 TG 积累的分子机制。最后，在目标 4 中，我们将建立小鼠模型来研究 PNPLA3 导致肝脏炎症和纤维化的机制。 通过阐明 PNPLA3 的生物学作用以及 I148M 突变导致脂肪肝易感性的机制，本提案中概述的实验将为了解患病率持续增加的主要人类疾病的发病机制提供新的见解。我们的最终目标是开发新的方法和策略来诊断、预防和治疗 NAFLD。

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem that affects one-third of adults and an increasing number of children in the U.S. The disease process begins with the accumulation of triglyceride (TG) in the liver (steatosis), which in some individuals elicits an inflammatory response (steatohepatitis) that can progress to cirrhosis, and possibly liver cancer. Although various factors (e.g., obesity, insulin resistance) are associated with NAFLD in cross-sectional studies, the pathogenesis of NAFLD remains poorly understood and therapeutic options are currently very limited. Our group has taken a genetic approach to identify causal factors that contribute to NAFLD. Recently, we identified a missense mutation (I148M) in patatin-like phospholipase domain-containing protein, PNPLA3 that is strongly associated with both hepatic TG content and hepatic injury. The variant is most common in Hispanics, the group with the greatest prevalence of hepatic steatosis and least common in African-Americans who have the lowest frequency of steatosis. Subsequent studies have confirmed our findings and showed that the PNPLA3-I148M variant is enriched in subjects with biopsy-proven steatohepatitis and with alcohol-related cirrhosis. Thus, PNPLA3 is implicated as a contributing factor in the full spectrum of NAFLD as well as alcoholic cirrhosis. Basic questions remain regarding the physiological role of PNPLA3 and how genetic variation in this enzyme promotes hepatic TG accumulation, inflammation and fibrosis. The overall goal of this application is to elucidate the role of PNPLA3 in fatty liver disease. To this end, we will use a combination of classical biochemistry and physiology plus state-of-the-art mass-spectrometry in mice with genetically-defined changes in PNPLA3 function to identify the substrates and products of the enzyme, the role in PNPLA3 in lipid metabolism and the molecular basis for its association with TG accumulation and liver damage. Two complementary approaches will be used to identify the biological substrate(s) of PNPLA3: i) a candidate substrate approach using purified enzyme (Aim 1a) and a comparative lipidomic approach in genetically- modified mice (Aim 1b) to identify lipids that are altered by changes in PNPLA3 activity. In Aims 2 we will use our mouse models to examine effects of PNPLA3-I148M on hepatic lipid metabolism. Aim 3 focuses on identifying molecular mechanisms by which PNPLA3-I148M promotes TG accumulation in the liver. Finally, in Aim 4 we will establish a mouse model in which to investigate the mechanisms by which PNPLA3 contributes to hepatic inflammation and fibrosis. By elucidating the biological role of PNPLA3 and the mechanisms by which the I148M mutation confers susceptibility to fatty liver disease, the experiments outlined in this proposal will provide new insight into the pathogenesis of a major human disease that continues to increase in prevalence. Our ultimate goal is to develop new approaches and strategies to diagnose, prevent and treat NAFLD.