Hepatic Integration of Mitochondrial Oxidative Metabolism Pathways in Health and Disease

非酒精性脂肪性肝病（NAFLD），包括非酒精性脂肪性肝炎（NASH），是最常见的 在美国，它是肝脏疾病的一种，它增加了肝硬化和肝细胞癌的风险。之前 研究表明肝脏线粒体氧化代谢途径中失调的脂质分配[即， 三羧酸（TCA）循环、氧化磷酸化（OXPHOS）和生酮]是肝脏的基础 脂肪变性和氧化应激。线粒体脂质分配的传统观点是， 当超过末端氧化（TCA循环和OXPHOS）的能力时，脂质注定要生酮， 但当TCA循环和OXPHOS可以支持末端氧化或处于 肝脏能量需求高。然而，这种脂质分配的二元论观点未能描述 线粒体在NAFLD谱中的功能，并揭示了我们对如何理解的知识差距 肝线粒体可协调脂质代谢。本项目的目标是测试创新的前提 酮生成积极支持TCA循环功能，这种有益偶联的丧失有助于 NAFLD。这是基于我们来自不同NAFLD严重程度的小鼠模型的初步数据。初步研究 使用磷脂酰乙醇胺N-甲基转移酶（PEMT）缺失的小鼠，其由于减少的 磷脂酰胆碱，其是人NASH的特征。PEMT缺失小鼠显示较低的肝脏NAD+和 然而，终末氧化的分子指标，体内TCA循环通量不变，生酮作用 增加小鼠中的这种表型模拟了我们在患有NASH的人类中的初步数据， 酮生成和保存的TCA循环通量。值得注意的是，PEMT缺失小鼠喂食高脂肪饮食，野生型小鼠喂食 生酮饮食具有较低的肝脏烟酰胺N-甲基转移酶（NNMT），这可能导致更大的NAD+ 进行抢救，以促进否则会受损的三醋酸循环通量。此外，生酮蛋白的敲低 高脂饮食小鼠的3-羟甲基戊二酰辅酶A合酶2（HMGCS 2）导致肝脏 生酮（如预期）和NAD+。我们的中心假设是，酮生成通过以下途径增强TCA循环通量： 急性和慢性NAD+供应过量脂质可用性的条件下。这一假设将得到检验 两个具体目标。目的1将证明酮体-NNMT-NAD+轴通过以下方式减轻肝脏脂肪变性： 促进NASH中TCA循环通量。缺乏肝脏HMGCS 2的小鼠将接受Gubra Amylin NASH（GAN）饮食 并将独立地与肝脏特异性NNMT敲除小鼠杂交或提供烟酰胺核苷， 增加NAD+。目的2将确定由生酮支持的增加的NAD+/NADH比率促进 运动后的脂质代谢HMGCS 2和β-羟基丁酸脱氢酶1将在 喂食GAN饮食以阻碍酮生成和NAD+供应的小鼠的肝脏。急性和慢性运动方案 将完成。影响：该项目将通过推进我们的 了解生酮如何促进脂质处置，并为预防NASH的新疗法提供信息。

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), is the most common liver disease in the United States and it increases the risk for cirrhosis and hepatocellular carcinoma. Prior research shows that dysregulated lipid partitioning in liver mitochondrial oxidative metabolism pathways [i.e., tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and ketogenesis] is fundamental to liver steatosis and oxidative stress that underlie NAFLD. A traditional view of mitochondrial lipid partitioning is that lipids are fated to ketogenesis when the capacity of terminal oxidation (TCA cycle and OXPHOS) is exceeded, but ketogenesis is limited when the TCA cycle and OXPHOS can support terminal oxidation or under states of high hepatic energy demand. However, this dualist view of lipid partitioning fails to describe heterogeneity in mitochondrial function across the NAFLD spectrum and reveals knowledge gaps in our understanding of how liver mitochondria may coordinate lipid catabolism. The objective of this project is to test the innovate premise that ketogenesis actively supports TCA cycle function and that loss of this salutary coupling contributes to NAFLD. This is based on our preliminary data from mouse models of varying NAFLD severity. Initial studies used phosphatidylethanolamine N-methyltransferase (PEMT)-null mice that exhibit NASH owing to reduced phosphatidylcholine, which is characteristic of human NASH. PEMT-null mice showed lower liver NAD+ and molecular indices of terminal oxidation, however, in vivo TCA cycle flux was unaltered and ketogenesis was increased. This phenotype in mice mimics our preliminary data in humans with NASH which showed elevated ketogenesis and preserved TCA cycle flux. Notably, PEMT-null mice fed a high-fat diet and wild type mice fed a ketogenic diet had lower liver nicotinamide N-methyltransferase (NNMT), which may lead to greater NAD+ salvage to facilitate TCA cycle flux that would otherwise be impaired. In addition, knockdown of ketogenic enzyme 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in mice on a high-fat diet resulted in lower liver ketogenesis (as expected) and NAD+. Our central hypothesis is that ketogenesis enhances TCA cycle flux via acute and chronic NAD+ provision under conditions of excess lipid availability. This hypothesis will be tested via two Specific Aims. Aim 1 will demonstrate that a ketone body-NNMT-NAD+ axis mitigates liver steatosis by promoting TCA cycle flux in NASH. Mice lacking liver HMGCS2 will receive a Gubra Amylin NASH (GAN) diet and will be independently crossed with liver-specific NNMT knockout mice or provided nicotinamide riboside to increase NAD+. Aim 2 will determine that an increased NAD+/NADH ratio supported by ketogenesis facilitates lipid disposal in response to exercise. HMGCS2 and β-hydroxybutyrate dehydrogenase 1 will be deleted in livers of mice fed a GAN diet to impede ketogenesis and NAD+ provision. Acute and chronic exercise protocols will be completed. Impact: This project will lead progress in creating a paradigm shift by advancing our understanding of how ketogenesis facilitates lipid disposal and informing new therapies for preventing NASH.