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，这会增加患上肝硬变和肝细胞癌的风险。之前 研究表明，肝脏线粒体氧化代谢途径中的脂分配失调[即， 三羧酸(TCA)循环、氧化磷酸化(OXPHOS)和酮类合成]是肝脏的基础 脂肪变性和氧化应激是NAFLD的基础。关于线粒体脂质划分的传统观点是 当超过末端氧化(TCA循环和OXPHOS)的能力时，脂类注定要生酮， 但是，当TCA循环和OXPHOS能够支持末端氧化或处于以下状态时，酮的生成是有限的 肝脏能量需求高。然而，这种对脂质分配的二元论观点未能描述 线粒体在NAFLD谱系中的功能，并揭示了我们在理解如何 肝脏线粒体可能协调脂质分解代谢。本项目的目标是检验创新的前提 酮类生成积极支持TCA循环功能，而这种有益偶联的丧失有助于 NAFLD。这是基于我们从不同严重程度的NAFLD小鼠模型中获得的初步数据。初步研究 用过的磷脂酰乙醇胺N-甲基转移酶(PEMT)缺失的小鼠，由于减少而表现出NASH 磷脂酰胆碱，这是人类NASH的特征。PEMT缺失的小鼠表现出较低的肝脏NAD和 然而，在活体内，末端氧化的分子指标TCA循环通量没有改变，而酮的生成 增加了。小鼠的这种表型与我们在患有NASH的人类中的初步数据相似，显示NASH升高 酮的生成和保存的TCA循环通量。值得注意的是，PEMT基因缺失的小鼠喂高脂饮食，野生型小鼠喂食 生酮饮食有较低的肝脏烟酰胺N-甲基转移酶(NNMT)，这可能导致较大的NAD 打捞以促进本来会受到损害的三氯乙烷循环通量。此外，生酮基因敲除 高脂饮食小鼠肝脏3-羟甲基戊二酰辅酶A合成酶2(HMGCS2)的表达 酮体生成(如预期)和NAD。我们的中心假设是酮的生成通过 在脂质供应过剩的情况下提供急性和慢性NAD。这一假设将得到检验。 通过两个具体目标。目标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中的TCA循环通量。缺乏肝脏HMGCS2的小鼠将接受Gubra Amylin Nash(GAN)饮食 并将与肝脏特异性NNMT基因敲除小鼠独立杂交，或提供烟酰胺核苷 增加NAD。目标2将确定由酮类生成支持的NAD/NADH比率的增加有助于 因运动而产生的脂肪代谢。HMGCS2和β-羟丁酸脱氢酶1将在#年删除 饲喂GaN饲料以阻滞酮的生成和NAD供应的小鼠的肝脏。急性和慢性运动方案 将会完成。影响：该项目将通过推进我们的 了解酮体生成如何促进脂质处置，并为预防NASH提供新的治疗方法。

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.