Ketone Body Metabolism and Integrated Metabolic Homeostasis

非酒精性脂肪性肝病（NAFLD），包括非酒精性脂肪性肝炎（NASH），是西方国家最常见的肝病原因。人类NAFLD的严重程度与全身性胰岛素抵抗相关; 2型糖尿病的风险;并驱动下游并发症，包括心血管疾病，肝硬化，需要肝移植和肝癌。随着NAFLD的进展，线粒体氧化功能障碍成为一个突出的特征，并且人NAFLD表现出进行性生酮缺陷。肝细胞酮生成产生酮体乙酰乙酸酯（AcAc）和D-b-羟基丁酸酯（D-bOHB），这是脂肪不完全氧化的产物。生酮的测量通常被用作肝脂肪氧化的代表，但是这些测量不能完全报告肝脂肪氧化，因为生酮对三羧酸（TCA）循环中的完全脂肪氧化是盲目的，其也在NAFLD的过程中变化。在上一个资助周期中产生的已发表和未发表的观察结果表明，酮生成提供了协调整体肝细胞能量供需的重要反馈。当小鼠被遗传编程为缺乏所有生酮时，肝脏通过增加TCA循环通量进行补偿，但易发生高脂肪饮食诱导的纤维化，这是NASH中最能预测不良结局的特征。相反，当小鼠被遗传编程为仅产生AcAc而不产生D-bOHB时[通过敲除NAD+/NADH D-bOHB脱氢酶（BDH 1）]，肝脏通过减少TCA循环通量进行补偿，并保护其免受高脂肪饮食诱导的纤维化。这些发现揭示了肝脏能量供应和需求之间的意外关系，这可能对如何在NAFLD中考虑代谢药物靶点产生深远影响。此外，最近的观察表明，肝细胞衍生的AcAc通过在相邻巨噬细胞中的氧化来防止纤维化。因此，该建议的中心假设是肝酮代谢通过（a）调节肝细胞能量供需平衡和（B）肝巨噬细胞线粒体中AcAc的代谢来调节肝纤维化。这一假设将通过两个具体目标进行检验。首先，为了揭示BDH 1在NASH相关肝损伤中的作用，将使用基于示踪剂的质谱法、核磁共振和线粒体生物能量学研究来询问肝细胞中选择性缺乏BDH 1的小鼠，这些研究一起支持碳、电子、质子和氧通量的复杂定量，以构建线粒体效率与NAFLD样发病机制之间的关系。在第二个目标中，示踪剂和基于通量的方法将使用缺乏琥珀酰-CoA：3-含氧酸-CoA转移酶（SCOT）的小鼠量化肝细胞和邻近巨噬细胞之间的酮体交换在NASH样发病机制中的作用，所述转移酶是巨噬细胞中AcAc氧化所需的。总之，拟议的实验将确定酮代谢可以用来保护肝脏免受NAFLD损伤恶化的机制，这是一个明显未满足的需求，对公共卫生的影响不断升级。

Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH) is the most common cause of liver disease in Western countries. The severity of NAFLD in humans correlates with systemic insulin resistance; risk of type 2 diabetes; and drives downstream complications, including cardiovascular disease, cirrhosis, need for liver transplantation, and liver cancer. As NAFLD progresses, mitochondrial oxidative dysfunction becomes a prominent feature, and human NAFLD exhibits progressive ketogenic deficits. Hepatocyte ketogenesis produces the ketone bodies acetoacetate (AcAc) and D-b-hydroxybutyrate (D-bOHB), which are products of incomplete fat oxidation. Measurements of ketogenesis are often used as a proxy for hepatic fat oxidation, but these measures fail to fully report hepatic fat oxidation, because ketogenesis is blind to complete fat oxidation in the tricarboxylic acid (TCA) cycle, which also varies over the course of NAFLD. Published and unpublished observations generated during the previous funding cycle suggest that ketogenesis provides vital feedback coordinating overall hepatocyte energy supply and demand. When mice are genetically programmed to be devoid of all ketogenesis, the liver compensates by increasing TCA cycle flux, but is predisposed to high fat diet-induced fibrosis, the feature most predictive of adverse outcomes in NASH. Conversely, when mice are genetically programmed to produce only AcAc, but not D-bOHB [via knockout of NAD+/NADH D-bOHB dehydrogenase (BDH1)], the liver compensates by decreasing TCA cycle flux, and is protected from high fat diet-induced fibrosis. These findings reveal unexpected relationships between energy supply and demand in liver that may have profound impact on how metabolic drug targets should be considered in NAFLD. Moreover, recent observations indicate that hepatocyte derived AcAc protects against fibrosis through oxidation in neighboring macrophages. Therefore, the central hypothesis of this proposal is that liver ketone metabolism modulates hepatic fibrogenesis through (a) tuning hepatocyte energy supply/demand balance and (b) metabolism of AcAc in the mitochondria of liver macrophages. This hypothesis will be tested through two Specific Aims. First, to reveal the role of BDH1 in NASH-relevant liver injury, mice lacking BDH1 selectively in hepatocytes will be interrogated using tracer-based mass spectrometry, nuclear magnetic resonance, and mitochondrial bioenergetics studies, together supporting sophisticated quantifications of carbon, electron, proton, and oxygen fluxes to construct relationships between mitochondrial efficiency and NAFLD-like pathogenesis. In the Second Aim, tracer and flux-based approaches will quantify the effects of ketone body exchange between hepatocytes and neighboring macrophages in NASH-like pathogenesis, using mice that lack succinyl-CoA:3-oxoacid-CoA transferase (SCOT), which is required for AcAc oxidation in macrophages. Together, the proposed experiments will define mechanisms through which ketone metabolism can be leveraged to protect the liver from worsening NAFLD injury, a clear unmet need with escalating public health implications.