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的严重程度与全身胰岛素抵抗、2型糖尿病风险相关，并导致下游并发症，包括心血管疾病、肝硬变、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显著的特征，人类NAFLD表现出进行性的生酮缺陷。肝细胞酮的发生产生酮体乙酰乙酸酯(Acac)和D-b-羟基丁酸酯(D-bOHB)，这是不完全脂肪氧化的产物。酮体生成的测量通常被用作肝脏脂肪氧化的替代指标，但这些方法不能完全报告肝脏脂肪氧化，因为酮体生成在三羧酸(TCA)循环中对脂肪的完全氧化视而不见，三羧酸循环也随NAFLD的进程而变化。在前一个资金周期中发表和未发表的观察结果表明，酮的生成提供了重要的反馈，协调了整个肝细胞的能量供需。当小鼠被遗传编程为缺乏酮生成时，肝脏通过增加TCA循环流量进行补偿，但容易发生高脂饮食诱导的纤维化，这是最能预测NASH不良后果的特征。相反，当小鼠被基因编程为只产生acac，而不产生D-bOHB[通过敲除NAD+/NADH D-bOHB脱氢酶(BDH1)]时，肝脏通过减少TCA循环流量来补偿，并防止高脂饮食诱导的纤维化。这些发现揭示了肝脏能量供需之间意想不到的关系，这可能对NAFLD应如何考虑代谢药物靶点产生深远影响。此外，最近的观察表明，肝细胞来源的acac通过氧化邻近的巨噬细胞来保护肝纤维化。因此，这一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)肝巨噬细胞线粒体内acac的代谢来调节肝纤维化的形成。这一假设将通过两个具体目标进行检验。首先，为了揭示BDH1在NASH相关肝损伤中的作用，将使用基于示踪剂的质谱学、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失BDH1的小鼠，并支持复杂的碳、电子、质子和氧通量的量化，以建立线粒体效率与NAFLD样发病之间的关系。在第二个目标中，示踪剂和基于助熔剂的方法将使用缺乏琥珀酰辅酶A：3-氧代酸-辅酶A转移酶(SCOT)的小鼠来量化肝细胞和邻近巨噬细胞之间的酮体交换在NASH样发病中的影响。总之，拟议的实验将定义酮代谢可以被用来保护肝脏免受不断恶化的NAFLD损伤的机制，这是一个明显未得到满足的需求，对公共健康的影响不断升级。

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 Published and unpublished observations generated during the previous funding cycle suggest that ketogenesis provides vital feedback coordinating overall hepatocyte energy supply and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.