In Vivo 2H/13C Metabolic Flux Analysis of NASH Pathogenesis

 描述(申请人提供)：尽管非酒精性脂肪性肝病在西方社会日益流行，但控制非酒精性脂肪性肝病(NAFLD)发展为非酒精性脂肪性肝炎(NASH)的潜在分子机制仍然知之甚少。目前提案的总体目标是应用体内2H/13C代谢通量分析(MFA)和代谢组学分析来确定加速从NAFLD向NASH转变的肝脏表型，并评估体内对旨在抑制这一转变的药物和遗传干预的反应。这些研究将使用黑素皮质素-4受体基因敲除(MC4R-/-)小鼠模型，该模型先前已被证明在西方饮食喂养下迅速和自发地发展出人类NASH的特征。为了能够分析这个和其他转基因小鼠模型中的中间肝脏通量，最近开发了一种新的基于GC-MS的MFA方法，该方法只需要40μL的血浆，而且可以以与最先进的基于核磁共振的方法相当的精度测定体内的通量。这一方法将被应用于同时评估柠檬酸循环(CAC)和糖异生通量的变化，以响应慢性或急性脂毒治疗。中心假说是，内质网钙释放异常促进线粒体代谢途径的过度激活和组织氧化损伤，导致NASH。这项研究的基本原理是，确定区分单纯性脂肪变性和NASH的体内代谢途径改变将使更有针对性的方法预防或治疗NASH。这项研究涉及三个具体目标。第一个目标是确定与西方饮食诱导的肝损伤相关的代谢表型。工作假设是，在NAFLD的背景下，线粒体CAC通量的过度激活加速了肝细胞的脂毒性。第二个目标是确定抑制线粒体代谢在多大程度上可以防止脂肪肝的氧化应激。工作假设是，对肝脏线粒体代谢的药物抑制将剂量依赖性地减少NAFLD小鼠模型的氧化组织损伤。第三个目的是确定抑制内质网钙释放在多大程度上可以逆转脂肪肝线粒体的过度激活。工作假说是，增强内质网隔离钙的能力将使线粒体代谢正常化，并降低肝细胞的脂毒性。这项拟议的研究具有特别的创新性，因为它应用了一种新开发的基于GC-MS的MFA方法，该方法比基于核磁共振的方法更快、更便宜，需要的样本量更少。预计这项研究将取得以下关键成果。首先，它将确定促进单纯性肝脂肪变性向进行性NASH转变的代谢表型。其次，它将确定抑制这一转变的潜在治疗靶点。第三，它将建立一种可扩展的体内MFA方法，适合于常规的小鼠肝脏表型。这项研究意义重大，因为它将确定可以靶向抑制从NAFLD向NASH进展的失调的代谢途径。

 DESCRIPTION (provided by applicant): Despite its growing prevalence in Western society, the underlying molecular mechanisms that control the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) remain poorly understood. The overall objective of the current proposal is to apply in vivo 2H/13C metabolic flux analysis (MFA) and metabolomics profiling to identify liver phenotypes that accelerate the transition from NAFLD to NASH and to assess in vivo responses to pharmacologic and genetic interventions designed to inhibit this transition. These studies will use a melanocortin-4 receptor knockout (MC4R-/-) mouse model that has been previously shown to rapidly and spontaneously develop characteristics of human NASH upon Western diet feeding. To enable the analysis of intermediary liver fluxes in this and other transgenic mouse models, a novel GC-MS-based MFA approach has been recently developed that requires only 40 μL of blood plasma, yet can determine in vivo fluxes with precision equivalent to state-of-the-art NMR-based approaches. This approach will be applied to simultaneously assess changes in citric acid cycle (CAC) and gluconeogenesis fluxes in response to chronic or acute lipotoxic treatments. The central hypothesis is that abnormal ER calcium release promotes overactivation of mitochondrial metabolic pathways and oxidative tissue injury leading to NASH. The rationale for this research is that identifying in vivo metabolic pathway alterations that differentiate simple steatosis from NASH will enable better targeted approaches for NASH prevention or treatment. The research involves three specific aims. The first aim is to identify metabolic phenotypes associated with Western diet-induced liver injury. The working hypothesis is that overactivation of mitochondrial CAC flux accelerates hepatocyte lipotoxicity in the context of NAFLD. The second aim is to determine the extent to which inhibition of mitochondrial metabolism can prevent oxidative stress in fatty liver. The working hypothesis is that pharmacological inhibition of mitochondrial metabolism in liver will dose-dependently reduce oxidative tissue injury in NAFLD mouse models. The third aim is to determine the extent to which inhibition of ER calcium release can reverse mitochondrial overactivation in fatty liver. The working hypothesis is that enhancing the ability of ER to sequester calcium will normalize mitochondrial metabolism and reduce hepatocyte lipotoxicity. The proposed research is exceptionally innovative because it applies a newly developed GC-MS-based MFA approach that is faster, less expensive, and requires less sample volume than NMR-based approaches. The research is expected to achieve the following key outcomes. First, it will identify metabolic phenotypes that promote the transition from simple hepatic steatosis to progressive NASH. Second, it will identify potential therapeutic targets for inhibiting this transition. Third, it will establish a scalable in vivo MFA approach that is suitabe for routine mouse liver phenotyping. This research is significant because it will identify dysregulated metabolic pathways that can be targeted to inhibit the progression from NAFLD to NASH.