Targeting hepatic mitochondrial function in humans with NAFLD using insulin sensitizers

项目总结/摘要 非酒精性脂肪性肝病（NAFLD）描述了一系列肝脏异常，从相对良性的 非酒精性脂肪肝（NAFL）中过量脂肪的储存，以及更严重的非酒精性脂肪性肝炎（NASH）， 其特征在于肝细胞损伤、坏死性炎症和纤维化。NASH中肝损害最快 肝细胞癌的生长原因，现在是肝移植的最常见的原因， 妇女NAFLD的患病率与肥胖、胰岛素抵抗和2型糖尿病（T2 D）的患病率相当， 患有这些合并症的患者发生危及生命的肝脏结局的风险更大。但没有 批准的NAFLD治疗方法，迫切需要新的策略来抑制这种新兴的肾上腺素依赖性脂肪肝。 demic。我们的目标是在人类受试者中采用创新和高度整合的方法， 深入了解NAFLD的发病机制，这将促进开发新的治疗方法， 潜在的病理过程。在NAFL的早期过程中，肝脏脂质递送增加， 高脂饮食的顺序、脂肪组织胰岛素抵抗以及脂质的摄取和从头合成增加。 这种代谢环境增加了NAFL肝脏中的线粒体密度、氧化能力和TCA循环通量， 其最终可能压倒抗氧化防御机制以促进NASH的发展。在 我们的临床前研究观察到胰岛素增敏剂和PPARγ激动剂吡格列酮直接抑制了 这些NAFL肝脏中肝线粒体氧化能力和TCA循环活性升高。因此在 在三个具体目标之后，我们将利用稳定同位素示踪方法来检验中心假设， 肝线粒体功能是人类NAFLD的靶向特征。在第一阶段，我们首先要做的是量化-- 使用三重稳定同位素示踪法（[U-100]）测定NAFLD谱中的肝线粒体通量。 13 C]丙酸酯、[3，4 - 13 C2]葡萄糖和2 H2O）偶联到2 H和13 C NMR分析上，以同时询问 明确表征的NAFL和NASH患者的肝葡萄糖代谢和线粒体通量。接下来， 在具体目标2中，我们将评估肝线粒体功能作为人类胰岛素增敏剂的靶点， 将询问外周PPARy激动对肝线粒体通量和葡萄糖代谢的贡献， NAFLD中的禁忌。最后，在具体目标3中，我们将确定线粒体机制， 吡格列酮对NAFLD的影响：在NAFLD小鼠中使用单核基因组学和鸟枪法联合研究 脂质组学方法来检查实质和非实质细胞转录组学，并定量mi- 在慢性吡格列酮治疗后获得的肝活检组织中，分别观察到脂质重塑。 总的来说，这些研究将确定未来可以利用的机制，以促进 安全靶向T2 D和NAFLD的新疗法。

PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of liver abnormalities from the relatively benign storage of excess fat in nonalcoholic fatty liver (NAFL) to the more ominous nonalcoholic steatohepatitis (NASH), characterized by hepatocellular injury, necroinflammation, and fibrosis. Liver damage in NASH is the fastest growing cause of hepatocellular carcinoma and is now the most common reason for liver transplantation in women. The prevalence of NAFLD parallels that of obesity, insulin resistance and type 2 diabetes (T2D), and patients with these comorbidities are at greater risk of life-threatening liver outcomes. However, there are no approved treatments for NAFLD and there is an urgent need for new strategies to suppress this emerging epi- demic. Our goal is to employ innovative and highly integrative approaches in human subjects to gain mechanistic insight into the pathogenesis of NAFLD that will foster the development of new therapeutics that address the underlying pathological processes. Early in the course of NAFL, hepatic lipid delivery is increased as a conse- quence of high fat diets, adipose tissue insulin resistance and increased uptake and de novo synthesis of lipids. This metabolic milieu increases mitochondrial density, oxidative capacity and TCA cycle flux in NAFL livers, which eventually may overwhelm antioxidant defense mechanisms to promote the development of NASH. In preclinical studies we observed that the insulin sensitizer and PPARγ agonist pioglitazone directly suppressed elevated hepatic mitochondrial oxidative capacity and TCA cycle activity in these NAFL livers. Therefore, in the following three specific aims we will utilize stable isotope tracer methodology to test the central hypothesis that hepatic mitochondrial function is a targetable feature of NAFLD in humans. In Specific Aim 1 we will first quanti- tate hepatic mitochondrial fluxes across the NAFLD spectrum using a triple stable isotope tracer approach ([U- 13C]propionate, [3,4-13C2]glucose and 2H2O) coupled to 2H and 13C NMR analysis to simultaneously interrogate hepatic glucose metabolism and mitochondrial fluxes in well characterized patients with NAFL and NASH. Next, in Specific Aim 2 we will evaluate hepatic mitochondrial function as a target of insulin sensitizers in humans and will interrogate the contribution of peripheral PPARy agonism on hepatic mitochondrial fluxes and glucose me- tabolism in NAFLD. Finally, in Specific Aim 3 we will determine the mitochondrial mechanisms governing the effect of pioglitazone on NAFLD: In mice with NAFLD using a combination of single-nuclei genomics and shotgun lipidomics approaches to examine parenchymal and non-parenchymal cell transcriptomics and to quantitate mi- tochondrial lipid remodeling, respectively, in liver biopsies obtained following chronic pioglitazone treatment. Collectively, these studies will identify mechanisms that can be exploited in the future to foster the design of novel therapies that safely target T2D and NAFLD.