Mechanisms of insulin resistance related to nonalcoholic steatohepatitis

该项目的长期目标是了解导致肥胖/2型糖尿病中脂肪肝（NAFLD）和非酒精性脂肪性肝炎（NASH）的潜在机制，以及这些机制如何与全身胰岛素抵抗相关。我们还试图解开一个悖论，即虽然NASH与肥胖人类和小鼠的胰岛素抵抗密切相关，但这两种综合征在某些基因KO小鼠模型中明显分离。该提议的中心假设通过假定肝细胞胞质乙酰辅酶A水平通过产生棕榈酸/胆固醇毒性促进NAFLD和NASH，而肝细胞线粒体乙酰辅酶A水平通过刺激丙酮酸羧化酶和胰岛素生成来驱动胰岛素抵抗来解决该谜题。因此，我们提出，虽然肝细胞乙酰辅酶A池通常协调升高，但它们在某些肥胖和脂肪肝遗传小鼠模型中可以断开。为了测试我们的假设并直接解决这个问题，我们应用了新的基因沉默技术，该技术将独特的RNA修饰和GalNAC定向的肝细胞靶向结合在“自我递送”RNAi（sdRNA）化合物中。这些化合物可以在小鼠中单次皮下注射后沉默单个或多个靶向肝细胞基因2个月或更长时间。使用GalNAC-sdRNA，我们可以选择性地靶向和沉默每个 在产生胞质乙酰辅酶A的多种途径中（例如，ACLY和ACSS 2途径）与线粒体乙酰辅酶A（例如，FATP 2/5途径），同时避免了产生多基因KO小鼠的高昂成本和时间。在目的1中，我们将这种强大的RNAi技术与一种新的方法相结合，该方法定量肝细胞线粒体乙酰辅酶A与总细胞乙酰辅酶A，以确定ACLY，ACSS 2和FATP 2，FATP 5对瘦小鼠和HFD小鼠中这些特定肝细胞乙酰辅酶A库的相对贡献。在目标2中，我们提出通过从目标1中学习的适当GalNAC-sdRNA基因靶向来消耗NAFLD/NASH小鼠模型中的肝细胞胞质乙酰CoA，并确定其对肝脏甘油三酯、炎症、纤维化和葡萄糖耐量的影响以及其对枯否细胞和星状细胞功能障碍的影响。例如，我们将测试NASH小鼠模型中肝细胞乙酰辅酶A水平的消耗是否通过肝细胞转录因子TAZ的下调来减弱星状细胞的胶原蛋白产生，TAZ驱动肝细胞印第安刺猬（IHH）分泌和星状细胞活化。这些研究还将解决Kupffer和星状细胞功能障碍是由肝细胞NAFLD驱动还是由循环因子独立促进或两者兼而有之的关键问题。最后，在目标3中，我们将测试一种潜在的治疗策略， 确定靶向多个肝细胞基因的GalNAC-sdRNA是否将同时减轻肥胖/2型糖尿病中的NAFLD、NASH和胰岛素抵抗的所有三种综合征。这种方法具有主要的临床优势，因为针对不同基因的多种GalNAC-sdRNA由相同的化学组成组成，并且与小分子不同，被评估用于临床作为单一治疗剂。

The long term goal of this project is to understand the underlying mechanisms that cause fatty liver (NAFLD) and nonalcoholic steatohepatitis (NASH) in obesity/type 2 diabetes, and how such mechanisms relate to systemic insulin resistance. We also seek to unravel the paradox that while NASH is tightly correlated with insulin resistance in obese humans and mice, these two syndromes are clearly dissociated in certain gene KO mouse models. The central hypothesis of this proposal solves this riddle by positing that hepatocyte cytosolic Acetyl CoA levels promote NAFLD and NASH through producing palmitate/cholesterol toxicity, while hepatocyte mitochondrial Acetyl CoA levels drive insulin resistance by stimulating pyruvate carboxylase and gluconeogenesis. Thus, we propose that while hepatocyte Acetyl CoA pools are often coordinately elevated, they can be disconnected in certain genetic mouse models of obesity and fatty liver. In order to test our hypothesis and attack this problem directly, we apply novel gene silencing technology that combines unique RNA modifications and GalNAC-directed hepatocyte targeting in “self delivery” RNAi (sdRNA) compounds. These compounds can silence single or multiple targeted hepatocyte genes for 2 months or more after a single subcutaneous injection in mice. Using GalNAC-sdRNA, we can selectively target and silence each of the multiple pathways that produce cytosolic Acetyl CoA (e.g., ACLY and ACSS2 pathways) versus mitochondrial Acetyl CoA(e.g., FATP2/5 pathway), while avoiding prohibitive costs and time in generating multiple gene KO mice. In Aim 1, we couple this powerful RNAi technology with a novel method that quantifies hepatocyte mitochondrial Acetyl CoA vs total cellular Acetyl CoA to determine the relative contributions of ACLY, ACSS2 and FATP2, FATP5 to these specific hepatocyte Acetyl CoA pools in lean and HFD mice. In Aim 2 we propose to deplete hepatocyte cytosolic Acetyl CoA in NAFLD/NASH mouse models by appropriate GalNAC-sdRNA gene targeting learned from Aim 1, and determine its impact on liver triglyceride, inflammation, fibrosis and glucose tolerance as well as its impact on Kupffer and Stellate cell dysfunction. For example, we will test whether depletion of hepatocyte Acetyl CoA levels in NASH mouse models attenuates collagen production by Stellate cells through downregulation of hepatocyte transcription factor TAZ, which drives hepatocyte Indian hedgehog (IHH) secretion and Stellate activation. These studies will also resolve the key question whether Kupffer and Stellate cell dysfunction is driven by hepatocyte NAFLD versus independently promoted by circulating factors, or both. Finally, in Aim 3 we will test a potential therapeutic strategy by determining whether GalNAC-sdRNAs targeting multiple hepatocyte genes will simultaneously alleviate all three syndromes of NAFLD, NASH and insulin resistance in obesity/type 2 diabetes. This approach has major clinical advantages since multiple GalNAC-sdRNAs against different genes consist of the same chemical composition and, unlike small molecules, are evaluated for use in the clinic as a single therapeutic agent.