Mechanisms of insulin resistance related to nonalcoholic steatohepatitis

该项目的长期目标是了解肥胖/2型糖尿病患者脂肪肝(NAFLD)和非酒精性脂肪性肝炎(NASH)的潜在机制，以及这些机制与系统性胰岛素抵抗的关系。我们还试图揭开这样一个悖论：虽然NASH与肥胖的人类和小鼠的胰岛素抵抗密切相关，但在某些基因KO小鼠模型中，这两种综合征显然是分离的。这一建议的中心假设通过假设肝细胞胞浆乙酰辅酶A水平通过产生棕榈酸/胆固醇毒性来促进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糖异生来促进胰岛素抵抗来解决这一谜题。因此，我们认为，虽然肝细胞乙酰辅酶A池经常协调上升，但在某些肥胖和脂肪肝的遗传性小鼠模型中，它们可能是分离的。为了验证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，该技术结合了独特的RNA修饰和GalNAc定向的肝细胞靶向在“自我递送”RNAi(SdRNA)化合物中。这些化合物在小鼠皮下注射一次后，可以使单个或多个靶向肝细胞基因沉默2个月或更长时间。使用GalNAc-sdRNA，我们可以选择性地针对每个人并使其沉默 在产生胞质乙酰辅酶A的多种途径(例如ACLY和ACSS2途径)与线粒体乙酰辅酶A(例如FATP2/5途径)之间进行比较，同时避免产生多基因KO小鼠的高昂成本和时间。在目标1中，我们将这一强大的RNAi技术与一种新的方法相结合，该方法可以量化肝细胞线粒体乙酰辅酶A和总细胞乙酰辅酶A，以确定ACLY、ACSS2和FATP2、FATP5在LEAN和HFD小鼠中对这些特定的肝细胞乙酰辅酶A池的相对贡献。在目标2中，我们建议通过向目标1学习适当的GalNAc-sdRNA基因靶向来清除NAFLD/NASH小鼠模型肝细胞胞浆乙酰辅酶A，并确定其对肝脏甘油三酯、炎症、纤维化和糖耐量的影响以及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水平的降低是否通过下调肝细胞转录因子TAZ来减少星状细胞产生的胶原，TAZ驱动肝细胞印度刺猬(IHH)的分泌和星状细胞的激活。这些研究还将解决Kupffer和Stellate细胞功能障碍是由肝细胞NAFLD驱动还是由循环因素独立促进的关键问题，或者两者兼而有之。最后，在目标3中，我们将通过以下方式测试潜在的治疗策略 确定针对多个肝细胞基因的GalNAc-sdRNAs是否将同时缓解肥胖症/2型糖尿病的NAFLD、NASH和胰岛素抵抗的所有三种症状。这种方法具有主要的临床优势，因为针对不同基因的多个GalNAc-sdRNAs由相同的化学成分组成，并且与小分子不同，作为单一治疗剂在临床上使用进行评估。

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.