Mechanisms of insulin resistance related to nonalcoholic steatohepatitis

该项目的长期目标是了解肥胖/ 2型糖尿病患者引起脂肪肝（NAFLD）和非酒精性脂肪性肝炎（NASH）的潜在机制，以及这些机制与全身胰岛素抵抗的关系。我们还试图解开一个悖论，即尽管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。该提案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驱动胰岛素抵抗，从而解决了这个谜题。因此，我们提出，虽然肝细胞乙酰辅酶a池经常协调升高，但在某些肥胖和脂肪肝的遗传小鼠模型中，它们可能断开。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导向的肝细胞靶向“自我递送”RNAi （sdRNA）化合物。这些化合物可以在小鼠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它们

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.