Hepatic insulin resistance and metabolic disease

描述(由申请人提供)：这是一项题为“肝脏胰岛素抵抗和代谢性疾病”的新提案，基于我们实验室在过去5年中对LDKO(肝脏特异性IRS1-/-“Irs2-/-)和LTKO(肝脏特异性IRS1-/-”Irs2-/-“FoxO1-/-)小鼠所做的工作。LDKO-小鼠表现出全身代谢失调，包括肝脏和外周胰岛素抵抗、高血糖、中度高胰岛素血症和进行性NAFLD(非酒精性脂肪性肝病)。NAFLD的病因源于胰岛素抵抗、肥胖、高脂血症和糖尿病。由于LTKO-小鼠表现出正常的营养动态平衡，我们假设激活的肝脏FoxO1，而不仅仅是肝脏甘油三酯的积累，促进炎症进展到危及生命的坏死/凋亡和再生循环，最终以损害-NAFLD进展为NASH(非酒精性脂肪性肝炎)和肝细胞癌(肝细胞癌)的特征。为了研究潜在的病理生理学，我们将重点放在线粒体功能障碍上，这是由于长期激活的FoxO1增加了数百个基因的表达，包括Hmox1编码的血红素加氧酶-1和Ppif编码的亲环素D。为了探讨分子机制，我们建议使用靶向siRNA的纳米颗粒递送来抑制NAFLD起始(10周龄)和进展(10个月龄)期间肝脏FoxO1、Hmox1或Ppif的表达。为了在我们的实验室中使用这项技术，我们与东北大学药学院的Amiji小组达成了一项“联盟协议”。本实验旨在探讨FoxO1介导的肝线粒体功能障碍与进行性NAFLD/NASH在全身炎症和肌肉胰岛素作用中的关系，从而促进糖尿病的发生：i)通过调节FoxO1、Hmox1或Ppif的表达来恢复线粒体功能，减轻肝脏炎症，NASH及其进展为肝细胞癌，从而在持续的肝脏胰岛素抵抗过程中研究FoxO1介导的线粒体功能障碍。Ii)研究LDKO-小鼠骨骼肌胰岛素抵抗，以确定肝脏炎症与骨骼肌胰岛素作用和代谢紊乱之间的关系。Iii)在肝脏线粒体功能障碍和通过抑制FoxO1、Hmox1或Ppif抑制NAFLD前后，使用亚磷酸盐特异性单抗文库，对LDKO小鼠肌肉中IRS1的Ser/Thr磷酸化进行定量。由于LDKO小鼠不会受到肥胖/肥胖小鼠下丘脑型肥胖的显性影响，因此我们的实验主要集中在肝脏胰岛素抵抗和NAFLD之间的关系，以及它发展为全身性胰岛素抵抗和糖尿病。

DESCRIPTION (provided by applicant): This is a new proposal entitled "Hepatic insulin resistance and metabolic disease" that is based upon work conducted in our laboratory during the past 5 years with LDKO (hepatic-specific Irs1-/-"Irs2-/-) and LTKO (hepatic-specific Irs1-/-"Irs2-/-"FoxO1-/-) mice. LDKO-mice display systemic metabolic dysregulation, including hepatic and peripheral insulin resistance, hyperglycemia, moderate hyperinsulinemia, and progressive NAFLD (nonalcoholic fatty liver disease). The etiology of NAFLD is rooted in insulin resistance, obesity, hyperlipidemia, and diabetes. As LTKO-mice display normal nutrient homeostasis, we posit that activated hepatic FoxO1, rather than the mere accumulation of hepatic triglyceride, promotes inflammation that progresses to life- threatening necrosis/apoptosis and cycles of regeneration that culminate with damage-characteristic of the progression of NAFLD to NASH (nonalcoholic steatohepatitis) and HCC (hepatocellular carcinoma). To investigate the underlying pathophysiology, we focus upon mitochondrial dysfunction that develops as a result of chronically activated FoxO1 that increases the expression of hundreds of genes, including hemeoxygenase- 1 (encoded by Hmox1) and cyclophilin D (encoded by Ppif). To interrogate the molecular mechanisms, we propose to use nanoparticle delivery of targeted siRNA to suppress the expression of hepatic FoxO1, Hmox1, or Ppif during initiation (10 weeks of age) and progression (10 months of age) of NAFLD. To enable this technology in our laboratory, we have formed a 'Consortium Agreement' with the Amiji group in the School of Pharmacy at Northeastern University. The mouse-based experiments will investigate the relation between FoxO1-mediated hepatic mitochondrial dysfunction and progressive NAFLD/NASH upon systemic inflammation and muscle insulin action that contributes to diabetes in the following Specific Aims: i) Investigate FoxO1-mediated mitochondrial dysfunction in LDKO-mice by modulating the expression of FoxO1, Hmox1 or Ppif to restore mitochondrial function and attenuate hepatic inflammation, NASH and its progression to HCC during persistent hepatic insulin resistance. ii) Investigate skeletal muscle insulin resistance in LDKO-mice to establish the relation between hepatic inflammation and dysregulated skeletal muscle insulin action and metabolism. iii) Quantify Ser/Thr-phosphorylation of IRS1 in muscle of LDKO-mice using a library of phosphosite-specific monoclonal antibodies before and after resolution of hepatic mitochondrial dysfunction and NAFLD by suppression of FoxO1, Hmox1 or Ppif. Since the LDKO-mice are uncomplicated by the dominant effects of hypothalamic-based obesity encountered with ob/ob-mice, our experiments focus squarely upon the relation between hepatic insulin resistance and NAFLD, and its progression to systemic insulin resistance and diabetes.