凝血酶敏感蛋白（TSP-1）对肝脏胰岛素抵抗的调控及作用机制研究

The liver is a central organ for glucose and lipid metabolism, and thus hepatic insulin resistance plays a key role in the development and progress of Type 2 diabetes. However, the mechanism of hepatic insulin resistance remains to be clarified. We have recently performed a proteomics-based study and, for the first time, found that the serum levels of TSP-1（Thrombospondin-1）were significantly reduced in the patients of NAFLD who had a significant improvement in hepatic fat content and glucose metabolic profiles. Furthermore, hepatic expression of TSP-1 was significantly elevated in the fatty liver of obese rats and in steatotic hepatocytes. The siRNA-mediated knockdown of TSP-1 and its receptor, CD47, significantly prevented palmitate-induced insulin resistance and increased insulin-dependent Akt-FoxO1 activities in hepatocytes. Therefore, we hypothesize that TSP-1 is a novel cytokine that can induces hepatic insulin resistance. For clarifying this hypothesis, the project aims to investigate the mechanisms underlying TSP-1 negatively regulates hepatic insulin signaling by using different cellular and animal models of hepatic steatosis. We will also utilize different methods to overexpress or down-regulate TSP-1 and/or CD47 in these steatotic models to establish its role in the regulation of hepatic insulin signalling and hemostasis of glucose and lipid metabolism. By completing this project, we would be able to identify TSP-1 being a new factor of inducing hepatic insulin resistance, and expect that TSP-1 might be a potential therapeutic target for the treatment of Type 2 diabetes and fatty liver diseases and also clarify that TSP-1 could be a usful bio-marker for therapeutic evaluation or diagnosis of the metabolic diseases.

肝脏是糖脂代谢中枢器官，因此肝脏胰岛素抵抗在2型糖尿病发病中起到重要作用。然而，迄今仍缺乏对肝脏胰岛素抵抗的充分认识和有效防治措施。本研究基于前期蛋白质组学结果，首次发现NAFLD患者治疗后，血清凝血酶敏感蛋白（Thrombospondin-1，TSP-1）随着肝脂含量下降和糖代谢改善而显著降低。进一步研究显示，在肥胖大鼠的脂肪肝和脂肪变性肝细胞模型中TSP-1表达显著升高；利用siRNA沉默TSP-1及其特异性受体CD47的表达，明显改善肝细胞胰岛素敏感性。由此推测，TSP-1可能是引起肝脏胰岛素抵抗的重要因子。本研究将利用不同胰岛素抵抗动物及细胞模型，通过TSP-1过表达或沉默等分子生物学手段，深入探索TSP-1及与CD47的相互作用对肝脏胰岛素信号的负向调节机制，进一步完善肝脏胰岛素抵抗发生机理，并使TSP-1可能成为2型糖尿病治疗潜在靶点和用于临床诊断和评估预后标志物。

凝血酶敏感蛋白（Thrombosponsin, Thbs）是一类细胞外基质蛋白，在组织损伤或细胞应激时分泌。然而，Thbs的生物学功能尚不清楚。在这里，我们首次发现了肝活检确诊的NAFLD患者血清Thbs1水平较健康人群明显升高；经过降肝脂治疗后，随着肝脏脂肪含量降低病人血清Thbs1水平随之降低。为了进一步探讨Thbs1对胰岛素抵抗小鼠肝脏脂肪变性的影响，课题组采用高脂高蔗糖饮食构建小鼠肝脏脂肪沉积模型，并给予重组Thbs1蛋白注射，发现外源给予Thbs1可以改善小鼠肝脏脂质沉积。随后，采用高糖高胰岛素诱导原代肝细胞或HepG2细胞的脂质积聚，并用重组人Thbs1蛋白或稳定高表达Thbs1质粒干预，在细胞层面上发现 Thbs1可以抑制肝细胞脂质从头合成。在机制上，我们发现Thbs1可以抑制肝脂生成基因的表达，导致肝脂堆积减少，而在CD36受体敲除的肝细胞中这种抑制作用消失，证明Thbs1对肝脂的调节作用是通过CD36受体实现的。此外，我们还发现注射Thbs1蛋白改善小鼠葡萄糖代谢，在细胞层面Thbs1抑制原代肝细胞糖异生基因表达，进而抑制葡萄糖输出，从而改善高血糖和葡萄糖代谢。最后，我们还采用Thbs1的一种小分子多肽类似物ABT-526处理肝细胞，发现该小分子多肽类似物可以抑制肝细胞脂质从头合成及肝细胞葡萄糖生成。以上这些结果证实Thbs1是NAFLD的一种新型生物标志物，针对调节Thbs1的药理改造可能对肝脂肪变性和2型糖尿病具有潜在的治疗作用。