KLF14靶向负调节SREBP-1c在脂肪肝发病中的作用及其机制研究

Nonalcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disease and strongly associated with the metabolic syndrome. However, the molecular determinants of NAFLD remain largely unknown. Although human genetic studies indicate that the Krüppel-like factor 14 (KLF14) is associated with obesity and type 2 diabetes, but the underlying pathogenesis is still unclear. In our previous study, we identified that the hepatic KLF14 mRNA levels were reduced in type 2 diabetic mice and high-fat diet-induced obese mice compared with mice fed normal chow. Meanwhile, our data show that KLF14 suppressed the critical lipogenesis regulator SREBP-1c promoter activity in HepG2 cells. Chromatin Immunoprecipitation (ChIP) confirmed that KLF14 proteins bound to the potential element of the SREBP-1c promoter. Furthermore, Adenovirus-mediated overexpression of mouse KLF14 decreased mRNA and protein expression levels of SREBP-1c and its target genes and intracellular triglyceride content in primary hepatocytes. These findings imply a strong correlation between KLF14 expression and lipid metabolism in liver. The present project will be focused on the role of KLF14 in the regulation of hepatic lipid homeostasis using different mouse models. And we expect that up-regulation of KLF14 will rectify abnormal lipid metabolism and maintain lipid homeostasis in type 2 diabetic and high-fat diet-induced obese mice. The performance of this project might explore a novel potential target for curing NAFLD and type 2 diabetes.

非酒精性脂肪肝疾病（NAFLD）是与代谢综合征紧密相关的一种最常见慢性肝脏疾病，严重危害人类健康，其发病机制不清。遗传学研究提示KLF14与肥胖及2型糖尿病的发生、发展相关。我们前期研究发现在2型糖尿病小鼠和肥胖小鼠肝组织中KLF14的表达水平显著下调；ChIP和报告基因实验结果证实KLF14可以直接结合在脂肪合成关键调节因子SREBP-1c的启动子区并抑制其活性；并且上调KLF14表达显著降低SREBP-1c及其下游靶基因表达水平，从而降低原代肝细胞合成和分泌甘油三酯的能力。由此推测KLF14可能参与机体脂代谢的调节。本研究旨在阐明KLF14参与肝脏脂代谢调节作用中的机制，通过构建肝脏特异性敲除KLF14基因小鼠，并上调2型糖尿病小鼠和肥胖小鼠肝脏中KLF14的表达检测能否缓解机体脂代谢的紊乱，维持脂代谢稳态。本项目的开展有望为防治NAFLD和2型糖尿病提供新靶点。

在本研究中，我们探讨了转录因子KLF14（Krüppel-like factor 14，KLF14）在肝脏糖脂代谢中的作用及其调节机制。通过干预C57BL/6J、db/db糖尿病小鼠或者高脂饮食（HFD）诱导的肥胖（DIO）小鼠中KLF14的表达，检测小鼠糖脂代谢相关基因表达、葡萄糖耐受、胰岛素耐受和胰岛素抵抗的情况；并且在小鼠肝脏原代细胞中通过腺病毒上调或者下调KLF14的表达，检测肝脏糖异生相关基因表达、肝细胞合成和分泌葡萄糖情况以及对胰岛素信号通路的影响。研究结果证实，KLF14的表达受机体营养状况调控，正常C57BL/6J小鼠禁食后肝脏KLF14表达上升，同样在db/db小鼠以及ob/ob小鼠肝脏中KLF14表达与对照小鼠相比也显著上升；这表明KLF14可能参与了机体代谢的调控。由腺病毒介导的在小鼠肝脏原代细胞中上调KLF14的表达能够增加糖异生关键调控因子转录共激活因子过氧化物酶体增殖体激活受体γ共激活因子1-α（PGC-1α）及其下游靶基因磷酸烯醇式丙酮酸羧激酶（PEPCK）和葡萄糖-6-磷酸酶（G6pc）的mRNA和蛋白的表达水平，从而增加细胞葡萄糖产出。相反，下调KLF14的表达则会导致PGC-1α表达水平降低，从而降低肝脏原代细胞中的葡萄糖产出。双萤光素酶报告基因实验和ChIP实验结果证实KLF14可以和PGC-1α基因启动子上的KLF14潜在的结合元件CACCC相结合，并促进该启动子的活性。在体内实验中，我们发现在正常C57BL/6J小鼠肝脏中过表达KLF14会增加饥饿后小鼠血糖水平、损害葡萄糖耐受和胰岛素敏感性；相反，在糖尿病小鼠肝脏中敲低KLF14基因则会降低血糖水平，并改善葡萄糖耐受和胰岛素敏感性。综上所述，我们的研究证实KLF14能够通过调节PGC-1α的表达从而参与肝脏糖异生的调节。KLF14表达水平的上升可能与2型糖尿病的发生和发展密切相关。