ANGPTL8经ERK/NF-kB信号通路调控2型糖尿病脂代谢紊乱作用机制

ANGPTL8 is a newly identified hormone abnormally highly expressed in the plasma of type 2 diabetes patients with insulin resistance. It may regulate the metabolism of both glucose and lipid, especially resulting in significantly increased content of triglycerides (TG) in the plasma. However, the involved molecular mechanism is still far from understood. Our previous study indicated that ANGPTL8 could up-regulate the phosphorylation level of ERK protein. Based on the relationship of ANGPTL8 gene with lipid metabolism and ERK, we speculated that ANGPTL8 may regulate the lipid metabolism via ERK/NF-kB signaling pathway during the occurrence and development of type 2 diabetes. In this study, we plan to over-express and knock out ANGPTL8 gene in the liver of mice using adeno-associated virus and CRISPR/CAS9 technique respectively to observe both in vitro and in vivo how it regulates the lipid metabolism before and after insulin resistance occurrence. Then, the blocking or over-expressing genes in the pathway, immune co-precipitation, point mutations, fragment cut-off and other methods will also be applied to study the molecular regulating mechanism of ANGPTL8 via ERK/NF-kB signaling pathway. This study will elucidate the role and the molecular regulating mechanism of ANGPTL8 in Type 2 Diabetes with disordered lipid metabolism. A novel targeting site may therefore be provided for clinical treatment of the disease.

ANGPTL8是在胰岛素抵抗2型糖尿病患者血浆中新发现的一种异常高表达激素，它可同时调控糖脂代谢，尤其能使血浆中甘油三脂（TG）含量显著升高，但其作用机制尚不清楚。前期研究发现，ANGPTL8可上调ERK蛋白磷酸化。根据ANGPTL8基因与脂代谢及ERK的密切关系，我们推测该基因可能是通过介导ERK/NF-kB信号通路来调控2型糖尿病脂代谢紊乱发生和发展。因此本项目拟通过构建ANGPTL8腺相关病毒过表达和基因敲除小鼠模型，分析该基因在2型糖尿病脂代谢紊乱形成中作用；体内外实验研究该基因在正常和胰岛素抵抗条件下对ERK/NF-kB信号通路影响及其对下游脂代谢相关基因表达调控；利用阻断或激活基因表达、免疫共沉淀、点突变、片段截短等方法分析该基因调控ERK/NF-kB信号通路的分子机制。由此阐明ANGPTL8在2型糖尿病脂代谢紊乱形成中作用及分子机制，为该疾病的临床治疗提供可能的新作用靶点。

高脂摄入是导致非酒精性脂肪性肝纤维化主要原因之一，脂代谢异常和慢性炎症与非酒精性脂肪性肝纤维化形成密切相关，但具体机制尚不明了。血管生成素样蛋白8(ANGPTL8)是新发现一种在人肝脏高表达分泌性蛋白，前期发现ANGPTL8在非酒精性脂肪肝病患者的血浆中含量显著升高，但具体作用需深入研究。通过ELISA分析在肝硬化及肝癌患者的血清中ANGPTL8的含量显著升高。高脂中OA和PA可以诱导ANGPTL8与炎症因子表达升高，炎症因子可促进ANGPTL8高表达，ANGPTL8缺失可抑制进餐诱导的炎症因子升高。ANGPTL8可促进HepG2细胞的脂质沉积，并促进HFD、HFHC诱导的脂肪肝形成。高脂喂养24w（周）及36w的非酒精性脂肪性肝纤维化小鼠中，ANGPTL8 KO小鼠的肝纤维化程度明显轻于WT小鼠。同样的，ANGPTL8 KO小鼠在CCL4诱导的肝纤维化模型中的肝纤维化程度明显轻于WT小鼠。ANGPTL8通过增强高脂诱导的炎症和免疫反应，并调控TGFβ1及下游ERK/NF-κB信号通路促进非酒精性脂肪性肝纤维化的形成。炎症联合高脂可刺激ANGPTL8分泌增加，分泌性ANGPTL8直接影响肝星状细胞形态、增殖能力和储脂能力；ANGPTL8蛋白与LILRB2受体相互作用后促进LILRB2表达，并上调ERK磷酸化激活自噬从而直接活化肝星状细胞。二甲双胍可降低ANGPTL8的表达，高脂喂养小鼠24w再经二甲双胍治疗4w后，ANGPTL8 KO小鼠脂肪肝和肝纤维化明显轻于WT小鼠；二甲双胍可增加肝星状细胞的脂滴抑制肝星状细胞失活，ANGPTL8抑制二甲双胍对肝星状细胞的失活作用。以上结果表明ANGPTL8促进高脂诱导的非酒精性脂肪性肝纤维化的形成；ANGPTL8可调节进食诱导的炎症反应，通过与LILRB2受体相互作用，进而激活TGFβ1/ERK介导的自噬途径促进非酒精性脂肪性肝纤维化的形成；ANGPTL8是二甲双胍治疗非酒精性脂肪性肝纤维化的新靶点。