GLP-1R结合蛋白SERP1抑制内质网应激所致肝脏脂质沉积的作用及机制

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with a variety of cardiovascular disease、cerebrovascular disease and cancer. Hepatic steatosis is the main characteristic of it and endoplasmic reticulum stress is essential for its development. Currently, Glucagon-like peptide 1(GLP-1) has been demonstrated to treat NAFLD effectively for steatosis. In order to improve the efficacy, GLP-1 receptor auxiliary binding proteins were screened by the membrane-based yeast two-hybrid system(MYTH). SERP1 was one of them and found that it could promote the receptor glycosylation and inhibit the process of endoplasmic reticulum stress. So SERP1 was supposed to play an important role in the occurrence and development of hepatic steatosis. Therefore, our work is aimed to (1) clarify the molecular mechanisms of SERP1 on GLP-1 and endoplasmic reticulum stress regulating triglyceride accumulation by establishing an overexpression/knockdown of SERP1 gene hepatocyte model; (2) analysis phenotype and molecular mechanisms of liver specific SERP1 knockout mouse model and SERP1-overexpression Ob/Ob mouse. Through this project, SERP1 is expected to improve GLP-1 treatment efficacy and specificity as new intervention target, also to provide a basis of accurate treatment for NAFLD.

非酒精性脂肪肝（NAFLD）和心脑血管疾病、恶性肿瘤密切相关。肝脏脂质沉积是其重要的病理特征，内质网应激在其发生发展中发挥关键作用。胰高血糖素样肽1（GLP-1）已被证实可减轻肝脏脂质沉积，但有效率尚需提高,因此深入机制探索尤为重要。本课题组前期筛选出胰高血糖素样肽1受体（GLP-1R）结合蛋白SERP1，首次发现其在肝细胞可促进GLP-1R糖基化和抑制内质网应激。本研究将：（1）建立沉默/过表达SERP1肝细胞模型，探讨SERP1在内质网应激和GLP-1作用下对甘油三酯合成的影响及分子路径；（2）采用肝脏特异性敲除小鼠和Ob/Ob小鼠过表达SERP1基因模型，观察不同条件下小鼠肝脏脂质代谢表型以及信号通路的变化；从细胞和动物水平阐明SERP1对肝脏脂质沉积的影响并解析相关机制。SERP1有望作为新的干预靶点提升GLP1类药物治疗的有效性和特异性，也为实现NAFLD的精准治疗提供理论依据。

本课题组前期应用膜酵母双杂交系统(Membrane-based yeast two-hybrid system，MYTH) 从人肝脏cDNA文库中筛选出34种GLP-1R结合蛋白， SERP1是其中之一。通过免疫共沉淀及免疫荧光法证实GLP-1R与 SERP1蛋白相互结合并定位于细胞膜，并发现SERP1在肝细胞可促进GLP-1R糖基化。在处于内质网应激状态的肝细胞将SERP1过表达后使用Exendin-4刺激发现GLP-1R下游的第二信使cAMP生成水平明显升高。说明SERP1在促进GLP-1R糖基化修饰维持受体功能中有重要作用。.本研究通过油酸（PA）和游离脂肪酸（FFA）处理的HepG2、L02肝细胞模型以及高脂饮食诱导脂肪肝模型小鼠、Ob/Ob小鼠肝脏组织样本均检测到SERP1蛋白水平明显下降。进一步建立敲低SERP1的肝细胞模型，发现脂肪合成相关基因PLIN和SCD表达明显增加，同时内质网应激标志物HSPA5和DDIT3升高。推测各种因素导致肝脏内质网应激发生后，通过抑制SERP1表达使脂肪合成相关基因上调来促进肝脏脂质沉积。此外予衣霉素刺激的HepG2细胞过表达SERP1后表现出内质网应激标志物CHOP，GRP78，Calnexin表达降低，提示SERP1高表达可有效缓解HepG2细胞中的内质网应激过程。目前GLP1类药物干预后可以改善非酒精性脂肪肝（NAFLD）已有较多的循证证据，但具体作用机制尚未明晰。SERP1有望提供新的视角，为突破NAFLD的治疗瓶颈提供数据支撑。.在积极探索SERP1参与NAFLD发生、发展的分子病理机制的同时，本课题组收集临床人群队列，利用代谢手术逆转NAFLD发生、发展、改善预后的数据，发现肌肉分泌因子与肝脏的脂肪沉积存在密切联系，较低的血肌酐与胱抑素C比值（CCR）水平与NAFLD风险增加独立相关，可以作为早期识别NAFLD高风险人群的新型血清学指标。