BAP31在非酒精性脂肪肝和糖代谢中的作用研究

BAP31/BCAP31 (B cell receptor association protein 31) is an evolutionarily conserved and ubiquitously expressed membrane protein of the endoplasmic reticulum (ER). Originally, it was identified as an associated protein of B cell antigen receptor, which may contribute to any functional differences between membrane IgM and IgD. Currently, most studies have focused BAP31 function on apoptosis, protein sorting, retention, survival or degradation. Some literature reported that BAP31 modulated VLDL assembly and secretion, suggested that BAP31 may play some roles on lipid metabolism in liver. In the previous study, we have generated the first line of liver-specific BAP31conditional knockout mice. And the preliminary results revealed hyperglycemia, hypertriglycemia, enhanced hepatic lipid accumulation, and reduced Akt phosphorylation in BAP31-deficient mice, suggested BAP31 may influence lipid metabolism and insulin signaling in liver. In the current project, BAP31-deficient mice and the littermate controls will be treated with high-fat diet (HFD) and induced to Non-Alcoholic Fatty Liver Disease (NAFLD); the primary hepatocytes will also be isolated and treated with high concentration of fatty acids or glucose, to evaluate the function of BAP31 on the development of NAFLD and on the regulation of hepatic lipid accumulation. Additionally, the experiments of glucose tolerance test, insulin tolerance test and insulin sensitivity assay will be performed, to evaluate the function on insulin signaling and insulin resistance. The function of BAP31 on gluconeogenesis will also be evaluated, eventually to explore the function of BAP31 on glucose metabolism in liver. Furthermore, Chip-on-chip assay will be performed and the downstream genes of BAP31 will be identified, which help to explain how BAP31 regulates lipid and glucose metabolism, and then finally reveal the underlying molecular mechanism. Overall, we would like to clarify the biological function of BAP31 on regulating the development of NAFLD and glucose metabolism from the current study, and find some effective and safe options to prevent the development of NAFLD.

BAP31是一种广泛表达的内质网膜蛋白，其在细胞凋亡、蛋白质运输和降解等的作用研究较多，而在肝脏脂肪代谢和脂质沉积等方面研究尚未系统展开。通过前期研究，我们构建了首例肝脏条件性BAP31基因敲除小鼠，发现BAP31基因敲除后可诱导血糖和血液甘油三酯升高，肝脏脂质沉积增加，并降低Akt磷酸化水平，提示其对肝脏脂肪代谢和胰岛素信号通路有影响。本研究拟使用BAP31基因敲除小鼠动物模型，通过高脂肪饮食诱导非酒精性脂肪肝形成，分离肝脏原代细胞并诱导脂质沉积，阐明BAP31基因敲除对肝脏脂质沉积的影响，明确其对非酒精性脂肪肝形成和肝脏脂肪代谢的调节作用；进一步探讨对葡萄糖耐受、胰岛素抵抗和肝脏糖异生功能的影响，明确其对肝脏糖代谢的调节机制；并利用Chip-on-chip技术筛选下游靶基因，探讨深层次分子机制，最终阐明BAP31在糖脂代谢方面的生物学功能，并寻找出一条有效途径抑制非酒精性脂肪肝形成。

BAP31是一种在多种组织中广泛表达的内质网膜整合蛋白，在细胞凋亡、蛋白质运输和降解、蛋白质内质网滞留等方面的研究较多。有临床研究报告显示，BAP31基因组序列突变或缺失可诱导肝脏脂肪变性，而有关其在肝脏脂质代谢和葡萄糖代谢等方面的系统研究尚未见报道。本项目使用了肝脏细胞特异性敲除BAP31基因缺陷小鼠，通过饲喂高脂肪饮食诱导肝脏脂肪变性和肥胖发生，并检测BAP31基因敲除对脂肪肝形成和胰岛素抵抗等方面的调节作用。研究结果显示，BAP31基因敲除促进了SREBP1蛋白向细胞核聚集及其信号通路的激活，促进与脂质合成相关基因表达增加，促使肝脏脂质合成和脂质沉积增加，加速脂肪肝形成；并降低机体葡萄糖耐受和胰岛素敏感性，加剧肝脏炎症反应，导致胰岛素抵抗产生。BAP31基因敲除小鼠对饥饿诱导的相关效应不敏感，24小时饥饿处理后，BAP31基因敲除小鼠血清β-羟丁酸和甘油含量减低，肝脏油脂含量升高。饥饿诱导的相关效应受PPARα信号通路所介导。研究发现，BAP31基因敲除通过抑制PPARα信号通路激活，降低与脂肪酸氧化相关基因表达，抑制脂肪酸氧化，导致肝脏组织脂肪消耗减少，促使肝脏脂质沉积增加和脂肪肝形成。BAP31基因敲除抑制了SIRT1信号通路激活，降低与肝脏糖异生相关基因表达，导致肝脏细胞葡萄糖合成能力减弱。BAP31基因敲除促进了内质网应激增加，加剧Tunicamycin诱导的肝脏损伤和肝脏脂肪变性。同时，BAP31基因敲除加剧了Acetaminophen诱导的肝脏损伤，可能通过抑制Nrf2信号通路，进而削弱BAP31基因敲除小鼠肝脏保护作用机制。本项目系统阐述了BAP31基因在肝脏组织脂质代谢和葡萄糖代谢等方面的生物学功能，扩充了BAP31基因在能量代谢方面的生物学研究。现有研究认为，肥胖刺激抑制BAP31基因表达，降低了BAP31基因在肝脏组织中的保护作用。本研究为临床上寻找可以特异性激活BAP31基因表达的化合物激动剂，阻止由于肥胖刺激诱导的BAP31基因表达降低及保护功能减弱，增强机体保护作用机制，减弱肥胖引起的相关病症，提供了有利理论依据。