去唾液酸糖蛋白受体1(ASGR1)调控脂质转运的分子机制及其在非酒精性脂肪肝炎中的作用研究

Asialoglycoprotein receptor 1 (ASGR1), which is specifically expressed in hepatocytes, has a function of mediating endocytosis and degradation of glycoproteins. It is reported that in human beings , heterozygous carriers of a deletion mutation had a lower level of lipid content in serum than noncarriers, but the intrahepatic lipid level and lipid transport status were unclear. We found that Asgr1 knockout mice had lower serum lipid levels, decreased lipid secretion, and decreased expression of lipoprotein assembly-related proteins in hepatocytes compared with wild-type mice. After a high fat high cholesterol diet, homozygous knockout mice developed severe nonalcoholic steatohepatitis (NASH) symptoms. We hypothesis that ASGR1 influences serum lipid level by regulating lipoprotein endocytosis and secretion. Based on preliminary data, we will observe the effects of ASGR1 deletion on lipoprotein endocytosis, assembly, metabolism and transport means firstly, confirming that it is a key target for regulating intracellular lipid transport. Secondly, to find potential interacting proteins of ASGR1 and related cell signaling pathway by proteome analysis and transcriptomics analysis. Finally, to verify interaction effects between two proteins and carry out the functional complementation analysis in vivo and in vitro to elucidate the molecular mechanism by which ASGR1 deletion leads to intrahepatic lipid accumulation. An in-depth understanding of the physiological functions of ASGR1 in lipid transport will accumulate data for new approaches to the development of interventions in NASH and metabolic diseases.

肝细胞特异表达的去唾液酸糖蛋白受体1（ASGR1）参与糖蛋白的内吞及降解。ASGR1杂合缺失突变携带者血脂水平显著降低，但其肝内脂质水平和脂质转运状况尚不清楚。我们发现Asgr1敲除鼠的血脂水平下降，脂质分泌的能力降低，肝细胞中脂蛋白组装相关蛋白表达量下降；在短暂给予高脂高胆固醇饮食后，纯合敲除小鼠出现严重的非酒精性脂肪肝炎（NASH）症状。我们推测ASGR1调控肝内脂蛋白的内吞或分泌过程，从而调节血脂水平。基于前期实验基础，本项目将首先观察ASGR1缺失对脂蛋白吸收、代谢、组转及转运方式的影响，确证其是调控细胞内脂质转运的关键靶标；其次通过蛋白质谱和转录组学分析寻找ASGR1的潜在互作蛋白及相关信号通路；最后在细胞和小鼠敲除模型中进行验证及回复实验，阐明ASGR1缺失导致肝内脂质积累的分子机制。深入认识ASGR1在脂质转运过程中的生理功能，将为研发干预NASH和代谢性疾病提供新思路。

大规模的群体遗传研究发现，ASGR1突变携带者血液中的非HDL-C水平和心血管疾病患病风险降低。然而，具体机制尚不清楚。本研究构建了Asgr1基因敲除小鼠，发现基因缺失小鼠血液中的非HDL-C和甘油三酯水平显著降低。进一步分析发现，小鼠血脂水平降低是由于肝脏对VLDL/LDL分泌减少而摄取增加引起的。这两种表型均与SREBPs的两个关键靶基因——MTTP和PCSK9的表达降低有关。此外，ASGR1缺失上调INSIG1的表达水平，从而将SREBPs锚定在内质网上，抑制其剪切激活。最后，在ASGR1缺失细胞中进行了两次拯救实验。INSIG1敲降和ASGR1回复表达都独立地逆转了ASGR1突变的表型，恢复了SREBP信号，表现为APOB分泌的增加和LDL摄取的减少。我们的研究揭示了ASGR1- INSIG1 – SREBPs调节血脂的作用机制，ASGR1缺失通过抑制nSREBPs剪切激活，导致非HDL-C的显著降低。这些研究结果提示，ASGR可以作为开发降脂药物的潜在靶点。