探讨溶酶体膜蛋白Sidt2通过自噬影响肝脏脂肪代谢的机制

Sidt2 has been identified by mass spectrometry in the mouse lysosomal membrane fraction and was later found to be a resident lysosomal membrane protein with nine transmembrane domains. Since the function of Sidt2 is unknown, it has been the subject of our two previous grants from National Natural Science Foundation of China. One has been completed in 2011, and the other was obtained in 2012. In those projects, we had developed a global Sidt2 knockout mouse, which had elevated serum triglyceride and alanine aminotransferase, hepatocellular ballooning and liver triglyceride storge resembling human nonalcoholic fatty liver disease, besides an elevated fast glucose and an impaired glucose tolerance. The lipid metabolic disturbance were even remarkable after a high-fat diet. Compared with their wild-type littermates, the global sidt2 knockout mice displayed reduced serum β-hydroxybutyric acid indicating a lower level of fatty acid catabolism by β-oxidation. Concurrently, the global Sidt2 knockout mice displayed a decreased LC3-II level, which is known to be an autophagy marker, and an increased p62, which was regarded as a substrate of autophagic degradation, both indicating an impaired autophagic process. These results suggest Sidt2 may regulate liver lipid metabolism via autophagy. To answer the question whether the hepatic lipid storge in the global Sidt2 knockout mice was the direct impact of Sidt2 on the liver lipid metabolism or was secondary to the impaired glucose tolerance, we plan to raise liver-specific Sidt2 knockout mice by breeding the Sidt2 Flox/Flox with mice carring the Cre recombinase gene driven by the liver albumin promoter. Lipid and glucose metabolism would be investigated in the liver-specific Sidt2 knockout mice and would be compared with those from global Sidt2 knockout mice. Sidt2 regulation of liver lipid metabolism via autophagy would be studied by method of cellular biology and application of nuclide tracing technique, with the focus on the passways of both hepatic lipid metabolism and autophagy signaling. Through the high throughput whole exon sequencing, an exonic point mutation at a conserved amino acid site of Sidt2 has been identified in a woman with severe obesity accompanied by type 2 diabetes and nonalcoholic fatty liver disease. In this study, we would design a plasmid containing the mutation and evaluate the consequence of this mutant at a cellular level. Through this study, we expect to discover that sidt2 is a new therapeutic target of nonalcoholic fatty liver disease.

Sidt2是申请人2011年结题NSFC青年项目和2012年面上项目的研究主体。我们培育了Sidt2全身剔除小鼠，该鼠肝脏脂质贮积，血清肝酶甘油三酯水平升高，类似人类非酒精性脂肪肝病，高脂饲养后上述异常更加显著。该鼠血清β-羟丁酸显著减少，提示脂肪降解通路脂肪酸β-氧化水平下降；同时自噬标记蛋白LC3-II减少，而自噬底物p62蓄积，提示自噬水平下降。本项目拟在已有Sidt2 Flox小鼠基础上，培育仅肝脏Sidt2剔除小鼠，观察是否同样有脂质代谢紊乱，以明确Sidt2是否直接影响肝脏脂质代谢。并用分子生物学及核素标记等技术，在动物和细胞水平研究Sidt2通过自噬信号通路调节肝脏脂质代谢，明确Sidt2缺陷引起脂肪肝的机制。并拟细胞水平研究在一肥胖患者发现的人类Sidt2基因遗传保守位点的点突变是否对脂质代谢及自噬有影响。通过该项目的研究，预期能发现Sidt2是干预治疗脂肪肝病的新靶点。

目的:本研究前期利用Cre/Loxp系统成功构建了Sidt2全身敲减小鼠，研究发现小鼠sidt2基因全身敲除存在肝脏脂质代谢障碍。为排除其他组织器官的影响对肝脏脂质代谢产生继发性影响，本项目拟建立肝脏组织特异性剔除小鼠模型，研究酶体膜蛋白Sidt2调控肝脏脂肪代谢的机制。.方法:构建肝脏特异敲除Sidt2小鼠及其对照组小鼠，普食和高脂喂养下观察两组小鼠体重增长情况，进食量，肝脏重量；HE染色和油红Ｏ染色观察小鼠肝脏病理差异；生化检测小鼠肝脏甘油三脂，胆固醇含量，血清游离脂肪酸含量；全自动生化检测仪分析小鼠血清甘油三酯，胆固醇，谷丙转氨酶和谷草转氨酶含量；micro-CT检测小鼠脂肪组织体积；WB检测自噬相关蛋白LC3Ⅱ，P62的表达水平；电镜观察小鼠自噬囊泡；原代培养肝细胞检测BafA1处理条件下LC3Ⅱ，P62的表达水平。Co-IP检测Sidt2的相互作用蛋白。.结果:普食和高脂喂养条件下肝脏特异敲除小鼠体重、进食量、肝脏重量均无明显差异；HE染色和油红O染色显示肝脏特异敲除小鼠肝脏脂质贮积，肝脏甘油三酯含量增加，高脂喂养差距更加明显，且转氨酶明显升高。WB检测肝脏LC3Ⅱ，P62水平增高。电镜下脂滴增多，自噬囊泡增多。肝脏特异性敲除Sidt2小鼠来源肝细胞LC3Ⅱ增高。高脂刺激下，mTOR信号通路激活受阻。.结论 本研究证实了Sidt2是肝脏脂质代谢的一个重要蛋白。Sidt2可能通过mTOR信号通路参与自噬影响脂质代谢。我们的研究结果丰富了对 Sidt2蛋白功能的认识，补充了自噬溶酶体相关蛋白的功能，为未来脂质代谢类疾病提供了理论基础。