内源性apoO调控脂肪分泌功能对动脉粥样硬化影响的研究

Apolipoprotein O (apoO) is a newly discovered apolipoprotein, and its biological function has not been fully elucidated. According to our previous studies, we have proposed for the first time that apoO has functions within the cells it is expressed in. Our following in vivo gain-of-function and in vitro loss-of-function studies identified apoO as an important inflammatory modulator in adipose tissue. In this item, we aim to generate fat tissue-specific apoO knockout mice and apoE double knockout mice to ascertain the intracellular function of apoO in adipokines secrection and atherosclerosis. Then,we are going to confirm the intracellular interaction between apoO and caveolin-1 by confocal laser scanning and co-immunoprecipitation; to make sure the key sequence in apoO that binding to caveolin-1 by site-directed mutagenesis and direct binding test; to explore the pivotal roles of TLR/NF-κB and GPRC5B/Fyn signaling in the caveolin-1-mediated action of apoO. This project will clarify the mechanism of intracellular action of apoO in adipocytes, and bring about new intervention target for atherosclerosis.

载脂蛋白O（apoO）是近年发现的一种新的载脂蛋白，其生理功能尚未完全阐明。据前期研究结果，本研究组首次提出“apoO主要在细胞内发挥功能”的观点，并通过体内gain-of-function和体外loss-of-function研究明确apoO在脂肪组织具有抗炎表型。本研究拟首先构建apoO脂肪特异性敲除小鼠及与apoE双敲除的小鼠模型，在体内明确apoO对脂肪分泌功能及动脉粥样硬化的影响。进一步体外利用基因转染改变apoO在脂肪细胞内的表达，借助激光共聚焦和免疫共沉淀明确apoO与caveolin-1的生物结合作用；采用多位点定点突变确定两者相互作用的关键序列；并借助体外实验探讨TLR/NF-κB和GPRC5B/Fyn信号通路在caveolin-1介导apoO调节脂肪分泌功能中的关键作用。通过实施本课题，可首次阐明apoO调节脂肪分泌功能的机制，为动脉粥样硬化的防治寻找到新的靶点。

脂肪细胞功能异常是肥胖重要的病理生理变化，也是肥胖导致动脉粥样硬化的关键环节。载脂蛋白O（apolipoprotein O, apoO）尽管为分泌型蛋白，但其非糖基化形式可存在于线粒体嵴MICOS复合体中，参与线粒体功能调节。该研究通过构建apoO整体敲除小鼠，体内表型研究未发现脂肪炎症因子的显著改变，但apoO敲除促进年龄和高脂饮食所致的肥胖和代谢紊乱，并导致动脉粥样硬化病变加重。考虑到apoO作为线粒体蛋白在棕色脂肪组织高表达，进一步构建脂肪组织特异性敲除小鼠模型，同样发现apoO特异性敲除促进高脂饮食所致的肥胖，同时棕色脂肪白色化明显。进一步机制研究发现棕色脂肪体内产热功能下降，与apoO敲除后致线粒体形态和功能改变相关。ApoO为线粒体内膜MICOS复合体的一部分，该复合体与线粒体外膜SAM复合体组成MIB复合物，介导线粒体蛋白转运，apoO缺失后，可能导致线粒体蛋白转运障碍，从而引起线粒体功能缺失，下一步研究拟通过线粒体蛋白组学筛选转运障碍的蛋白质，并探讨对线粒体功能影响的具体机制。总之，通过本研究，首次阐明apoO可能调节脂肪细胞内脂质代谢和肥胖的分子机制，并可能为肥胖相关的代谢性疾病和心血管疾病带来新的干预靶点。