LAMP3在肝自噬相关脂分解中的调控作用及其机制研究

Previous researches have reported a critical function for autophagy in lipid metabolism, but the role of lysosome, the main site of autophagy, in autophagy-associated lipid metabolism is unclear. In the microenvironment of hypoxia, lysosome-associated membrane protein 3 (LAMP3) was remarkably upregulated. While autophagy and lipolysis were also accelerated when hypoxia. Our previous results showed that LAMP3 expressed relative highly in liver cell lines (LO2, HepG2 and Huh7). In high fatty acid treated hepatocytes, and liver tissues of HFD and ob/ob mice, LAMP3 expression were significantly higher than matched controls. After LAMP3 overexpressed in HepG2 cells, autophagy was activated, and the intracellular lipids induced by free fatty acid were decreased. It suggested that LAMP3 may play an important role in autophagy-associated lipolysis. In this project, we will combine the overexpression and knockdown of LAMP3 with autophagy specific inhibitor to elucidate the effect of LAMP3 on autophagy-associated lipolysis. Genome-wide mRNA expression profile detected by microarray and bioinformatics analysis strategy will be used to comprehensively screen downstream targets and the key signaling pathway of LAMP3. Then the modulation mechanism of LAMP3 on autophagy-associated lipolysis will be validated through specific compound targeting LAMP3 and recombinant adeno-associated virus in HFD and ob/ob mice. The objective of this study is to elucidate the regulation function of LAMP3 on autophagy-associated lipolysis and uncover its molecular mechanism. It might provide a potential therapeutic target for lipid metabolism disorder-associated diseases.

近年发现自噬参与肝脏脂代谢过程，但自噬完成的主要场所溶酶体在自噬相关脂代谢中的作用并不清楚。项目基于在缺氧条件下，溶酶体膜蛋白LAMP3表达上调；而缺氧时也表现自噬激活、脂滴分解加速的事实，结合课题组前期发现：LAMP3主要在肝细胞表达，在高脂环境下其表达下调；上调肝细胞LAMP3的表达，可激活自噬，减少游离脂肪酸诱导的脂质沉积，推测LAMP3可能在肝自噬相关脂分解中发挥重要调控作用。故项目拟通过双向调节LAMP3，结合自噬抑制剂使用，明确LAMP3在肝自噬相关脂分解中作用；经表达谱芯片与生物信息学分析筛选LAMP3关键靶分子，解析其下游途径；进而以小鼠脂肪肝和肥胖为模型，以LAMP3特异激动小分子和腺相关病毒为干预手段，探讨其成为恢复脂代谢自稳态干预靶点的可能性。从而阐明LAMP3与肝自噬相关脂分解的关联，揭示其信号级联机制，旨在深入理解脂代谢紊乱形成机理，为其干预提供新的策略与依据。

溶酶体相关膜蛋白3（Lysosome associated membrane protein 3, LAMP3）是一种高度糖基化的蛋白，是LAMPs家族成员之一。LAMPs家族在自噬溶酶体的融合过程中起着至关重要的作用。最近，自噬被证实可以调节肝脏脂肪分解；然而，LAMP3在脂质代谢中的生理功能尚不清楚。在该研究中，我们发现在非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）患者、高脂饮食和ob/ob小鼠的肝组织中，LAMP3的表达水平显著高于相应对照组；经游离脂肪酸处理后，肝癌细胞HepG2和QGY-7703中LAMP3的表达也明显增加。此外，在HepG2和QGY-7703细胞中过表达LAMP3，细胞内脂滴和甘油三酯明显沉积。进一步的研究表明，LAMP3的过表达可激活Akt，上调HepG2细胞中脂合成酶FASN和SCD-1的表达。此外，通过小分子抑制剂阻断PI3K/Akt信号通路，可削弱LAMP3过表达引起的甘油三酯含量增加。我们的研究结果表明LAMP3是肝脏脂合成代谢的重要调控因子，这为LAMP3成为脂质代谢紊乱相关疾病（如NAFLD和肥胖）的药物干预靶点提供了实验依据。

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