线粒体蛋白SIRT5对氧化/硝化应激诱导胰岛beta细胞损伤的调控作用及机制研究

Silent information regulators- - Sirtuins (SIRTs) are a family of nicotinamide adenine dinucleotide (NAD+)-depended deacetylases. In mammals, seven genes have been identified as SIRT 1-7. SIRT5, primarily localized in the mitochondrial matrix, regulating the metabolic enzymes activity and oxidative stress pathway. Cytochorme C (Cyto C) as one of the SIRT5 substrates, is a conserved mitochondrial intermembrane space protein, and has a central function in oxidative metabolism as well as apoptosis initiation. SIRT5 exhibited deacetylase activity against acetylated Cyto C, suggesting a role of regulating stress-induced mitochondrial-dependent apoptosis. .In the last five years, some evidences had indicated that SIRT5 played an important role in controlling the development of atherosclerosis, alcoholic steatohepatitis and age-related disease. However few studies have examined the direct association between the biological function of mitochondrial SIRT5 and diabetes..Our previous studies found that SIRT5 was also expressed in the mice pancreatic tissue, and the expression level of SIRT5 is variant in pancreas between the normal and diabetic mice.Therfore, we hypothesized that mitochondrial SIRT5 may play a critical role during the pancreatic beta cell apoptosis induced by elevated ROS/RNS in diabetes. To elucidate whether SIRT5 is required for apoptosis and the mechanism underlying the role of SIRT5 in regulating beta cell apoptosis in diabetic state, we sought to detecte the off-target and overexpression effects of SIRT5 on ROS/RNS-induced apoptosis in beta cell lines in vitro. The interactions between SIRT5 and pro-survival or apoptosis members in mitochondrion would also be detected..The aim of the present study is to investigate whether SIRT5 is a critical regulator of mitochondrion-controlled apoptosis signaling in the islet beta cells. Our findings will generate new functions of SIRT5 in beta cell, which may contribute to explore a new potential therapeutic target for protecting beta cells apoptosis in diabetes.

沉默信息调节因子SIRT5是线粒体内的一种NAD+依赖的蛋白去乙酰化酶。它的作用底物之一是线粒体诱导细胞凋亡过程中的重要因子细胞色素C，但其确切的调控机制仍未阐明。目前，对线粒体蛋白SIRT5在胰岛beta细胞中的作用和调节机制国内外尚无报道。前期研究中，我们发现SIRT5在正常和糖尿病小鼠的胰腺中均有表达且存在显著差异，其表达水平与几种线粒体凋亡关键因子的变化呈现一定相关性。因此，我们推测："线粒体蛋白SIRT5可能参与调控氧化/硝化应激诱导的beta细胞损伤"。为证实该假说，本项目拟采用过表达、基因沉默和免疫共沉淀等技术方法，从整体和细胞多个层面，考察SIRT5在beta细胞损伤及凋亡进程中的重要生理功能并探讨其作用机制。本研究将揭示SIRT5与beta细胞功能受损的关系及调控方式，从新的视角阐明beta细胞损伤的分子机制，并为寻找药物作用新靶点和拓展糖尿病治疗新思路奠定实验基础。

糖尿病状态下，胰岛β细胞应激导致的线粒体功能异常与胰岛素的分泌及细胞凋亡有密切的关系。尽管已有研究表明线粒体损伤以及功能异常参与糖尿病胰岛β细胞凋亡的发生、发展过程，但迄今尚未明确线粒体中的何种调控因子在凋亡过程中发挥关键作用。沉默信息调节因子SIRT（silent information regulator, SIRT）是一种NAD+依赖的蛋白去乙酰化酶，该家族成员线粒体蛋白Sirtuin 5（SIRT5）可对特定的线粒体蛋白中不同赖氨酸残基进行多种翻译后修饰。SIRT5的作用底物之一是参与线粒体介导细胞凋亡中的重要因子——细胞色素C。SIRT5在多个代谢通路中起到关键作用，是能量稳态调节的潜在作用因子之一，也是氧化还原代谢通路关键位点的重要调控分子。而氧化应激和线粒体功能异常导致的β细胞损伤及功能紊乱在糖尿病发生发展的过程中起到了重要推动作用。因此，SIRT5作为一个重要的线粒体蛋白功能调节因子，可能对胰岛beta细胞的存活和功能产生重要调节作用，但目前对其功能的探索和研究尚不十分深入。本课题在探索SIRT5调节胰岛素分泌和beta细胞损伤的作用机理方面取得了突破性进展。本研究首次观察到SIRT5在糖尿病模型动物胰腺中的表达水平发生显著变化，并进一步在胰岛beta细胞株中，采用基因工程手段过表达或降低SIRT5的表达水平，对其可能调控的通路中相关因子、胰岛素分泌功能的变化进行分析和探讨，以确定其对胰岛beta细胞可能具有的调控作用。具体研究成果汇总如下：在多种属来源的胰岛beta细胞株中（大鼠、小鼠及人源），采用葡萄糖、棕榈酸共同诱导形成糖脂毒性造成的细胞凋亡，利用 TUNEL-DAPI双染及细胞生长实时监测系统对相应生化指标进行监测。与阴性对照组相比，过表达SIRT5可显著减少糖脂诱导的凋亡细胞数，并抑制Caspase家族关键蛋白活性并下调其表达水平。同时，SIRT5可提高胰岛beta细胞对于葡萄糖刺激的反应性，一定程度上改善胰岛beta细胞分泌功能。另外，本研究首次发现，SIRT5可能通过与抗凋亡蛋白Bcl-XL间的相互作用产生抑制胰岛beta细胞凋亡的作用。本研究揭示了SIRT5与胰岛beta细胞功能受损发生发展的关系及其调节机制，为拓展糖尿病治疗新思路和发现药物作用新靶点奠定了实验基础。