SOD2的O-GlcNAc修饰与胰岛素抵抗中的线粒体氧化应激：从营养传感到抗氧化防御？

Protein post-translational O-GlcNAc modification has been recognized as the core linking nutritional stress to insulin resistance (IR). Mitochondrial oxidative stress and dysfunction is intimately involved in IR. Mitochondrial proteome has recently been demonstrated to be O-GlcNAcylation thereby O-GlcNAcylation can regulate mitochondrial function. We recently observed association of increased O-GlcNAcylation and mitochondrial oxidative stress in skeletal muscle of IR mouse model. Superoxide Dismutase 2 (SOD2), the key enzyme eliminating ROS in mitochondria, has been reported to be O-GlcNAcylated, however the mechanisms remain unknown. Here we propose to explore whether O-GlcNAcylation can regulate mitochondrial antioxidant defense via targeting specific sites of SOD2 to contribute to IR. We’ve analyzed and predicted the O-GlcNAcylation of SOD2 by bioinformatics method, and will detect the O-GlcNAcylation of SOD2 in animal and cellular IR models with mass spectrometry as well as biochemical and molecular methods. We will also elucidate the specific sites, manners and function of O-GlcNAcylation on SOD2, and further validate and reveal the role and regulatory mechanism of O-GlcNAcylation on key sites of SOD2 in mitochondrial oxidative stress and IR. Those efforts shall promote the study of the mechanisms underlying IR from the view of linking nutrient sensing to antioxidant defense, and answer the question of whether targeting O-GlcNAcylation of SOD2 to regulate mitochondrial redox system could be potential new strategy for prevention and treatment of IR.

氧-糖苷键链接氮-乙酰葡糖胺(O-GlcNAc)修饰是连接营养过剩与胰岛素抵抗(IR)的核心环节。线粒体氧化应激及功能障碍与IR密切相关。新发现线粒体蛋白广泛发生O-GlcNAc修饰且线粒体功能受其调控。我们近期观察到IR模型小鼠骨骼肌线粒体中O-GlcNAc修饰升高、氧化应激增加。清除自由基的关键酶线粒体超氧化物歧化酶(SOD2)被提示存在O-GlcNAc修饰但机制不明。本项目意在探究SOD2关键位点的O-GlcNAc修饰是否可调控线粒体氧化应激并参与IR。我们用生物信息学方法对SOD2的O-GlcNAc修饰进行了分析预测；将在动物及细胞IR模型中用质谱及生化与分子生物学方法检测SOD2的O-GlcNAc修饰，阐明修饰的关键位点、方式、功能，再到IR模型中验证并揭示其调控线粒体氧化应激参与IR的机制。这将从营养传感到抗氧化防御角度进一步完善IR机理的研究，并为防治IR提供新策略和新靶点。

能量摄入过剩是当前代谢综合征（MS）高发的重要因素。MS的核心是胰岛素抵抗（insulin resistance, IR），其潜在机制之一是线粒体功能障碍（MD）与氧化应激（OS）。蛋白质翻译后修饰，氧-糖苷键链接氮-乙酰葡糖胺(O-GlcNAc)修饰，是一种营养和环境压力的细胞内传感机制。近年发现线粒体蛋白组广泛发生O-GlcNAc修饰且线粒体功能受其调控；但线粒体内最关键抗氧化酶超氧化物歧化酶(SOD2)是否发生O-GlcNAc修饰、其功能是否受O-GlcNAc修饰调控，及其在IR中的作用尚不明确。我们首先观察到MS模型小鼠肝脏中存在总体O-GlcNAc修饰增加、MD及OS。在细胞模型中发现高葡萄糖浓度或棕榈酸刺激均可诱导IR，并致SOD2酶活下降；我们用蛋白质免疫沉淀/共沉淀方法证明SOD2可发生响应于营养因素的O-GlcNAc修饰；SOD2与介导O-GlcNAc修饰的加载及移除的两个酶OGT、OGA之间均存在物理相互作用，且其结合强度对营养因素敏感。借助化学交联剂，我们发现SOD2的O-GlcNAc修饰可能负调控SOD2的同源四聚化程度，其机制可能是通过调节SOD2的68位赖氨酸残基（K-68）乙酰化水平。我们用生信法预测了几个可能的SOD2 O-GlcNAc修饰位点，定点突变实验显示这些位点可不同程度降低SOD2的O-GlcNAc修饰水平，且可调控SOD2 K-68乙酰化水平及同源四聚化程度。这些结果提示SOD2可发生O-GlcNAc修饰，且可能通过调节K-68乙酰化水平影响SOD2同源四聚化进而影响其酶活。本研究揭示了营养过载（高糖或高脂）的信号通过蛋白质翻译后O-GlcNAc修饰的营养传感机制传递给线粒体内最重要的抗氧化防预酶SOD2，可能调节K-68位乙酰化并干扰SOD2同源四聚化、抑制SOD2酶活，进而导致MD和OS，这从营养传感到线粒体抗氧化防御角度进一步阐释了营养过载导致IR的机理，为理解IR机制及发展防治IR新策略提供基础。.此外还发现①自噬缺陷可通过p62-FOXO1/3轴来负调控内源性抗氧化防御能力包括SOD1、SOD2、Catalase的表达和酶活力，从而加剧OS和MD；②L-阿拉伯糖可通过调控小鼠肠道内产氢菌和耗氢菌的相对丰度诱导氢气释放，调控关键代谢组织中的脂代谢基因的表达、改善线粒体功能，从而改善高脂饮食诱导的MS。