基于TFEB调控溶酶体功能研究维生素D改善肝脏胰岛素抵抗的相关机制

Hepatic insulin resistance is a key link of the development of type 2 diabetes mellitus (T2DM), but the related mechanisms are still far from clear. Lysosomal damage and its mediated autophagy dysfunction may be an important mechanism of hepatic insulin resistance induced by glucolipotoxicity. Based on the new finding of TFEB, which is the core regulatory molecule of lysosomal biosynthesis and function, our present project is designed to analysis systematically the mTORC1 and ERK2-mediated TFEB nuclear translocation and SUMOylation-mediated transcriptional activity by animal and cell experiments combined with gene transfection and silence, pathway’s exciting or blocking and other molecular biology techniques, and reveal the underlying mechanisms of glucolipotoxicity caused hepatic insulin resistance especially in VD deficiency and its pathophysiological significance. In addition, the 1,25 (OH) 2VD3 is selected for intervention due to its active role. The effect and potential target of 1,25 (OH) 2VD3 on TFEB translocation and transcriptional activity will be discussed to elucidate the molecular mechanisms of 1,25 (OH) 2VD3 against hepatic insulin resistance from a new perspective. Thus, this project will not only enrich the basic theory of IR and T2DM, but also provide reference for T2DM nutritional intervention, the prevention of VD on hepatic insulin resistance and lysosome injury related diseases, which has a very important application value.

肝脏IR是T2DM发生发展的关键环节，相关机制远未阐明。溶酶体损伤及其介导的自噬功能障碍是糖脂毒性所致肝脏IR的重要机制。本项目以新近发现的溶酶体生物合成及功能的核心调控分子TFEB为切入点，将动物整体实验与细胞水平的机制研究相结合，借助基因转染与沉默、通路激动与阻断等多种分子生物学手段，对mTORC1与ERK2介导TFEB核转位及SUMO化修饰介导的转录活性等进行层层剖析与验证，阐明糖脂毒性尤其是在VD缺乏状态下导致肝脏IR发生发展的关键机制及病理生理学意义。在此基础上，选择发挥活性作用的1,25(OH)2VD3进行干预，分析其对TFEB核转位与转录活性的调控及潜在作用靶点，从全新的角度深入揭示其拮抗肝脏IR的分子机制。本项目的实施，不仅可望丰富IR、T2DM发生发展的基础理论，还可为T2DM的营养干预及VD在肝脏IR相关疾病、溶酶体损伤相关疾病的防治提供借鉴，具有非常重要的应用价值。

肝脏胰岛素抵抗（IR）是2型糖尿病（T2DM）发生发展的关键环节，但具体机制和信号通路的研究仍远未阐明。溶酶体损伤及其介导的自噬功能障碍是糖脂毒性所致肝脏IR的关键机制，而TFEB是溶酶体功能及自噬作用的核心调控分子，深入研究溶酶体损伤在糖脂毒性导致的肝脏IR中的作用对于深入阐明肝脏IR发生发展的潜在机制具有非常重要的病理生理学意义。同时，改善T2DM具有明显作用的维生素D 对于糖脂毒性引起的肝脏IR是否与其调控TFEB改善溶酶体功能相关，如何调控，尚待研究。在此背景下，本项目以自发性2型糖尿病大鼠（ZDF大鼠）为研究对象，建立维生素D缺乏的肝脏胰岛素抵抗模型，同时选择发挥活性作用的1,25(OH)2D3进行干预，并建立胰岛素抵抗的大鼠原代肝细胞模型，对1,25(OH)2D3干预改善肝脏IR的相关机制进行验证。本研究成功建立了动物模型和细胞模型，并发现维生素D缺乏会加快和加重ZDF大鼠的病程，引起更为严重的肝脏IR。溶酶体损伤及其介导的自噬功能障碍在糖脂毒性所致肝脏IR中发挥着关键作用，高糖高脂会抑制TFEB的核转位及其转录活性而造成损伤的溶酶体不能及时修复进而导致肝细胞自噬功能受阻，肝细胞严重损伤。动物实验初步结果及细胞实验验证结果显示，1,25(OH)2D3干预可通过抑制mTORC1、ERK1/2活性促进TFEB的核转位以及促进SUMO-1介导的TFEB SUMO化修饰增强转录活性，促进溶酶体的生物合成并增强自噬作用，从而缓解肝脏IR。本项目研究结果丰富了T2DM、肝脏IR发生发展的分子机制，为胰岛素抵抗相关疾病的防治提供有效的干预靶点，也为维生素D改善T2DM、胰岛素抵抗相关疾病、溶酶体损伤相关疾病的营养防治提供重要的理论支撑。