肠能量感受分子mTOR对GLP-1生成的调控作用及分子机制

Glucogon-like peptide (GLP-1), an intestinal incretin produced by L cells,is secreted after nutrient ingestion and acts on endocrine pancreas beta cells to enhance insulin secretion. However, the molecular mechanisms by which L cells sense nutrient intake and fuel status at the organism level to regulate GLP-1 expression and secretion are currently unknown. Mammalian target of rapamycin (mTOR), a highly conserved serine-threonine kinase, has been reported to serve as an intracellular fuel sensor. Our previous study shows intestinal mTOR upregulates GLP-1 production and secretion,however,the molecular mechanism is still unkown.The major focus of this proposal is to investigate the molecular mechanism of the intestinal mTOR pathway regulates the production and secretion of GLP-1 and thus its effect on blood glucose control and energy expenditure. We propose to use cell biological and molecular biological techniques to achieve these goals. Completion of this proposal will advance our understanding on how energy supply affects intracellular processes in the production of GLP-1, and will provide new information on the interaction between intestinal endocrine cells and nutrients. Results of this project will therefore yield new insights relevant to treatment strategies for diabetes.

肠腔内营养物质刺激L细胞分泌胰高血糖素样肽-1（GLP-1）入血，后者继而发挥促进胰岛素分泌以及葡萄糖浓度依赖性降糖作用。mTOR （Mammalian Target of Rapamycin）是细胞内重要的能量感受分子，能够感受营养物质的刺激。肠道L细胞是否存在能量感受分子mTOR，以及肠道mTOR如何通过感受机体能量状态来调控GLP-1合成分泌的分子机制尚未见报道。申请人前期预实验提示肠道存在能量感受分子mTOR，肠mTOR分子正性调节GLP-1合成与分泌但具体分子机制尚不清楚。本课题采用药物干预以及基因过表达、基因干扰、免疫荧光染色、免疫蛋白印迹、ChIP等分子生物学技术从从整体动物和原代分离胎鼠肠细胞以及STC-1 细胞系模型上探讨肠mTOR信号分子感受机体能量状态、调控GLP-1合成分泌的作用以及分子机制。为今后筛选以肠道为靶点，控制能量代谢、防治糖尿病药物提供理论依据。

肠腔内营养物质刺激L细胞分泌胰高血糖素样肽（GLP-1）入血，后者继而发挥促进胰岛素分泌以及葡萄糖浓度依赖性降糖作用。mTOR （Mammalian Target of Rapamycin）是细胞内重要的能量感受分子，能够感受营养物质的刺激。肠道L细胞是否存在能量感受分子mTOR，以及肠道mTOR如何通过感受机体能量状态来调控GLP-1合成分泌的分子机制尚未见报道。我们采用正常C57/BL6小鼠、高脂饮食诱导的糖尿病小鼠、db/db小鼠、Neurog3-TSC1-/-（内分泌细胞特异性TSC1基因缺失小鼠）转基因小鼠以及STC-1细胞株作为研究对象。我们的研究发现：饥饿显著抑制正常C57小鼠回肠mTOR信号通路活性的同时抑制回肠Proglucagon基因的表达和GLP-1的释放，而饥饿后重饲具有逆转作用。与正常C57小鼠相比，高脂饮食诱导的糖尿病小鼠回肠mTOR信号通路活性、GLP-1合成和释放均呈现较低水平。雷帕霉素在抑制正常、糖尿病小鼠回肠mTOR信号通路活性的同时抑制回肠Proglucagon基因的表达和GLP-1的释放以及诱导了葡萄糖不耐受和胰岛素抵抗的发生。亮氨酸灌胃、TSC1基因敲除均可刺激小鼠体内GLP-1合成和释放。体外在STC-1细胞实验发现，过表达mTOR质粒具有刺激Proglucagon基因启动子活性和GLP-1生成的作用，过表达TSC1和TSC2质粒具有抑制mTOR活性和抑制GLP-1生成的作用。总之，肠道mTOR信号分子可通过感受机体能量状态来调控肠源性激素GLP-1的合成、分泌。