雷帕霉素不敏感mTOR伴随蛋白（Rictor）调节胰岛β细胞功能的分子机制

Pancreatic islet β cell mass and function defects are the major pathophysiological mechanisms of Type 2 Diabetes. Our previous published study （Diabetes 2011.03）firstly demonstrated that the kinase for pAKTS473, mTORC2 is essential for maintaining β cell proliferation and mass. However, we simultaneously found that in in vitro islet perfusion, deletion Rictor also significantly inhibited GSIS to 30% of normal level which made us thought that Rictor might also play an important role in β cell function. Our preliminary data showed that glucose stimulated intracellular calcium concentration was dramatically decreased by knocking down Rictor and that AKT phosphorylation，PKC expression and intracellular localization were significantly changed in β cell lack of Rictor. PKC and AKT are the major downstream substrates of Rictor/mTORC2. They are reported to be involved in glucose sensing ,ion channels activities and insulin granules movement. Therefore, Rictor could also modulate glucose stimulated intracellular concentration to promote insulin secretion through controlling glucose sensing and insulin granules movement in addition to ion channel activities. In this project, we will take advantage of the mice with β cell specific knockout Rictor, mating them with R26GFP mice. Using purified β cell by FACs, we will employ the ex vivo perifusion, primary β cell membrane clamp and virus transduction, to investigate the underlying mechanism Rictor mediated by to regulate β cell GSIS in the hope to find the potential target for regulating both β cell function and proliferation.

胰岛β细胞数量和功能性代偿缺陷是2型糖尿病重要致病机制。我们前期研究首次证明Rictor/mTORC2活性是维持β细胞增殖的重要因子(Diabetes,2011.03)。我们还发现β细胞特异敲除Rictor后可显著抑制胰岛70%的葡萄糖刺激的胰岛素分泌（GSIS），提示Rictor/mTORC2在β细胞功能调节中亦十分重要。进一步研究发现敲除Rictor显著降低β细胞葡萄糖刺激的胞内钙离子浓度[Ca2+]i，Rictor主要下游底物AKT和PKC的表达和活性受抑。Rictor可能通过这两个信号通路调控β细胞葡萄糖感受，离子通道活性和胰岛素囊泡运动影响 [Ca2+]i及GSIS。因此本课题将采用β细胞特异敲除Rictor小鼠模型，分选纯化GFP标记活体β细胞；用离体灌流、细胞膜片钳和病毒转染等方法阐明Rictor调节β细胞GSIS的具体分子机制，希望找到促进β细胞增殖和功能的双重调节靶点。

雷帕霉素不敏感mTOR伴随蛋白）是mTORC2（mTOR蛋白复合体2）的关键架构蛋白。我们前期研究首次发现，小鼠β细胞内特异敲除Rictor后小鼠β细胞增殖和β细胞质量（β cell mass）显著降低。同时敲除Rictor小鼠胰岛的GSIS(Glucose Stimulated Insulin Secretion，葡萄糖刺激的胰岛素分泌)功能也受到影响。本课题进一步研究Rictor/mTORC2在β细胞功能调节中的作用环节及具体机制。一系列体外促泌，电生理学膜片钳，免疫荧光以及投射电子显微镜实验发现mTORC2缺失未对β细胞的糖感受、离子通道活性及胰岛素合成等产生明显影响,而可能作用于胰岛素囊泡分泌环节。通过活体β细胞GFP标记的FACs（流式活细胞分选）出纯化的β细胞进行转录组学的研究发现：与磷脂代谢,囊泡运动以及细胞内钙离子信号调控相关的分子如：如Plcb1（Phospholipase C, Beta 1），Chrm1（Cholinergic Receptor, Muscarinic 1），Cacna1a（Calcium Channel, Voltage-Dependent, P/Q Type, Alpha 1A Subunit），Pde1a（Phosphodiesterase 1A, Calmodulin-Dependent），Vamp1（Vesicle-Associated Membrane Protein 1）及Stx11（Syntaxin 11）等相关基因在敲除小鼠的β细胞内表达显著下降。而这些基因都参与 Rictor/mTORC2特异底物PKC信号系统调控。我们在Rictor特异敲除的小鼠胰岛中发现PKCα和PKCε蛋白水平和/或细胞内分布因Rictor敲除而明显下降。敲除mTORC2/Rictor后的胰岛对PKC通路激动剂PMA的刺激的反应几被乎完全抑制。TM模拟磷酸化（Rictor 激活位点）的PKCα（AdPKCα-HA-T638D）腺病毒感染离体胰岛发使PMA刺激下的胰岛素分泌缺陷得到一定程度的逆转。mTORC2/PKCα信号在高脂喂养的小鼠胰岛中显著升高，且PMA依赖的胰岛素分泌也得到加强。因此本课题发现，mTORC2/Rictor/PKC 信号系统可能介导了β细胞在代谢刺激下维持功能代偿所必须的信号途径。