TBC1D1/14-3-3相互作用在四型葡萄糖转运体细胞质膜转移过程中的功能研究

Insulin stimulates uptake of glucose into skeletal muscle mainly through glucose transporter 4 (GLUT4) that translocates from its intracellular storage vesicles (GSVs) onto the plasma membrane upon insulin. Deregulation of this process is directly linked to the pathogenesis of type II diabetes. Besides insulin, other signals such as exercise and pharmaceutical AMPK activators (i.e. metformin，a drug for treatment of type II diabetes) can also stimulate GLUT4 translocation in an insulin-independent manner, which requires LKB1/AMPK pathway instead. Recent studies show that two related RabGAPs, AS160 and TBC1D1, may play important roles in regulating GLUT4 trafficking. AS160 and TBC1D1 are both regulated by protein phosphorylation and interaction with regulatory proteins named as 14-3-3 although some key regulatory details for these two RabGAPs are distinct. Insulin stimulates AS160 Thr642 phosphorylation and subsequently increases its interaction with 14-3-3 proteins while AMPK activators induce TBC1D1 Ser237 phosphorylation and up-regulate its binding to 14-3-3 proteins. The applicant has recently generated a kockin mouse model in which AS160 Thr642 is substituted by a non-phosphorylatable alanine, and showed that AS160 Thr642 phosphorylation and/or its binding to 14-3-3s play an important role in regulating insulin-stimulated GLUT4 trafficking in muscle and whole body glucose homeostasis. The applicant also showed that AS160 Thr642 phosphorylation and/or its binding to 14-3-3s are not required for AMPK-dependent GLUT4 trafficking and whole body glucose homeostasis. In this proposal, the applicant hypothesizes that TBC1D1 Ser237 phosphorylation and its interaction with 14-3-3s mediate AMPK-dependent GLUT4 trafficking and whole body glucose homeostasis. Towards addressing this hypothesis, a novel knockin mouse model has been generated, in which TBC1D1 Ser237 is mutated to a non-phosphorylatable alanine. This TBC1D1 knockin mouse will be scrutinised in respect of AMPK-dependent GLUT4 trafficking and whole body glucose homeostasis.

胰岛素和AMPK信号通路均可促进肌肉中四型葡萄糖转运体（GLUT4）从细胞内转移到细胞表面，从而诱导葡萄糖吸收。两种类似的RabGAP蛋白AS160和TBC1D1可能介导这两种通路对GLUT4细胞质膜转移的调控。申请人发现14-3-3蛋白会结合AS160和TBC1D1，而这分别受胰岛素和AMPK通路的调控。申请人最近证明了AS160/14-3-3的结合调控胰岛素诱导的GLUT4的细胞质膜转移，这填补了胰岛素调控的GLUT4质膜转移分子机制中的一个重要环节。关于TBC1D1/14-3-3相互作用的功能，申请人提出如下工作假设：TBC1D1/14-3-3的结合介导AMPK依赖的GLUT4细胞质膜转移，进而调控肌肉糖吸收和机体糖平衡。本申请拟利用基因敲入小鼠技术结合分子生理学手段研究这一假设，并阐明其分子机制。本项研究预期将填补AMPK通路调控的GLUT4细胞质膜转移分子机制中的一个关键环节。

生活方式和饮食结构的改变导致近年来我国肥胖发病率急剧增加，而肥胖是诱发糖尿病、脂肪肝、高血压、心脑血管疾病等许多慢性病的重要危险因素。缺乏运动以及高能量食物摄入会改变身体内能量状态，最终导致肥胖和2型糖尿病，但这背后的分子机制尚不完全清楚。.本项目利用细胞生物学和生物化学方法结合基因工程小鼠模型，首先对能量过剩导致肥胖的分子机制进行了系统研究。腺苷酸激活的蛋白激酶（AMPK）是细胞的能量感受器：能量过剩时如缺乏运动或过多能量摄入，AMPK活性会被抑制；而能量缺乏时，AMPK会被激活。本研究发现AMPK激活可抑制细胞分泌胰岛素样生长因子－1（IGF-1）。其机制是AMPK磷酸化RabGAP蛋白TBC1D1上的丝氨酸237位点（Ser237），抑制其GAP活性，从而减少下游分子开关Rab8a的GDP装载形式；而GDP装载的Rab8a可以促进细胞分泌IGF-1。当在小鼠体内利用基因工程手段将TBC1D1上237位的丝氨酸突变为非磷酸化的丙氨酸时，IGF-1分泌增加致使小鼠中IGF1R－PKB－mTOR通路活化，进而促进生长和脂质合成，导致小鼠罹患肥胖；而肥胖又进一步致使TBC1D1Ser237Ala基因敲入小鼠发生糖尿病和脂肪肝。该研究阐明了AMPK－TBC1D1－Rab8a－IGF-1通路在体内能量状态感知与肥胖发生中的作用机制，为今后干预和治疗肥胖提供了新靶点和新思路。.另外，能量感受器AMPK可感知身体能量状态，在葡萄糖稳态维持中也起重要作用，并且它的效应不依赖于胰岛素，目前一线2型糖尿病治疗药物二甲双胍的作用靶点之一就是AMPK。在本研究中，团队利用TBC1D1Ser237Ala基因敲入小鼠，发现AMPK－TBC1D1信号通路调控骨骼肌中4型葡萄糖转运体（GLUT4）的细胞质膜转位，进而介导非胰岛素依赖的骨骼肌葡萄糖吸收。当阻断内AMPK－TBC1D1信号通路后，AMPK激动剂AICAR的降血糖效应会显著减弱。这一研究阐明了非胰岛素依赖途径AMPK调控葡萄糖稳态的新机理，为将来开发以AMPK－TBC1D1信号通路为药物靶点的2型糖尿病治疗策略提供了理论基础。.