AS160在葡萄糖代谢稳态调控中的功能及作用机理研究

AS160是一种含多功能域的RabGAP蛋白，在身体葡萄糖代谢稳态调控中起着重要作用。AS160缺失会导致骨骼肌和脂肪中GLUT4表达显著下降，这在临床上会导致二型糖尿病，而在AS160基因敲除小鼠中也会引起胰岛素抵抗。但AS160缺失导致GLUT4表达下降的原因并不清楚。我们最近利用基因敲入技术在小鼠体内通过引入R917K点突变使AS160的GAP活性丧失，前期工作证明了AS160通过其GAP活性调控GLUT4的表达。因此我们提出如下工作假设：AS160蛋白通过其GAP功能控制下游Rab-X调控骨骼肌和脂肪中GLUT4蛋白内吞后与溶酶体融合降解的过程，从而调控GLUT4蛋白的表达水平，进而调控全身葡萄糖代谢稳态。本申请拟通过制备和深入分析AS160R917K基因敲入和Rab-X基因敲除小鼠来研究上述工作假设。本研究将有助于阐明葡萄糖代谢稳态调控的新的分子机制，以及它们与二型糖尿病的关系。

AS160 (Akt substrate of 160 kD) is Rab GTPase activating protein (RabGAP) that plays important roles in regulating glucose homeostasis. Phosphorylation of AS160 has been shown to regulate insulin-stimulated translocation of glucose transporter GLUT4 in skeletal muscle. Besides its role in regulating GLUT4 trafficking, AS160 also controls the protein expression level of GLUT4. Deficiency of AS160 causes insulin resistance and type II diabetes in human and mice, which is probably due to significant decreases in GLUT4 protein levels in skeletal muscle and fat. How AS160 deficiency causes the decrease in GLUT4 protein expression remains unclear. AS160 contains multiple functional domains including the RabGAP domain at the C-terminus, two phospho-tyrosine binding domains at the N-terminus and a calmodulin binding motif in the middle region. We recently generated an AS160R917K knockin mouse in which the R917K mutation inactivates the GAP function of AS160. Our preliminary data show that the AS160R917K knockin mutation causes a significant decrease in GLUT4 protein levels, suggesting that AS160 regulates GLUT4 protein levels via its GAP function. Based on these observations, we hypothesize that AS160 regulates GLUT4 protein levels via lysosomal degradation by controlling a yet to be identified Rab-X protein. We will utilise the AS160R917K knockin mice to decipher molecular mechanisms regulating GLUT4 degradation via AS160, including identification of Rab-X downstream of AS160 and the regulatory site in GLUT4 degradation controlled by the AS160/Rab-X nexus. We will then generate Rab-X knockout and AS160R917K/Rab-X-/- mice to further validate the AS160/Rab-X nexus in regulating GLUT4 degradation and investigate its role in the pathogenesis of type II diabetes. We believe that the proposed study will help to elucidate molecular mechanisms regulating GLUT4 degradation controlled by the AS160/Rab-X nexus and to reveal novel mechanisms in the pathogenesis of type II diabetes. This study will also help to develop novel therapeutic strategies to treat patients bearing AS160 null mutations.