Axin1和Tiam1在运动/肌肉收缩促进骨骼肌摄取葡萄糖和改善胰岛素抵抗机制中的作用研究

Exercise/muscle contraction stimulates skeletal muscle glucose uptake through different signaling pathways compared to insulin action. Researchers and our group have previously reported that AMP-activated protein kinase (AMPK) and Rac1 mediate contraction-stimulated glucose uptake, however, the detailed mechanisms are not clear yet. A recent study illustrated that Axin1 regulated insulin-stimulated glucose uptake in adipocytes. An Axin1-AMPK complex is also involved in the activation of liver AMPK during low-energy conditions. Our preliminary data demonstrate that contraction up-regulates Axin1 protein levels in skeletal muscle. In addition, our findings also implicate AMPK activation of Rac1 via the Rac1-GEF, Tiam1. Therefore, we hypothesize that an AMPK/Axin1-Tiam1-Rac1 signaling pathway mediates contraction-promoted skeletal muscle glucose uptake. Interestingly, exercise/muscle contraction can also enhance muscle insulin sensitivity. But the insulin signaling molecules that are amplified by exercise are not known. We previously reported that both contraction and insulin shared Rac1and Rabs 8a, 13 and 14 to stimulate skeletal muscle glucose uptake. Our preliminary data illustrate that some of these Rabs, Rac1 and Tiam1 are down-regulated in skeletal muscle of high-fat diet-induced obesity in mice, and these changes are reversed by exercise. We hypothesize that some of these molecules are involved in the mechanism of exercise-improved insulin sensitivity. In this study, we will utilize muscle cells stably expressing epitope-tagged GLUT4, mice with skeletal muscle-specific knockout of Axin1 and other mouse models to interrogate our hypotheses. The completion of this project will further illustrate the mechanisms of exercise-regulated whole-body glucose homeostasis and insulin sensitivity. It could also provide new drug targets to prevent and treat type 2 diabetes.

运动/肌肉收缩以不同于胰岛素的信号通路促进骨骼肌摄取葡萄糖（glucose uptake, GU），文献和我们报道AMPK和Rac1介导此作用，但详细机制不明。近来报道Axin1介导胰岛素促进的脂肪细胞GU，在肝细胞低能量时参与激活AMPK，我们预实验发现收缩上调Axin1，参与激活AMPK。此外，预实验提示AMPK通过Tiam1激活Rac1，推测AMPK/Axin1-Tiam1-Rac1通路介导收缩促进骨骼肌GU的作用。另一方面，运动还增加胰岛素敏感性，但尚未发现被运动放大的胰岛素信号。申请人报道收缩和胰岛素共用Rac1和几个Rab蛋白，预实验发现肥胖下调它们和Tiam1的表达，运动逆转此作用，推测它们参与运动增加胰岛素敏感性的作用。本研究拟应用我们建立的细胞株和敲除小鼠等动物模型验证我们的推测。本课题的完成将进一步阐明运动调节血糖的机制，发现新的药物靶点，有助于2型糖尿病的预防和治疗。

骨骼肌摄取血中的葡萄糖主要由细胞膜上的葡萄糖转运蛋白4（GLUT4）转运完成，运动和胰岛素均能促进GLUT4从胞浆转位到细胞膜上，转运摄取葡萄糖进入细胞，从而降低血糖。相比于胰岛素，运动/肌肉收缩调节骨骼肌摄取葡萄糖的机制远不明确。肝细胞低能量时Axin1参与LKB1激活AMPK的作用，本研究的体内体外实验发现，收缩上调骨骼肌Axin1的蛋白水平，AMPK介导此作用，Axin1介导收缩激活AMPK的作用，收缩促进Axin1/AMPK复合物的形成，并通过此复合物促进GLUT4转位到细胞表面膜上摄取葡萄糖。.我们前期报道AMPK和小G蛋白Rac1均介导收缩促进骨骼肌细胞GLUT4转位的作用。本研究体内体外实验发现，AMPK/Axin1复合物位于Rac1的上游，介导收缩激活Rac1的作用，收缩通过促进Axin1/AMPK复合物形成，增加Rac1活性，进而促进GLUT4转位和骨骼肌葡萄糖摄取。.AMPK是丝氨酸/苏氨酸激酶，不能直接激活Rac1，激活小G蛋白Rac1需要其调节蛋白，我们检测了包括鸟苷酸交换因子（GEF）Tiam1的作用，发现骨骼肌中Tiam1参与骨骼肌收缩刺激的Rac1激活、GLUT4转位和葡萄糖摄取作用；在骨骼肌肌管细胞和骨骼肌组织中，收缩通过AMPK上调Tiam1表达，进而激活Rac1，促进骨骼肌GLUT4转位和葡萄糖摄取。.随着研究的进行，我们拓展了研究内容，探讨了激酶CaMKII→Rac1-GEF（Kalirin）→Rac1通路在收缩促进骨骼肌摄取葡萄糖机制中的作用。发现CaMKII参与收缩激活Rac1促进骨骼肌细胞摄取葡萄糖的作用，收缩依赖CaMKII而不是AMPK上调Kalirin蛋白水平，Kalirin参与收缩激活Rac1促进骨骼肌细胞摄取葡萄糖的作用。我们还探讨了Rac1-GEF蛋白Kalirin在收缩促进骨骼肌摄取葡萄糖机制中的作用，以及收缩激活胰岛素信号分子Akt的机制。.主要结果为：.1.收缩作用通过AMPK上调Axin1蛋白水平，促进Axin1与AMPK相互作用，进而调节骨骼肌AMPK活性，通过Axin1/AMPK-Tiam1 -Rac1信号通路促进骨骼肌摄取葡萄糖。.2.CaMKII-Kalirin-Rac1通路也介导收缩促进骨骼肌摄取葡萄糖的作用，并激活胰岛素信号分子Akt。