Tunduguru Ragadeepthi, Chiu Tim T, Ramalingam Latha, Elmendorf Jeffrey S, Klip Amira, Thurmond Debbie C
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada.
Biochem Pharmacol. 2014 Nov 15;92(2):380-8. doi: 10.1016/j.bcp.2014.08.033. Epub 2014 Sep 6.
Skeletal muscle accounts for ∼ 80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1(-/-) knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.
骨骼肌占餐后葡萄糖清除率的约80%，骨骼肌葡萄糖清除对于维持胰岛素敏感性和血糖正常至关重要。胰岛素刺激的葡萄糖清除/摄取需要在一个需要皮质F-肌动蛋白重塑的过程中将葡萄糖转运蛋白4（GLUT4）募集到质膜（PM）；该过程在2型糖尿病中失调。最近的研究表明，PAK1是体内小鼠骨骼肌中GLUT4募集所需的一个元素，尽管其潜在的作用机制以及在葡萄糖摄取中的需求仍未确定。为此，我们在使用L6-GLUT4-myc肌肉细胞的研究中采用了PAK1抑制剂IPA3。IPA3完全消除了胰岛素刺激的GLUT4向质膜的转位，证实了在PAK1基因敲除小鼠的骨骼肌质膜中胰岛素刺激的GLUT4积累被消除的观察结果。IPA3处理也消除了胰岛素刺激的葡萄糖摄取到骨骼肌肌管中。从机制上讲，用IPA3处理的表达F-肌动蛋白生物传感器LifeAct-GFP的成肌细胞的活细胞成像显示，正常胰岛素诱导的皮质肌动蛋白重塑受到抑制。这种抑制是由IPA3处理的成肌细胞中正常胰岛素刺激的丝切蛋白去磷酸化的丧失所支撑的。这些发现扩展了现有的葡萄糖摄取中肌动蛋白重塑模型，将胰岛素刺激的PAK1信号传导作为促进肌动蛋白重塑和随后丝切蛋白去磷酸化的必需上游步骤。活跃的、去磷酸化的丝切蛋白然后为持续的F-肌动蛋白重塑提供G-肌动蛋白底物，以促进GLUT4囊泡转位，使葡萄糖摄取到骨骼肌细胞中。
