Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action
Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action
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DOI：
10.1016/j.cmet.2006.11.013
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发表时间：
2007-01-01
期刊：
影响因子：
29
通讯作者：
Xu, Tao
中科院分区：
文献类型：
--
作者：
Bai, Li;Wang, Yan;Xu, Tao
胰岛素刺激的GLUT4易位是葡萄糖稳态的核心。目前还缺乏区分GLUT4易位过程中各个步骤的功能分析，因此限制了阐明潜在分子机制的进展。在此，我们开发了一种鲁棒性的方法，该方法依赖于单个GLUT4存储囊泡（GSVs）的实时动态跟踪，用于解剖和系统分析GSVs与细胞表面的对接、启动和融合步骤。通过这种方法，我们发现在表面对接后制备具有融合能力的GSVs是胰岛素调控的关键步骤，而对接步骤是由PI3K及其下游效应物Rab GAP AS160调控的。这些数据表明，Akt依赖性的AS160磷酸化并不是GLUT4运输的主要调控步骤，这意味着在细胞表面对接的GSV下游的替代Akt底物或替代信号通路是主要调控节点。
Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.