Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action.
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.
Bai
Li
L
Joint Laboratory of Institute of Biophysics and Huazhong University of Science and Technology, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Wang
Yan
Y
Fan
Junmei
J
Chen
Yu
Y
Ji
Wei
W
Qu
Anlian
A
Xu
Pingyong
P
James
David E
DE
Xu
Tao
T
D017667:Adipocytes; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction
D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; C505647:Tbc1d4 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt