title: Microtubule network is required for insulin signaling through activation of Akt/protein kinase B: evidence that insulin stimulates vesicle docking/fusion but not intracellular mobility.
abstract: The microtubule network has been shown to be required for insulin-dependent GLUT4 redistribution; however, the precise molecular function has not been elucidated. In this article, we used fluorescence recovery after photobleaching (FRAP) to evaluate the role of microtubules in intracellular GLUT4 vesicle mobility. A comparison of the rate of fluorescence recovery (t((1/2))), and the maximum fluorescence recovered (F(max)) was made between basal and insulin-treated cells with or without nocodazole treatment to disrupt microtubules. We found that intracellular mobility of fluorescently tagged GLUT4 (HA-GLUT4-GFP) was high in basal cells. Mobility was not increased by insulin treatment. Basal mobility was dependent upon an intact microtubule network. Using a constitutively active Akt to signal GLUT4 redistribution, we found that microtubule-based GLUT4 vesicle mobility was not obligatory for GLUT4 plasma membrane insertion. Our findings suggest that microtubules organize the insulin-signaling complex and provide a surface for basal mobility of GLUT4 vesicles. Our data do not support an obligatory requirement for long range microtubule-based movement of GLUT4 vesicles for insulin-mediated GLUT4 redistribution to the cell surface. Taken together, these findings suggest a model in which insulin signaling targets membrane docking and/or fusion rather than GLUT4 trafficking to the cell surface.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Eyster
authors.forename: Quwanza S
authors.initials: QS
authors.lastname: Duggins
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Gorbsky
authors.forename: Ann Louise
authors.initials: AL
authors.lastname: Olson
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000970:Antineoplastic Agents; D051275:Glucose Transporter Type 4; D007328:Insulin; C495693:Slc2a4 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D015739:Nocodazole