title: GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein.
abstract: Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
authors.forename: P B
authors.initials: PB
authors.lastname: Oatey
authors.forename: D H
authors.initials: DH
authors.lastname: Van Weering
authors.forename: S P
authors.initials: SP
authors.lastname: Dobson
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
authors.forename: J M
authors.initials: JM
authors.lastname: Tavaré
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001692:Biological Transport; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; D049452:Green Fluorescent Proteins