title: Dual-mode of insulin action controls GLUT4 vesicle exocytosis.
abstract: Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Yingke
authors.initials: Y
authors.lastname: Xu
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Rubin
authors.forename: Charisse M
authors.initials: CM
authors.lastname: Orme
authors.forename: Alexander
authors.initials: A
authors.lastname: Karpikov
authors.forename: Chenfei
authors.initials: C
authors.lastname: Yu
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Bogan
authors.forename: Derek K
authors.initials: DK
authors.lastname: Toomre
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D005089:Exocytosis; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008561:Membrane Fusion; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D010739:Phospholipase D / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: C478539:Aspscr1 protein, mouse; D002352:Carrier Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C485187:PHluorin; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; D050988:Vesicle-Associated Membrane Protein 2; D049452:Green Fluorescent Proteins; D010739:Phospholipase D