title: Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering.
abstract: Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.
authors.affiliation: Laboratory of Cellular and Molecular Biophysics, Program on Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Lizunov
authors.forename: Ivonne
authors.initials: I
authors.lastname: Lisinski
authors.forename: Karin
authors.initials: K
authors.lastname: Stenkula
authors.forename: Joshua
authors.initials: J
authors.lastname: Zimmerberg
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Cushman
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D009154:Mutation; D051381:Rats; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C522783:Exo70 protein, mouse; C109901:Exoc7 protein, rat; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D033921:Vesicular Transport Proteins