title: Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation.
abstract: The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes. The magnitude of the insulin effect is much greater for GLUT4 than other recycling proteins such as the CD-MPR. In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of GLUT4. Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN. However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of GLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx. 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation.
authors.affiliation: Centre for Molecular and Cellular Biology & Dept Physiology and Pharmacology, University of Queensland, St Lucia, Brisbane, QLD 4072, Australia. s.martin@cmcb.uq.edu.au
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: C A
authors.initials: CA
authors.lastname: Millar
authors.forename: C T
authors.initials: CT
authors.lastname: Lyttle
authors.forename: T
authors.initials: T
authors.lastname: Meerloo
authors.forename: B J
authors.initials: BJ
authors.lastname: Marsh
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002458:Cell Fractionation; D005089:Exocytosis; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D021381:Protein Transport / Q000187:drug effects; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D017527:Receptor, IGF Type 2; C495693:Slc2a4 protein, mouse