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Journal of Histochemistry and Cytochemistry, Vol. 45, 1083-1096, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Immunocytochemical Evidence that GLUT4 Resides in a Specialized Translocation Post-endosomal VAMP2-positive Compartment in Rat Adipose Cells in the Absence of Insulin

Daniela Malideb, Nancy K. Dwyerc, E. Joan Blanchette-Mackiec, and Samuel W. Cushmanb
a Experimental Diabetes, Metabolism
b Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland
c Lipid Cell Biology Section, Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland

Correspondence to: Daniela Malide, EDMNS/DB/NIDDK/NIH, Bldg. 10, Rm. 5N102, 10 Center Drive, MSC 1420, Bethesda, MD 20892-1420.

Insulin stimulates glucose transport in rat adipose cells through the translocation of GLUT4 from a poorly defined intracellular compartment to the cell surface. We employed confocal microscopy to determine the in situ localization of GLUT4 relative to vesicle, Golgi, and endosomal proteins in these physiological insulin target cells. Three-dimensional analyses of GLUT4 immunostaining in basal cells revealed an intracellular punctate, patchy distribution both in the perinuclear region and scattered throughout the cytoplasm. VAMP2 closely associates with GLUT4 in many punctate vesicle-like structures. A small fraction of GLUT4 overlaps with TGN38-mannosidase ll, {gamma}-adaptin, and mannose-6-phosphate receptors in the perinuclear region, presumably corresponding to late endosome and trans-Golgi network structures. GLUT4 does not co-localize with transferrin receptors, clathrin, and lgp-120. After insulin treatment, GLUT4 partially redistributes to the cell surface and decreases in the perinuclear area. However, GLUT4 remains co-localized with TGN38-mannosidase ll and {gamma}-adaptin. Therefore, the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosomal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These results are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series. (J Histochem Cytochem 45:1083-1096, 1997)

Key Words: GLUT4, adipose cells, insulin, immunofluorescence


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