Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well as from the dynamic endosomal/trans-Golgi network in rat adipocytes.
Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-D resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system.
Ramm
G
G
Department of Cell Biology, Faculty of Medicine and Institute of Biomembranes, Utrecht University, 3584 CX Utrecht, The Netherlands.
Slot
J W
JW
James
D E
DE
Stoorvogel
W
W
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002451:Cell Compartmentation; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D050683:R-SNARE Proteins; D051381:Rats; D022161:Transport Vesicles / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism* / Q000648:ultrastructure
D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050683:R-SNARE Proteins; C495694:Slc2a4 protein, rat