The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.
Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle-associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP-2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.
Martin
S
S
Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Brisbane, Australia.
Tellam
J
J
Livingstone
C
C
Slot
J W
JW
Gould
G W
GW
James
D E
DE
D016475:3T3 Cells; D017667:Adipocytes; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary; D011992:Endosomes / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins; D050683:R-SNARE Proteins; D051381:Rats; D017422:Sequence Analysis, DNA; D051040:Vesicle-Associated Membrane Protein 3
D018076:DNA, Complementary; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050683:R-SNARE Proteins; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D051040:Vesicle-Associated Membrane Protein 3