Development of an in vitro reconstitution assay for glucose transporter 4 translocation.
In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca(2+)-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5'-[gamma-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein-protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.
Inoue
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G
Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02215, USA.
Cheatham
B
B
Kahn
C R
CR
D016475:3T3 Cells; D017667:Adipocytes; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002413:Cations, Divalent / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D009711:Nucleotides / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002413:Cations, Divalent; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009711:Nucleotides; D016271:Proto-Oncogene Proteins c-myc; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse