Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.
Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.
Laboratory of Molecular Cardiology, Diabetes Research Institute, Düsseldorf, Germany.
I
I
Uphues
T
T
Kolter
B
B
Goud
J
J
Eckel
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D011924:Rats, Zucker; D013347:Subcellular Fractions / Q000378:metabolism
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D019204:GTP-Binding Proteins