title: The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells.
abstract: We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.
authors.affiliation: Institute for Molecular Bioscience and the Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, Australia. J.Whitehead@imb.uq.edu.au
authors.forename: J P
authors.initials: JP
authors.lastname: Whitehead
authors.forename: J C
authors.initials: JC
authors.lastname: Molero
authors.forename: S
authors.initials: S
authors.lastname: Clark
authors.forename: S
authors.initials: S
authors.lastname: Martin
authors.forename: G
authors.initials: G
authors.lastname: Meneilly
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism* / Q000493:pharmacokinetics; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007477:Ions; D051379:Mice; D008856:Microscopy, Fluorescence; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D013347:Subcellular Fractions / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D002147:Calmodulin; D051275:Glucose Transporter Type 4; D007328:Insulin; D007476:Ionophores; D007477:Ions; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; C495693:Slc2a4 protein, mouse; D013449:Sulfonamides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000001:Calcimycin; D004533:Egtazic Acid; D015759:Ionomycin; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D005947:Glucose; D002118:Calcium