The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

Whitehead, Jonathan P., Molero, Juan Carlos, Clark, Sharon, Martin, Sally, Meneilly, Grady and James, David (2001) The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells. The Journal of Biological Chemistry, 276 30: 27816-27824. doi:10.1074/jbc.M011590200

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Author Whitehead, Jonathan P.
Molero, Juan Carlos
Clark, Sharon
Martin, Sally
Meneilly, Grady
James, David
Title The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells
Journal name The Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2001-07-27
Year available 2001
Sub-type Article (original research)
DOI 10.1074/jbc.M011590200
Open Access Status File (Publisher version)
Volume 276
Issue 30
Start page 27816
End page 27824
Total pages 9
Editor Herbert Tabor
Place of publication USA
Publisher The American Society for Biochemistry & Molecular Biology, Inc.
Language eng
Subject C1
320100 Medicine - General
780105 Biological sciences
321004 Endocrinology
320307 Medical Biochemistry - Other
270102 Cell Metabolism
Abstract We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) 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 A23187 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 degreesC, 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+/cahnodulin 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.
Keyword Biochemistry & Molecular Biology
Cytosolic Free Calcium
Isolated Skeletal-muscle
Glut4 Translocation
Phosphatidylinositol 3-kinase
Glucose-transporter-4 Translocation
3t3l1 Adipocytes
Hexose-transport
Rat Adipocytes
Kinase-b
Fusion
Q-Index Code C1
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Institute for Molecular Bioscience - Publications
UQ Diamantina Institute Publications
 
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Created: Wed, 15 Aug 2007, 02:58:32 EST