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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 E. 2001, The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells., Journal of biological chemistry, vol. 276, no. 30, pp. 27816-27824, doi: 10.1074/jbc.M011590200.

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Title The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells.
Author(s) Whitehead, Jonathan P.
Molero, Juan Carlos
Clark, Sharon
Martin, Sally
Meneilly, Grady
James, David E.
Journal name Journal of biological chemistry
Volume number 276
Issue number 30
Start page 27816
End page 27824
Publisher American Society for Biochemistry and Molecular Biology
Place of publication Baltimore, Md.
Publication date 2001-07-21
ISSN 0021-9258
1083-351X
Summary 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 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 °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.
Language eng
DOI 10.1074/jbc.M011590200
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2001, American Society for Biochemistry and Molecular Biology, Inc.
Persistent URL http://hdl.handle.net/10536/DRO/DU:30009187

Document type: Journal Article
Collection: School of Exercise and Nutrition Sciences
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