11818508

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We have examined the requirement for intracellular calcium (Ca(2+)) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca(2+) chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 microM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)beta. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca(2+) chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRbeta, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced. BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca(2+) depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca(2+) in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca(2+) is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca(2+) blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.

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378-89

pubmed-meshheading:11818508-3T3 Cells, pubmed-meshheading:11818508-Adipocytes, pubmed-meshheading:11818508-Animals, pubmed-meshheading:11818508-Cells, Cultured, pubmed-meshheading:11818508-Chelating Agents, pubmed-meshheading:11818508-Glucose, pubmed-meshheading:11818508-Glucose Transporter Type 4, pubmed-meshheading:11818508-Heat-Shock Response, pubmed-meshheading:11818508-Insulin, pubmed-meshheading:11818508-Intracellular Fluid, pubmed-meshheading:11818508-Mice, pubmed-meshheading:11818508-Monosaccharide Transport Proteins, pubmed-meshheading:11818508-Muscle Proteins, pubmed-meshheading:11818508-Phosphatidylinositol 3-Kinases, pubmed-meshheading:11818508-Phosphorylation, pubmed-meshheading:11818508-Phosphoserine, pubmed-meshheading:11818508-Phosphothreonine, pubmed-meshheading:11818508-Protein Binding, pubmed-meshheading:11818508-Protein Transport, pubmed-meshheading:11818508-Protein-Serine-Threonine Kinases, pubmed-meshheading:11818508-Proto-Oncogene Proteins, pubmed-meshheading:11818508-Proto-Oncogene Proteins c-akt, pubmed-meshheading:11818508-Signal Transduction

The effects of intracellular calcium depletion on insulin signaling in 3T3-L1 adipocytes.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.