Abstract
Insulin causes the activation of phosphatidylinositol 3-kinase (PI 3-kinase) through complexation of tyrosine-phosphorylated YMXM motifs on insulin receptor substrate 1 with the Src homology 2 domains of PI 3-kinase. Previous studies with inhibitors have indicated that activation of PI 3-kinase is necessary for the stimulation of glucose transport in adipocytes. Here, we investigate whether this activation is sufficient for this effect. Short peptides containing two tyrosine-phosphorylated or thiophosphorylated YMXM motifs potently activated PI 3-kinase in the cytosol from 3T3-L1 adipocytes. Introduction of the phosphatase-resistant thiophosphorylated peptide into 3T3-L1 adipocytes through permeabilization with Staphylococcus aureus alpha-toxin stimulated PI 3-kinase as strongly as insulin. However, under the same conditions the peptide increased glucose transport into the permeabilized cells only 20% as well as insulin. Determination of the distribution of the glucose transporter isotype GLUT4 by confocal immunofluorescence showed that GLUT4 translocation to the plasma membrane can account for the effect of the peptide. These results suggest that one or more other insulin-triggered signaling pathways, besides the PI 3-kinase one, participate in the stimulation of glucose transport.
Highlights
A major metabolic effect of insulin is the stimulation of glucose transport into muscle and adipose cells
Peptides Used—In our previous study, we found that peptides containing two tyrosine-phosphorylated YMXM motifs potently activate PI 3-kinase by binding simultaneously to the two Src homology 2 (SH2) domains on the 85-kDa regulatory subunit of this enzyme [7]
Peptide Binding to PI 3-Kinase SH2 Domains—To determine the effect of substituting the thiophosphoryl group for the phosphoryl group on binding to the PI 3-kinase SH2 domains, the Tyr(S) and Tyr(P) peptides were compared in a Biacore assay in which the effectiveness of the peptide to compete with immobilized Tyr(P) YZPZSPK peptide for binding a glutathione Stransferase fusion protein containing both SH2 domains was measured
Summary
Materials—2-Deoxy-D-[2,6-3H]glucose and [U-14C]sucrose were from Amersham. [␥-32P]ATP was from ICN. The medium containing ␣-toxin was removed, the cells were treated at 37 °C with insulin, GTP␥S, or peptide in 0.4 ml of ICR buffer for 15 min, or put in ICR buffer alone. Following this treatment, glucose transport into the permeabilized cells was measured by the uptake of 2-deoxyglucose, according to a method previously described for 3T3-L1 adipocytes permeabilized with streptolysin O [12]. The amount of sucrose remaining with the cells provided a measure of nonspecific trapping of and uptake from the medium, and the raw values for 2-deoxyglucose uptake were corrected by subtraction of the corresponding amount of this compound This correction amounted to approximately 30% of the 2-deoxyglucose uptake by adipocytes in the basal state.
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