Abstract
Glut4-containing vesicles immunoadsorbed from primary rat adipocytes possess endogenous protein kinase activity and phosphorylation substrates. Phosphorylation of several vesicle proteins including Glut4 itself is rapidly activated by insulin. Wortmannin blocks the effect of insulin when added to cells in vivo prior to insulin administration. By means of MonoQ chromatography and Western blot analysis, vesicle-associated protein kinase is identified as Akt-2, a lipid-binding protein kinase involved in insulin signaling. Akt-2 is found to be recruited to Glut4-containing vesicles in response to insulin.
Highlights
The regulation of postprandial blood glucose levels by insulin is achieved mainly by increased glucose transport into skeletal and cardiac muscle and fat [1, 2]
We show here that Glut4-containing vesicles immunoisolated from rat adipocytes possess a tightly associated protein kinase activity and several phosphorylation substrates
Glut4-containing Vesicles Contain an Insulin-stimulated Protein Kinase Activity and Phosphorylation Substrates— Glut4-containing vesicles were immunoadsorbed from intracellular membranes of rat adipose cells treated and not treated with insulin, and [␥-32P]ATP was added directly to the material adsorbed on the beads as described under “Materials and Methods.”
Summary
The regulation of postprandial blood glucose levels by insulin is achieved mainly by increased glucose transport into skeletal and cardiac muscle and fat [1, 2] These are the only tissues that express a specific isoform of the glucose transporter, Glut, which mediates the hormonal effect Insulin administration to cells may release this trafficking block by, for example, removal of an endogenous inhibitor from Glut4-containing vesicles, or by disassociating these vesicles from an intracellular anchor, leading to their default fusion with the plasma membrane. Phosphorylation of vesicle component proteins as well as artificial substrates by the vesicle-associated protein kinase is rapidly increased by insulin in a wortmannin-sensitive fashion These data may provide a missing link in the insulin signal transduction pathway by directly coupling Glut4-containing vesicles to the previously established enzymatic cascade
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