Abstract
Insulin stimulates glucose transport in its target cells by recruiting the glucose transporter Glut 4 from an intracellular compartment to the cell surface. Previous studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) is a necessary step in this insulin action. We have investigated whether PI 3-kinase activation is sufficient to promote Glut 4 translocation in transiently transfected adipocytes. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged Glut 4 and a constitutively active form of PI 3-kinase (p110*). The expression of p110* induced the appearance of epitope-tagged Glut 4 at the cell surface at a level similar to that obtained after insulin treatment, whereas a kinase-dead version of p110* had no effect. The p110* effect was observed over a wide range of the transfected cDNA. When subcellular fractionation of adipocytes was performed, p110* was found, similar to the endogenous PI 3-kinase, enriched in the low density microsomal compartment, which also contains the Glut 4 vesicles. This could suggest that a specific localization of PI 3-kinase in this compartment is required for the action on Glut 4. The observations made with PI 3-kinase are in contrast with those seen with the MAP kinase cascade. Indeed, a constitutively active form of MAP kinase kinase had no effect on Glut 4 translocation in basal conditions. At the highest degree of expression, the constitutively active form of MAP kinase kinase slightly inhibited the insulin stimulation of Glut 4 translocation. Taken together, our results indicate that Glut 4 translocation can be efficiently promoted by an active form of PI 3-kinase but not by the activation of the MAP kinase pathway.
Highlights
Insulin promotes glucose uptake in muscle and adipose tissue by increasing the translocation of the glucose transporter Glut 4 from an intracellular compartment to the plasma membrane, but the exact molecular mechanism is still undetermined [1, 2]
The p85 subunit of PI 3-kinase binds through its Src homology 2 (SH2) domains to the tyrosine phosphorylated YMXM motifs of IRS1 [3]; the consequences are an increase in the catalytic activity of the PI 3-kinase p110 subunit [4] and a rise in the intracellular level of PI 3,4-biphosphate and PI 3,4,5-triphosphate
It is possible that PI 3-kinase has to be activated in a specific subcellular compartment (11, 14 –16), an effect that could occur with insulin but not with platelet-derived growth factor or interleukin 4 (IL-4)
Summary
Insulin promotes glucose uptake in muscle and adipose tissue by increasing the translocation of the glucose transporter Glut 4 from an intracellular compartment to the plasma membrane, but the exact molecular mechanism is still undetermined [1, 2]. By the binding of an anti-Myc antibody to intact cells, Glut 4 translocation is exclusively studied in the small fraction of the cells that have been transfected This recently described system allows investigation of the signal transduction pathway involved in the translocation of Glut 4 because the epitope-tagged Glut 4 behaves to the endogenous Glut 4 in response to insulin [17, 18]. Taking advantage of this system, we looked directly at the effect of a constitutively active p110 PI 3-kinase (p110*) [19, 20] on Glut 4 translocation, and we determined whether its targeting into the cell was similar to that of the endogenous enzyme. PI 3-Kinase and Glut 4 Translocation the same intracellular compartment as the endogenous enzyme and is as efficient as insulin in promoting Glut 4 translocation
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