Abstract
Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. We found that PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDS-polyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 was without a major effect. PDGF pretreatment for 60-90 min led to a marked 80-90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. We examined the functional consequences of this decrease in IRS-1-associated PI3K activity. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport was unaffected by 60-90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70(s6kinase), kinases downstream of PI3K, was unaffected by PDGF pretreatment. Wortmannin was capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, 1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. 2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. 3) PDGF has no effect on insulin stimulation of Akt, p70(s6kinase), GLUT4 translocation, or glucose transport. 4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70(s6kinase); GLUT4 translocation; and glucose transport.
Highlights
Tyrosine kinase receptors such as the insulin receptor, the epidermal growth factor (EGF)1 receptor, and the platelet
Insulin stimulation leads to the tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS1/2) and the association of phosphatidylinositol 3-kinase (PI3K) with the Src-homology 2 (SH2) binding sites contained within these proteins
Because PDGF pretreatment has a major inhibitory effect on insulin-stimulated PI3K activity, we evaluated the impact of PDGF treatment on insulin-stimulated glucose transport. 3T3-L1 adipocytes were stimulated with insulin or PDGF or were pretreated with PDGF for various times followed by insulin stimulation
Summary
Vol 273, No 39, Issue of September 25, pp. 25139 –25147, 1998 Printed in U.S.A. Platelet-derived Growth Factor Inhibits Insulin Stimulation of Insulin Receptor Substrate-1-associated Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes without Affecting Glucose Transport*. Because the activation of PI3K has been shown to be necessary for the metabolic effects of insulin, such as the translocation of the insulin-stimulated glucose transporter, GLUT4, to the cell membrane, glucose uptake, and glycogen synthesis, it has been puzzling as to how necrosis factor ␣; pY, phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; PIP2, phosphatidylinositol 3,4-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; ATP, adenosine triphosphate. It appears that whereas PI3K activity is necessary for the metabolic effects of insulin, IRS-1 phosphorylation is not essential
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