Abstract
In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562-1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to approximately 25 microM H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBalpha and PKBgamma. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.
Highlights
In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562– 1569)
Oxidative Stress Impairs the Compartment-specific Activation of PI 3-Kinase by Insulin—In order to study the effect of oxidative stress on the ability of insulin to activate PI 3-kinase in various cellular fractions, fully differentiated 3T3-L1 adipocytes were exposed for 2 h to 27.4 Ϯ 0.8 M H2O2 continuously generated by addition of 100 milliunits/ml glucose oxidase to the culture medium
The data presented demonstrate that while certain insulinomimetic effects of micromolar H2O2 concentrations could be observed, oxidative stress impaired the compartment-specific activation of PI 3-kinase, insulin receptor substrate (IRS)-1 redistribution, and protein kinase B (PKB) activation induced by insulin
Summary
In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562– 1569). Two-hour exposure to ϳ25 M H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). Insulin was reported to activate an adipocyte membrane bound NADPH oxidase [6, 7], further supporting a potential role for reactive oxygen species as second messengers In agreement with this concept, direct exposure of cells to H2O2 has been demonstrated to result in insulinomimetic effects, as demonstrated both by mimicking the metabolic response to insulin as well as by activating components of its signal transduction machinery (8 –11). The tyrosine-phosphorylated IRS in the LDM was suggested to serve as a docking molecule for PI 3-kinase, leading to a rapid translocation of the p85 subunit from the cytosol to the LDM, resulting in increased PI 3-kinase activity in this fraction [21, 22]
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