Oxidative Stress Disrupts Insulin-induced Cellular Redistribution of Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes

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 ∼25 μm 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 PKBα and PKBγ. 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.

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H 2 O 2 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 ϳ25 M H 2 O 2 (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 PKB␣ and PKB␥. 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.
The information regarding the biological actions of reactive oxygen and nitrogen species has increased considerably in recent years, revealing diverse functions (1,2). Exposure of tissues to free radicals in a variety of experimental systems leads to apoptosis and to cell damage (2). Paradoxically, reactive oxygen species have been demonstrated to participate in normal cellular responses including in signal transduction path-ways and in gene regulation (1,3,4). H 2 O 2 , for example, has been shown to be produced intracellularly following stimulation with platelet-derived growth factor and to mediate the normal response to this growth factor (5). Similarly, 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 H 2 O 2 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). However, prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H 2 O 2 resulted in impaired insulin-stimulated lipogenesis, activation of glycogen synthase, glucose transport, and GLUT4 translocation to the plasma membrane (PM) 1 (12,13).
Insulin-stimulated GLUT4 translocation has been suggested to depend upon the activation of phosphatidylinositol 3-kinase (PI 3-kinase), which in fat cells occurs in various cellular fractions (14,15). Recently, the concept that the activation of PI 3-kinase in the low density microsomal fraction (LDM) or in GLUT4 containing vesicles is necessary for the specific ability of insulin to promote GLUT4 translocation has been suggested (16 -19). In addition, overexpression of a constitutively active p110 (the catalytic subunit of PI 3-kinase), which resulted in increased total and LDM PI 3-kinase activity in primary adipocytes or in 3T3-L1 adipocytes, dramatically stimulated glucose uptake and GLUT4 translocation (18,20). In a recent study we observed that impaired insulin-stimulated GLUT4 translocation following oxidation could not be attributed to defects in activation of PI 3-kinase as detected in total cell lysate (13). Thus, impaired compartment-specific activation of PI 3-kinase by insulin may represent a putative cellular mechanism for oxidation induced insulin resistance.
The events leading to the specific activation of PI 3-kinase in the LDM are not clear. Recently, insulin-induced redistribution of insulin receptor substrates (IRS) was suggested to play a role in the compartment-specific activation of PI 3-kinase in 3T3-L1 adipocytes (21). Insulin stimulation was found to induce a reduction in the amount of IRS1/2 in the LDM, while IRS tyrosine phosphorylation was elevated. 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).
Although the insulin signaling steps toward GLUT4 translocation distal to PI 3-kinase activation are currently not fully understood, a number of downstream targets for PI 3-kinase have been identified (23,24). Among them, a serine/threonine kinase of 60 kDa (24,25) termed protein kinase B (PKB) also known as RAC protein kinase or Akt (26 -28). PI 3-kinase is both necessary and sufficient for insulin-dependent phosphorylation and activation of PKB, although the exact mode of activation has not been fully elucidated (reviewed in Ref. 29). It was suggested that binding of phosphatidylinositol 3,4,5trisphosphate or phosphatidylinositol 3,4-bisphosphate to the pleckstrin homology domain of PKB and its translocation to the membranes are necessary for PKB phosphorylation on Thr 308 and Ser 473 by its upstream kinases PDK1 and PDK2, respectively. In the search for its relevant biological activity, PKB was found to mediate some of insulin's effects, such as the inhibition of glycogen synthase kinase-3 (30,31), the stimulation of glucose and amino acids uptake (23), and protein synthesis (32). Several lines of evidence strongly support a crucial role for PKB in mediating GLUT4 translocation. In agreement with this notion, insulin activation of PKB precedes the hormonal effect on glucose transport (33). Moreover, overexpression of a constitutively active form of PKB resulted in enhanced glucose transport and GLUT4 translocation (32)(33)(34)(35)(36), while inhibition of PKB activity by transfecting rat adipocytes with a dominant PKB-inactive mutant, significantly inhibited insulinstimulated GLUT4 translocation (34). The respective role of the different isoforms of PKB in mediating insulin-stimulated GLUT4 translocation in various cell types is as yet unclear.
Although the involvement of IRS, PI 3-kinase, and PKB cellular redistribution and activation in the normal response to insulin is increasingly recognized, their relevance for the understanding of the cellular mechanisms leading to insulin resistance is largely unclear. In this study we report that impaired insulin-stimulated GLUT4 translocation induced by oxidative stress is associated with disruption of insulin-induced IRS-1 and PI 3-kinase intracellular trafficking and with inhibition of PKB␣ and PKB␥ activation. This represents a novel cellular mechanism for the understanding of insulin resistance in response to a change in the extracellular environment.
Cell Culture-3T3-L1 pre-adipocytes (American Type Culture Collection) were grown to confluence in Dulbecco's modified Eagle's medium containing 25 mM glucose, as described previously (12). 48 h following confluence cells were induced to differentiate to adipocytes by changing the medium to Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 5 g/ml recombinant human insulin, 0.5 mM 3-isobutylmethylxanthine and 0.25 M dexamethasone for 48 -72 h. Cells were used 9 -10 days following differentiation induction when exhibiting Ͼ90% adipocyte phenotype. H 2 O 2 was generated by adding 100 milliunits/ml glucose oxidase (type II from Aspergillus niger, 20,000 units/g solid in non-oxygen-saturated conditions, Sigma) to serum-free Dulbecco's modified Eagle's medium supplemented with 0.5% radioimmunoassay grade bovine serum albumin. Cellular Membrane Preparations-Following treatment with or without glucose oxidase, cells were rinsed three times with phosphatebuffered saline (PBS) and incubated for 7 or 20 min with or without 100 nM insulin in freshly prepared medium supplemented with 0.5% bovine serum albumin (radioimmunoassay grade). Membrane preparations (4 ϫ 10-cm plates per condition) were performed following the procedure described by Clancy and Czech (39). The activity of the PM marker 5Ј-nucleotidase (EC 3.1.3.5) was determined as described previously (39). The specific activity (nmol/min/mg protein) of PM 5Ј-nucleotidase for control and glucose oxidase-treated cells was 28.1 Ϯ 3.3 and 27.9 Ϯ 1.9, respectively, which reflected purification of more then 10-fold versus homogenate. Protein recovery of the different membrane fractions was not altered by glucose oxidase treatment. The various cellular compartments were resuspended in the original fractionation buffer (255 mM sucrose, 20 mM HEPES, pH 7.4, 1 mM EDTA, 0.2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 M aprotonin, 1 M leupeptin, 1 M pepstatin A), with no addition of detergent, as described (13,39).
Plasma Membrane Lawn Preparation-Plasma membrane sheets were prepared as described by Chen (40). Differentiated 3T3-L1 adipocytes were washed with PBS and incubated for 1 min with 0.5 mg/ml poly-D-lysine followed by three washes with hypotonic buffer (23 mM KCl, 10 mM HEPES, pH 7.5, 1.7 mM MgCl 2 , 1 mM EGTA). The cells were then covered with sonication buffer (3 ϫ hypotonic buffer containing 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride) and sonicated with a probe membrane disrupter. Following sonication, the plasma membrane sheets were washed three times with sonication buffer and were used for immunoblotting as described below.
Immunoprecipitation and PI 3-Kinase Assay-Cells were lysed in ice-cold deoxycholate based buffer, as described above. For immunoprecipitation, either 0.5 mg protein of cell lysate and cytosols or 75 g of protein of LDM were used and assayed as described (13). PI 3-kinase assay was performed following the protocol described by Hadari et al. (10), using phosphatidylinositol and [␥-32 P]ATP as substrates. The phosphorylated phosphatidylinositols were separated using thin layer chromatography and quantified by video densitometry.
Hexose Transport Determinations-2-Deoxyglucose uptake measurements were performed as described previously (12). Assays were performed for 10 min using 50 M 2-deoxy-[ 3 H]glucose (1 Ci/ml). Nonspecific uptake (less than 10% of the total) was determined in the presence of cytochalasin B (50 M) and was subtracted from the total uptake.
Statistical Analysis-Data are expressed as mean Ϯ S.E. Each treatment was compared with control, and statistical significance between two groups was evaluated using the Student's t test. The criterion for significance was set at p Ͻ 0.01.

RESULTS
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 H 2 O 2 continuously generated by addition of 100 milliunits/ml glucose oxidase to the culture medium. In agreement with recent reports (16,21), insulin induced a 2.8-fold increase in p85 content in the LDM (Fig. 1A), which was associated with a 50% reduction in its abundance in the cytosolic fraction (Fig. 1C). PI 3-kinase activity measured in p85 immunoprecipitates revealed an approximately 7-fold increase by insulin in the LDM and a 50% reduction in the cytosolic fraction (Fig. 1, B and D, respectively). These results indicate that insulin-induced activation of PI 3-kinase in the LDM can be attributed to both translocation of this enzyme from the cytosol, as well as to its activation in the LDM fraction. Oxidative stress induced a 3-fold increase in PI 3-kinase activity in the LDM in the absence of insulin, which was not associated with increased LDM p85 content (Fig. 1, B and A). This may suggest that oxidative stress increased LDM PI 3-kinase activity primarily by inducing activation rather than translocation of this enzyme in the LDM. Yet, oxidative stress resulted in a significant reduction in the ability of insulin to increase p85 content in the LDM (Fig.  1A) or to reduce its cytosolic content (Fig. 1C). This was associated with inhibition of insulin-stimulated PI 3-kinase activation in the LDM (Fig. 1B), as well as in impaired insulin induced reduction in its cytosolic activity (Fig. 1D). In total cell lysates, both p85 content as well as the activation of p85-associated PI 3-kinase activity by insulin were not affected following oxidation (Fig. 1, E and F, respectively). Taken together these data suggest that both the insulin-stimulated translocation of PI 3-kinase to the LDM and its activation in this fraction are impaired following oxidative stress and are associated with retention of this enzyme in its cytosolic pool.
Oxidative Stress Alters the Pattern of Insulin-stimulated IRS-1 Redistribution-In 3T3-L1 adipocytes most of PI 3-kinase activation by insulin occurs via its interaction with tyrosine-phosphorylated IRS-1 (21). In order to assess whether altered IRS-1 cellular compartmentalization may account for the impaired PI 3-kinase activation in the LDM, IRS-1 content and interaction with PI 3-kinase were evaluated in the LDM and cytosolic fractions. In control cells, an approximately 50% reduction in IRS-1 LDM content following 7-min insulin stimulation was observed ( Fig. 2A). In oxidized cells, IRS-1 content in the LDM was significantly reduced by approximately 35%, with no further reduction following insulin stimulation. In control cells, insulin induced an inhibition in IRS-1 gel mobility ( Fig. 2A), which could be attributed to increased serine/threonine phosphorylation, since incubation of LDM with alkaline phosphatase diminished this change in gel mobility (data not shown). In addition, despite the reduction in LDM IRS-1 content ( Fig. 2A), insulin induced a marked elevation in IRS tyrosine phosphorylation in this fraction (Fig. 2B). In LDM prepared from oxidized cells, insulin stimulation failed to induce the change in IRS gel mobility ( Fig. 2A), suggesting inhibition of its insulin stimulated serine/threonine phosphorylation. Moreover, insulin-induced tyrosine phosphorylation of IRS-1 was significantly impaired following oxidation (Fig. 2B). In accordance with this finding, the 16-fold increase in IRS-1associated PI 3-kinase activity observed in the LDM of control cells was markedly impaired by oxidative stress (Fig. 2C).
In the cytosolic fraction, insulin stimulation of either control or oxidized cells did not result in changes in IRS-1 gel mobility, as in the LDM fraction (Fig. 2D). Yet, both the increase in cytosolic IRS-1 content induced by insulin (Fig. 2D), as well as the stimulation of IRS-1-associated PI 3-kinase activity in this fraction (Fig. 2E), were significantly impaired by oxidative stress. Taken together these results suggest that oxidation disrupted IRS-1 cellular redistribution and tyrosine phosphorylation in the LDM. This in turn resulted in the reduced ability of IRS-1 to serve as a LDM docking protein for PI 3-kinase.
FIG. 1. Insulin-induced PI 3-kinase redistribution and activation in cellular fractions of control and glucose oxidase-treated cells. Differentiated 3T3-L1 adipocytes were serum-starved for 16 h, followed by 2-h treatment without (Control) or with 100 milliunits/ml glucose oxidase (GO). Cells were then rinsed three times with PBS followed by a further 7-min incubation with (ϩ) or without (Ϫ) 100 nM insulin. Cells were then subjected to subcellular fractionation, and the LDM and cytosol fractions were collected, as described under "Experimental Procedures." Each panel contains a representative blot of at least three independent experiments, under which the results of densitometric analysis is presented. Values are the mean Ϯ S.E. of the densitometry values expressed in arbitrary units as compared with values obtained in unstimulated control cells. A, 40 g of LDM protein were subjected to polyacrylamide gel electrophoresis and immunoblotted with antibody against the p85 regulatory subunit of PI 3-kinase. B, 75 g of protein from LDM were subjected to immunoprecipitation with an antibody against p85 and assayed for PI 3-kinase activity using phosphatidylinositol as a substrate, as detailed under "Experimental Procedures". C, 500 g of protein from the cytosolic fractions were immunoprecipitated using anti-p85 antibody followed by Western blot analysis for the amount of p85 or assayed for PI 3-kinase activity (D). E, 50 g of protein of whole cell lysate was assayed for total p85 content by Western blot analysis. F, 500 g of protein of total cell lysate were subjected to immunoprecipitation using anti-p85 antibody, after which PI 3-kinase activity was assessed. *, p Ͻ 0.01 versus unstimulated control cells; **, p Ͻ 0.01 versus insulin-stimulated control cells.
Insulin-stimulated Activation of PKB␣ and PKB␥ Is Impaired following Oxidative Stress-To assess whether the defect in PI 3-kinase redistribution is associated with impairment in the activation of PKB by insulin, PKB phosphorylation and activation were evaluated following oxidative stress. Immunoblot analysis of total cell lysates using anti-phospho-Ser 473 PKB antibody demonstrated that oxidative stress induced a 3.47 Ϯ 0.85-fold increase in PKB phosphorylation, which was wortmannin-sensitive (Fig. 3A). Exposure to insulin led to a rapid, time-dependent stimulation of PKB phosphorylation reaching maximal stimulation by 10 min in control cells, while in oxidized cells PKB phosphorylation was dramatically reduced (Fig. 3B). To correlate changes in PKB phosphorylation with its activity, total cell lysates were subjected to immunoprecipitation using anti PKB antibodies raised against its Cterminal tail. The efficiency of the immunoprecipitation protocol was not affected by oxidation, as assessed by immunoblotting the supernatant above the immunoprecipitate (data not shown). Fig. 3C demonstrates a 12.2 Ϯ 2.3-fold increase in PKB activity following 7-min exposure to insulin. In accordance with the serine phosphorylation pattern, oxidative stress induced a 3.6 Ϯ 1.7-fold increase in PKB activity, while the net insulin effect on PKB activity was markedly reduced from 24.10 Ϯ 2.7 to 4.65 Ϯ 0.9 milliunits/mg of protein/min (p Ͻ 0.001). Both basal activation of PKB induced by oxidative stress, as well as its stimulation by insulin, were completely inhibited by wortmannin, indicating the involvement of PI 3-kinase in both processes.
To further characterize the effect of oxidative stress on basal and insulin stimulated PKB activity, the various isoforms of PKB were studied using isoform specific antibodies. In 3T3-L1 adipocytes, all known PKB isoforms were found to be expressed, and their total cellular content was not significantly altered by oxidation (Fig. 4A). PKB activity was measured in immunoprecipitates of the different PKB isoforms from total cell lysates of control and oxidized cells before and after exposure to 100 nM insulin for 7 min. Fig. 4B demonstrates that in 3T3-L1 adipocytes the basal activity of PKB␣, PKB␤, and PKB␥ was 1.87 Ϯ 0.25, 2.74 Ϯ 0.42, and 6.02 Ϯ 0.96 milliunits/mg of protein/min, respectively. Insulin increased the activity of PKB␣ and PKB␥ by approximately 13-and 6-fold, respectively, while exerting only a minor effect on PKB␤ activity. Exposure of the cells to oxidative stress resulted in a significant increase in basal PKB␣ activity. The ability of insulin to further activate PKB␣ was completely inhibited, while a significant reduction in insulin stimulated PKB␥ activity was observed (from 32.3 Ϯ 4.9 to 15.6 Ϯ 2.6 milliunits/mg of protein/ min, p Ͻ 0.01). These data demonstrate the role of PKB␣ in the induction of basal PKB activity observed in oxidized cells, while the reduction in insulin-stimulated PKB activity could be mainly related to PKB␣ and PKB␥ isoforms.
To assess whether oxidative stress also impairs the ability of PKB to be activated through non-PI 3-kinase-mediated pathways, the phosphorylation and activation of PKB by heat shock were studied in oxidized cells. In control cells, exposure of 3T3-L1 adipocytes for 20 min to 44°C resulted in a wortmannin-insensitive increase in both PKB serine phosphorylation (Fig. 5A) and in PKB activity (Fig. 5B). Cells treated for 2 h

FIG. 2. Insulin-induced IRS-1 redistribution in control and glucose oxidase-treated cells.
Cell treatment and cellular fraction isolation were identical to those described in the legend for Fig. 1. A, total IRS-1 content in the LDM was measured by Western blot analysis. B, tyrosine phosphorylation of LDM proteins was evaluated using anti-phosphotyrosine antibodies (4G10). C, 75 g of LDM were immunoprecipitated using anti-IRS-1 antibody, and the activity of PI 3-kinase co-precipitated with IRS-1 was assayed. D, 500 g of cytosol were subjected to immunoprecipitation using anti-IRS-1 antibody and assessed for IRS-1 content, as well as for PI 3-kinase activity using phosphatidylinositol as substrate (E). with glucose oxidase, and subsequently exposed to heat shock treatment, exhibited increased PKB phosphorylation and activation comparable with control cells. Taken together, these results suggest that oxidative stress does not interfere with activation of PKB through wortmannin-insensitive pathway(s).
Oxidative Stress-impaired Insulin-stimulated GLUT4 Translocation and Glucose Transport Activity-The metabolic relevance of the impaired insulin stimulation of PI 3-kinase redistribution and PKB activation was evaluated by assessing the effects of oxidative stress on glucose transport and GLUT4

FIG. 3. PKB phosphorylation and activation by insulin in control and oxidized cells.
A, cells were treated without or with 250 nM wortmannin for 15 min before and during the 2-h incubation without (C) or with glucose oxidase (GO). Subsequently, cells were lysed in PKB lysis buffer, and 50 g of protein were separated on SDS-polyacrylamide gel electrophoresis and immunoblotted with phosphoserine 473-specific PKB antibody, as described under "Experimental Procedures." B, control and oxidized cells were incubated without or with insulin for the indicated times, and the phosphorylation pattern was studied by Western blot analysis using anti-phospho-Ser 473 antibody. C, 200 g of cell lysate were subjected to immunoprecipitation with anti PKB (C-terminal) antibody and assayed for PKB activity using Crosstide as a substrate. Wortmannin treatment was as described above and also during the 7-min insulin stimulation. *, p Ͻ 0.01 as compared with unstimulated control cells; **, p Ͻ 0.01 versus insulin-stimulated control cells.

FIG. 4. The effect of oxidation on the PKB isoforms expression and regulation by insulin.
A, control or glucose oxidase (GO)-treated 3T3-L1 adipocytes were lysed using PKB lysis buffer, and 50 g of protein were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-PKB␣, anti-PKB␤, and anti-PKB␥ antibodies. Each blot is a representative of at least three independent experiments. B, 200 g of protein from control and GO-treated cells, stimulated without or with 100 nM insulin for 7 min, were lysed and subjected to immunoprecipitation using the specific PKB isoform antibodies. PKB activity was assayed on the immunoprecipitates using Crosstide as a substrate, as further detailed under "Experimental Procedures." Values are expressed as milliunits of activity/mg of protein and are the mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.01 as compared with unstimulated control cells; **, p Ͻ 0.01 versus insulin-stimulated control cells.
translocation. Fully differentiated 3T3-L1 adipocytes exhibited an approximately 12-fold increase in glucose transport activity following 20-min stimulation with 100 nM insulin (Fig. 6A). When cells were exposed for 2 h to glucose oxidase, a marked reduction in insulin-stimulated glucose transport activity was observed. This was associated with a significant 2.1 Ϯ 0.15-fold increase in basal glucose transport, resulting in a reduction in the net insulin effect on glucose transport activity above basal from 510 Ϯ 17 to 77 Ϯ 13 pmol/mg of protein/min. As observed with the activation of PKB (Fig. 3C), both the basal activation as well as the insulin stimulation of glucose transport were completely inhibited by wortmannin. Since insulin acutely activates glucose transport mainly by promoting the translocation of GLUT4 from internal membrane pools to the plasma membrane, GLUT4 translocation was assessed. PMs were prepared using the plasma membrane lawn technique, as described (40), and subjected to Western blot analysis (Fig. 6B,  left side). Using this method, insulin induced a 4.87 Ϯ 1.43-fold increase in PM GLUT4 content in control cells, while only a 1.6 Ϯ 0.3-fold increase was observed in cells treated with glucose oxidase prior to insulin stimulation. To further established whether oxidative stress inhibits translocation of GLUT4 from LDM to the PM, GLUT4 content was evaluated in the subcel-lular fractions using the sucrose cushion methods (39). As can be evaluated, insulin induces 2.55 Ϯ 0.35-fold increase in PM GLUT4 content in control cells (Fig. 6B, right side). This was associated with a 55 Ϯ 1.2% reduction in its abundance in the LDM, indicating insulin-stimulated GLUT4 translocation. In glucose oxidase-treated cells, no significant increase in PM GLUT4 content was observed with insulin, while a nonsignificant 15% reduction in LDM GLUT4 was detected. Taken together these results indicate that the changes in normal insulin-induced PI 3-kinase redistribution and PKB activation by oxidative stress correlate with impairment in insulin-stimulated GLUT4 translocation and activation of glucose transport activity.

DISCUSSION
This study was aimed at investigating the cellular mechanisms by which oxidative stress disrupts insulin action in 3T3-L1 adipocytes. The data presented demonstrate that while certain insulinomimetic effects of micromolar H 2 O 2 concentrations could be observed, oxidative stress impaired the compart-FIG. 5. The effect of oxidative stress on heat shock-induced PKB activation. Control and glucose oxidase (GO)-treated 3T3-L1 adipocytes were further incubated in fresh medium for 20 min at either 37 or 44°C (Heat shock). Heat shock-exposed cells were co-treated without or with 250 nM wortmannin as described in the legend for Fig.  3. A, 50 g of protein of whole cell lysate were subjected to Western blot analysis using anti-phospho-specific PKB antibody (raised against phospho-Ser 473 ). B, 200 g of cell lysate were subjected to immunoprecipitation with anti PKB (C-terminal) antibody and assayed for PKB activity. Values are expressed as milliunits of activity/mg of protein and represent the mean Ϯ S.E. of two independent experiments. *, p Ͻ 0.01 as compared with unstimulated control cells. ment-specific activation of PI 3-kinase, IRS-1 redistribution, and PKB activation induced by insulin. These provide a putative mechanism for impaired insulin-stimulated GLUT4 translocation and glucose transport activity following oxidative stress.
The concept of signal transduction events taking place in specific cellular compartments has been suggested as a plausible explanation for understanding the specificity of hormones and growth factors action. In isolated adipocytes and in 3T3-L1 adipocytes, the activation of PI 3-kinase in the LDM has been suggested as necessary for the specific ability of insulin to induce GLUT4 translocation to the PM, resulting in enhanced glucose transport activity (16 -19, 22, 42). Using the subcellular fractionation protocol on sucrose cushion, the LDM fraction has been shown to contain intracellular membranes comprising recycling endosomes, the Golgi apparatus, intracellular GLUT4 storage vesicles (43)(44)(45), as well as cytoskeleton components (22). Our findings of insulin-stimulated IRS-1 redistribution from the LDM to the cytosol, and of increased PI 3-kinase and tyrosyl-phosphorylated IRS-1 in this fraction following insulin, are in close agreement with previous reports (21,22,46). Whether the IRS-1-PI 3-kinase complex in the LDM directly interacts with the GLUT4 vesicles during the normal insulin signaling toward GLUT4 translocation is currently a subject of debate. Heller-Harrison et al. (19) reported a direct interaction that was demonstrated by insulin-stimulated IRS-1-associated PI 3-kinase activity in isolated GLUT4 vesicles. Yet, other reports challenged this view (15,22). Clark et al. (22) suggested that IRS-1 and PI 3-kinase could be recovered in cytoskeleton components that were nonsoluble by nonionic detergents, as opposed to the GLUT4 protein. The role of such interaction in the normal response to insulin is suggested by the demonstration that PI 3-kinase is necessary for insulininduced actin rearrangement (47,48) and by the observation that disruption of the actin filament network by cytochalasin D or by latrunculin B impairs insulin-stimulated GLUT4 translocation and glucose transport activity (49). Whether the impaired insulin-induced PI 3-kinase and IRS-1 redistribution in 3T3-L1 adipocytes exposed to oxidative stress (Figs. 1 and 2, respectively) represent a defect in their interaction with GLUT4 vesicles or cytoskeleton elements could not be determined by this study. Yet, these observations demonstrate that insulin stimulated PI 3-kinase translocation to and activation in the LDM, possibly through its interaction with tyrosyl phosphorylated IRS-1, are oxidation sensitive steps that may be essential for GLUT4 translocation. To the best of our knowledge, this is the first demonstration that changes in the extra cellular environment induce insulin resistance by impairing IRS-1 and/or PI 3-kinase trafficking, without affecting proximal insulin signaling events as detected in total cell lysates.
Insulin is known to induce both the tyrosine phosphorylation of IRS-1, as well as its phosphorylation on serine/threonine residues. The role and interrelation of these two processes is not fully understood. While the propagation of early steps in the insulin signal appears to depend on tyrosine phosphorylation (50), serine/threonine phosphorylation may be involved in its termination (51,52). Several serine/threonine kinases which are normally activated in response to insulin stimulation have been suggested to phosphorylate IRS-1, potentially representing an inherent shut-down mechanism for the insulin signal. These include ERK 1/2 MAP kinases (53), GSK3 (54), PKB (55), and certain PKC isoforms (56). Moreover, activation of insulin stimulated serine/threonine kinase(s) was suggested to play a role in the translocation of IRS-1 from the LDM fraction to the cytosol (21). In this study we demonstrate that the activation of the serine/threonine kinase PKB was dramat-ically inhibited by oxidative stress (Figs. 3 and 4). Insulin induced activation of the ERK1/2 MAP kinase was also impaired (unpublished results). Interestingly, insulin induced serine/threonine phosphorylation of IRS-1 in the LDM was markedly reduced by oxidation ( Fig. 2A), and was associated with inhibition of its translocation from the LDM to the cytosol. These results are consistent with the notion that serine/threonine phosphorylation of LDM IRS-1 may be needed for its normal cellular redistribution. Nevertheless, insulin stimulated tyrosine phosphorylation of IRS-1 was also impaired in the LDM following oxidation (Fig. 2B), and was associated with reduced PI 3-kinase activity in this fraction ( Fig. 1 and 2). Thus, the oxidation-induced reduction in tyrosine phosphorylated IRS-1 in the LDM may represent the cause rather than the effect of alterations in serine/threonine phosphorylation of LDM IRS-1. Moreover, reduced tyrosine-phosphorylated IRS-1 in the LDM appears to represent the limiting factor for the translocation and activation of PI 3-kinase in this fraction.
It is well established that PKB activation by insulin is dependent on PI 3-kinase (29). Yet whether insulin stimulation of PI 3-kinase in the LDM is required for the activation of the various known isoforms of PKB is largely unknown. Thus, the effect of oxidative stress on the basal and insulin-stimulated activity of PKB␣, PKB␤, and PKB␥ was evaluated. In 3T3-L1 adipocytes, similar to observations in L6 myotubes (23), most of the insulin-stimulated PKB activation could be attributed to PKB␣ and PKB␥ (Fig. 4). Surprisingly, insulin barely affected PKB␤ activity, which was demonstrated as the major PKB isoform regulated by insulin in primary rat adipocytes (57). Oxidative stress profoundly impaired both insulin-stimulated PKB␣ and PKB␥ activities (Fig. 4), resulting in an overall reduction of total cellular PKB activity. The striking similarity between the effect of oxidation on PKB (Figs. 3C and 4) and on glucose uptake (Fig. 6A) suggests a potential cause and effect relationship between the impairment in both insulin stimulated PKB activation and GLUT4 translocation.
The ability to activate PKB through wortmannin-insensitive pathway(s) following oxidative stress was evaluated to exclude a direct inhibitory effect of oxidation on either PKB or its immediate upstream kinases PDK1 and PDK2 (29,58). As demonstrated in COS-7 and NIH 3T3 cells (59), heat shock treatment of 3T3-L1 adipocytes induced phosphorylation and activation of PKB through wortmannin-insensitive mechanisms (Fig. 5, A and B, respectively). Following oxidative stress, this activation of PKB remained intact, supporting the notion that the impaired insulin-stimulated PKB activation induced by oxidation may be a consequence of the reduced ability of insulin to activate PI 3-kinase in the LDM. This, in turn, may offer the possibility that activation of PKB is also an insulin signaling event dependent on normal cellular compartmentalization of PI 3-kinase. In support of this concept is the observation that membrane-localized p110 was found to be sufficient to activate PKB in COS-7 cells (60).
This paper presents evidence that while micromolar concentrations of H 2 O 2 inhibits acute metabolic response to insulin, they also induce a certain insulinomimetic effect. Exposure to H 2 O 2 for 2 h resulted in wortmannin-sensitive increase in basal glucose transport and in PKB activities, as well as in basal PI 3-kinase activity in the LDM (Figs. 6A, 3C, and 1B, respectively). These seemingly opposing effects suggest several cellular targets for H 2 O 2 along the multistep insulin signaling network. While one or more targets may be activated by H 2 O 2 , other can eventually become rate-limiting for additional insulin stimulation.
The potential relevance of findings presented herein may be in the understanding of the cellular mechanisms leading to peripheral insulin resistance in various conditions. Increased oxidative stress has been suggested by various mechanisms to occur in diabetic or prediabetic individuals (61)(62)(63) and to contribute to the pathogenesis of diabetic late complications (64), as well as to peripheral insulin resistance (65,66). Reduced GLUT4 expression and impaired translocation in response to insulin stimulation were reported in adipocytes of insulin-resistant individuals (67). The impaired insulin response was suggested to represent both receptor and postreceptor mechanisms. This study offers a potential mechanism by which oxidative stress can contribute to the development of impaired insulin-stimulated GLUT4 translocation in adipocytes of diabetic subjects.