Insulin-Mediated Cellular Insulin Resistance Decreases Osmotic Shock-Induced Glucose Transport in 3T3-L1 Adipocytes

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0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 12 Printed in U.S.A.

Insulin-Mediated Cellular Insulin Resistance Decreases Osmotic Shock-Induced Glucose Transport in 3T3-L1 Adipocytes* ANDREJ JANEZ, DOROTHY SEARS WORRALL†,

JERROLD M. OLEFSKY

AND

Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093 ABSTRACT Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in re-

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OLECULAR DEFECTS IN insulin target tissues impede insulin action, inducing a state of cellular insulin resistance, as in obesity, type 2 diabetes, and other conditions (1). In these states, skeletal muscle and adipocytes become desensitized to the effects of insulin, leading to decreased insulin stimulated glucose transport in these tissues (2). The exact mechanisms and signaling cascades regulating these events have not been completely elucidated. At the molecular level, impaired insulin signaling results from various defects, including diminished insulin receptor function, disregulation of the GLUT4 transporter, and other alterations in postreceptor signaling steps (1, 3). It is known that hyperosmolarity has potent insulin-like properties on glucose metabolism, including activation of glucose transport in adipocytes and skeletal muscle (4, 5). Chen et al. (6) have shown that osmotic shock-stimulated GLUT4 translocation and glucose transport activity occur via a novel tyrosine kinase-dependent pathway. They have further shown that osmotic shock pretreatment of 3T3-L1 adipocytes induces resistance to insulin-stimulated GLUT4 translocation and glucose transport (7). Although insulin and osmotic shock use distinct signaling pathways to activate GLUT4 translocation and glucose transport, it is clear that the Received April 14, 2000. Address all correspondence and requests for reprints to: Jerrold M. Olefsky, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: [email protected]. * This work was supported by NIH Grant DK-33651 and the Veterans Administration Medical Research Service. Andrej Janez was supported by a grant from Slovenian Ministry of Science and Technology (sklad za mlade raziskovalce). † Supported by NIH/NIDDK Individual NRSA Grant DK-09595.

sponse to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction. (Endocrinology 141: 4657– 4663, 2000)

exposure of cells to osmotic shock attenuates the function of molecules required for insulin signal transduction. Specifically, osmotic shock pretreatment markedly inhibited insulin stimulation of Akt/PKB and p70S6 kinase activities; however, the precise mechanisms for these effects are unknown. Given the molecular cross-talk between the two pathways suggested by these data (7), we hypothesized that chronic insulin exposure would conversely induce cellular resistance to osmotic shock-induced glucose transport and GLUT4 translocation. In the present work, we provide evidence that prolonged insulin treatment of 3T3-L1 adipocytes decreases both insulin- and osmotic shock-induced glucose transport and GLUT4 translocation. Furthermore, it inhibits Gab-1 tyrosine phosphorylation and Gab-1 associated PI 3-kinase activity, events that are necessary for stimulation of glucose transport (8). Materials and Methods Materials DMEM and Glutamax were obtained from Life Technologies, Inc. (Rockville, MD). Penicillin-Streptomycin and FCS were obtained from Omega Scientific (Tarzana, CA). Insulin was a gift from Eli Lilly & Co. (Indianapolis, IN). Silica-coated TLC plates and all chemicals, unless otherwise noted were obtained from Sigma (St. Louis, MO). Gab-1, p85␣-N-SH3 antibodies, wortmannin, and recombinant protein Aagarose were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The GLUT4 antibody was obtained from Chemicon (Temecula, CA). Mouse monoclonal antiphosphotyrosine (PY-20) was from Transduction Laboratories, Inc. (San Diego, CA). Fluorescein isothiocyanate (FITC)-, tetramethyl-rodamine isothiocyanate (TRITC)-and aminomethylcoumarin acetate-conjugated antimouse were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All radioisotopes were obtained

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from NEN Life Science Products (Boston, MA). Enhanced chemiluminescence reagent was obtained from Pierce Chemical Co. (Rockford, IL).

Cell culture 3T3-L1 fibroblasts were maintained in DMEM, high glucose, containing 10% calf serum. Postconfluency fibroblasts were differentiated into adipocytes by changing the medium with DMEM, high glucose, containing 10% FCS, 1 ␮g/ml insulin, 0.1 ␮g/ml dexamethasone, and 112 ␮g/ml isobutylmethylxanthine. The medium was removed after 2 days and replaced with DMEM, low glucose, containing 10% FCS, Glutamax, and 1% penicillin-streptomycin. Three to 7 days after the addition of the differentiation mix, the cells were plated in 6- or 12-well dishes at densities of 8 ⫻ 105 and 4 ⫻ 105, respectively. The medium was changed every second day until use, 10 –12 days post differentiation. Approximately 90% of the cells exhibited large lipid droplets indicative of adipocytes. Twenty-four hours before the start of all experiments, cells were given fresh DMEM low glucose media. In the case of the chronic insulin treatment, fully differentiated cells were incubated with 10 nm insulin for 10 h in DMEM, low glucose media. After 10 h, cells were rinsed and placed in a Krebs-Ringer phosphate buffer (KRP) containing 0.1% BSA for 30 min at 37 C before being stimulated. This protocol was used in all experiments.

2-Deoxyglucose uptake in 3T3-L1 adipocytes Chronically insulin-treated and control 3T3-L1 adipocytes were treated as described above, followed by stimulation with 16.6 nm insulin or 600 mm sorbitol for 20 min at 37 C. Glucose transport was determined by the addition of 0.1 mm 2-deoxyglucose containing 0.2␮Ci of 2-[3H] deoxyglucose as described previously (9). Nonspecific uptake was assessed using 0.1 mm l-glucose containing 0.2 ␮Ci of l-[3H]glucose. The reaction was stopped after 10 min by aspiration, and extraneous glucose was removed by four washes with ice-cold PBS. Cells were lysed in 1 n NaOH, and glucose uptake was assessed by scintillation counting. Samples were normalized for protein content by Bradford protein assay.

Subcellular fractionation of 3T3-L1 adipocytes and GLUT4 immunodetection Cells from one 10-cm dish were incubated with 16.6 nm insulin or 600 mm sorbitol for 20 min at 37 C, and then washed three times with ice-cold PBS. Cells were scraped into ice-cold HES buffer (255 mm sucrose, 20 mm HEPES, 1 mm EDTA, pH 7.4) supplemented with protease inhibitors (1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 200 ␮m PMSF). Cells were then homogenized using an LSC homogenizer. Subcellular fractionation was carried out as described previously (10). Proteins from different fractions were were solubilized in Laemmli sample buffer and separated by SDS-PAGE with a 10% resolving gel. The proteins were then transferred to PVDF membrane and blotted overnight at 4 C with GLUT4 antibodies. After incubation with a secondary horseradish peroxidase-conjugated goat antirabbit antibody, the proteins were visualized by enhanced chemiluminescence, autoradiography, and densitometric quantitation.

Assessment of Gab-1 tyrosine phosporylation and association with p85 Adipocytes plated in six-well dishes were stimulated at 37 C with 16.6 nm insulin or 600 mm sorbitol for 20 min. Cells were lysed at 4 C in a lysis buffer containing 50 mm HEPES, 150 mm NaCl, 1% Triton X-100, 4 mm sodium orthovanadate, 20 mm sodium pyrophosphate, 200 mm sodium fluoride, 0.5 m EDTA, 80% glycerol, pH 7.4. Lysates were centrifuged at 14,000 ⫻ g for 10 min at 4 C. Supernatants were incubated with Gab-1 antibody and recombinant protein A-agarose overnight at 4 C. Pellets were washed three times in lysis buffer. Laemmli‘s buffer was added to the pellets and boiled for 5 min. Samples were separated by SDS-PAGE on 7.5% polyacrylamide gels. Proteins were transferred to PVDF membrane and blotted with PY20, p85, and Gab-1 antibodies according to the manufacturer‘s instructions. Following incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were visualized by enhanced chemiluminescence, autoradiography, and densitometric quantitation.

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Measurement of PI 3-kinase activity 3T3-L1 adipocytes were stimulated as described in the figure legends and then lysed as described above. Supernatants were incubated with Gab-1 antibody and recombinant protein A-agarose overnight at 4 C. Bead pellets were washed three times with Buffer A (Tris-buffered saline, pH 7.4, 1% Nonident P-40, and 100 ␮m Na3VO4), three times with Buffer B (100 mm Tris, pH 7.4, 500 mm LiCl2, and 100 ␮m Na3 VO4), and twice with Buffer C (10 mm Tris, pH 7.4, 100 mm NaCl, 1 mm EDTA and 100 ␮m Na3VO4). Pellets were resuspended in Buffer C without the Na3VO4. As described previously (11), PI 3-kinase activity was assessed by the phosphorylation of phosphatidylinositol in the presence of 20␮Ci of [␥-32P]ATP for 10 min. The reactions were stopped with 20 ␮l of 8 n HCl and 160 ␮l of CHCl3:methanol (1:1) and centrifuged. The lower organic phase was removed and applied to potassium oxalate (1%)coated silica gel TLC plates. Following the separation of lipids by TLC using the borate-buffered system (12), phosphatidylinositol 3-phosphate was visualized by autoradiography. National Institutes of Health Image scanning software was used for quantitation.

Actin localization As done previously (8, 9), 3T3-L1 adipocytes were stimulated with 16.6 nm insulin or 600 mm sorbitol for 10 min and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized in 0.2% Triton X-100 for 5 min, washed in PBS, and incubated at room temperature for 45 min with TRITC-phalloidin (0.5 ␮g/ml) in PBS together with FITC-antimouse antibodies (1:100) for 45 min. After staining, coverslips were washed successively in PBS and deionized water for 15 min and mounted with Gelvatol. Cytoskeletal changes were quantitated blindly, counting at least 100 cells in random fields. Cells that showed actin staining at the periphery were scored as positive for membrane ruffles. The percentage of total counted cells displaying membrane ruffles is represented as ruffling index. The cells were inspected and photographed with a Carl Zeiss (New York, NY) Axiophot fluorescence microscope.

Statistical analysis Values are expressed as means ⫾ sem. Results were analyzed by using the Student’s t test. A value of P ⬍ 0.05 was considered significant.

Results Chronic insulin treatment induces resistance to osmotic shock-stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes

Previous studies have shown that prolonged insulin treatment of 3T3-L1 adipocytes causes a state of insulin resistance as manifested by decreased GLUT4 translocation and glucose transport (13). Additionally, chronic insulin exposure (greater than 16 h) can result in decreased GLUT4 protein expression in 3T3-L1 adipocytes (13). We chose to use a 10-h insulin incubation for our studies because, at this time point, cells are rendered insulin resistant with respect to glucose transport but there is no loss of GLUT4 protein (Fig. 1A). Consistent with previous studies (6, 7), osmotic shock, induced by sorbitol treatment, resulted in a significant increase in 2-deoxyglucose uptake. Sorbitol treatment was not as effective as insulin but led to a 4-fold increase in glucose transport, approximately 60% the effect of insulin (Fig. 1B, open bars). In contrast, as seen in Fig. 1B (solid bars) after 10 h of chronic insulin treatment, osmotic shock and acute insulin stimulation of glucose transport were markedly reduced. In other words, the cells had become resistant to both osmotic shock and insulin. We observed a tendency for the basal glucose transport to be higher in resistant cells compared

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FIG. 1. Chronic insulin treatment induces osmotic shock resistance to glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were untreated or incubated with 10 nM insulin for 10 h at 37 C. Subsequently, cells were stimulated with 16.6 nM insulin for 20 min. Proteins were solubilized in Laemmli sample buffer and separated by 10% SDSPAGE and blotted with antibody to GLUT4. A typical autoradiogram is shown along with graphical representation of the densitometry data (expressed in arbitrary units). Data are representative of two independent experiments. B, 3T3-L1 adipocytes were untreated (open bars) or incubated with 10 nM insulin for 10 h at 37 C (solid bars). Cells were then untreated or stimulated with 16.6 nM insulin or 600 mM sorbitol for 20 min. 2-[3H]deoxyglucose uptake was determined as described in Materials and Methods. Mean ⫾ SEM of five experiments is shown. *, P ⬍ 0.01 (insulin-resistant vs. nonresistant); **, P ⬍ 0.05 (sorbitolresistant vs. nonresistant). C, Insulinresistant and nonresistant 3T3-L1 adipocytes were untreated or stimulated with 16.6 nM insulin or 600 mM sorbitol for 20 min. Cells were fractionated into low density microsomes (LDM) or plasma membranes (PM) as described in Materials and Methods. Proteins were separated by 10% SDS-PAGE and blotted with antibody to GLUT4. A typical autoradiogram is shown along with graphical representations of densitometry data which are the mean ⫾ SEM of three independent experiments (expressed as percent of maximum signal).

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with control cells; however, this difference did not reach statistical significance. We then investigated whether chronic insulin treatment affects GLUT4 translocation to the plasma membrane. 3T3-L1 adipocytes were incubated for 10 h with 10 nm insulin, washed and acutely stimulated with sorbitol or insulin for 20 min followed by isolation of low density microsomes (LDMs) and plasma membranes (PMs) and immunoblotted with GLUT4 antibodies (Fig. 1C). After insulin stimulation of control cells, the amount of GLUT4 in the LDM fraction decreased by 58%, and increased 7.8-fold in the PM fraction. Osmotic shock led to a 43% decrease in LDM GLUT4 and 2.2-fold increase in the PM GLUT4. In contrast, resistant cells show no significant effect of acute insulin or osmotic shock stimulation on GLUT4 localization to LDMs or PMs, compared with basal localization. Osmotic shock-induced Gab-1 phosphorylation is decreased in cells chronically treated with insulin

We have previously shown that osmotic shock leads to Gab-1 tyrosine phosphorylation with PI 3-kinase association and that Gab-1 is a key component of the shock-induced glucose transport signaling pathway (8). To determine whether Gab-1 phosphorylation following osmotic shock stimulation is altered in chronically insulin-treated cells, lysates from insulinor sorbitol-stimulated cells were immunoprecipitated with antiGab-1 antibody and immunoblotted with antiphosphotyrosine antibody. In control, nonresistant cells, an 8- to 10-fold increase in tyrosine phosphorylated Gab-1 was observed upon sorbitol stimulation compared with a 6-fold increase with insulin stimulation (Fig. 2, upper panel). In resistant cells, the phosphorylation of Gab-1 was markedly decreased in response to either osmotic shock or acute insulin stimulation. Changes in Gab-1 phosphorylation, were not due to change in the total amount of the Gab-1 protein, as detected by immunoblotting (Fig. 2, lower panel). Decreased Gab-1 binding and activation of PI 3-kinase upon osmotic shock stimulation in chronically insulin treated cells

To investigate whether chronic insulin treatment alters Gab-1 association with the p85 subunit of PI 3-kinase, we immunoprecipitated lysates from sorbitol- or insulin-stimulated cells with anti-Gab-1 antibodies and immunoblotted with anti-p85 antibody. As shown in Fig. 3, in control cells, sorbitol treatment led to a 10-fold increase in the association of Gab-1 with the p85 subunit of PI 3-kinase in contrast to a 4-fold increase upon insulin stimulation. In insulin resistant cells, this association was decreased by 54% upon sorbitol stimulation, and by 64% (mean ⫾ sem of three experiments) in response to an acute insulin stimulation. The levels of Gab-1 protein in these immunoprecipitates were similar (data not shown). We further determined the effect of chronic insulin treatment on Gab-1 associated PI 3-kinase activity in osmotic shock- and acutely insulin-stimulated cells. In control cells, acute insulin treatment induced a 2.7-fold increase in PI 3-kinase activity in anti-Gab-1 immunoprecipitates (Fig. 4A, lane 4), compared with an 7.8-fold increase upon sorbitol

FIG. 2. Osmotic shock-induced Gab-1 phosphorylation is decreased in insulin-resistant 3T3-L1 adipocytes. Insulin-resistant and nonresistant 3T3-L1 adipocytes were stimulated with 16.6 nM insulin or 600 mM sorbitol for 20 min. Lysates were immunoprecipitated with antiGab-1 antibodies. A, Immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and blotted with antiphosphotyrosine (pY) antibodies. Representative authoradiograph is shown (upper panel). The membrane was stripped and reblotted with anti-Gab-1 antibody to detect the levels of Gab-1 protein (lower panel). B, Graphical representation of phospho-Gab-1 densitometry data, which are the mean ⫾ SEM of three independent experiments represented in upper panel of (A) and expressed in arbitrary units.

stimulation (Fig. 4B, lane 4). As expected, addition of 100 nm wortmannin to the reaction mixture completely inhibited Gab-1-associated PI 3-kinase activity stimulated by either osmotic shock or insulin (Fig. 4, A and B, lane 5). In insulinresistant cells, the Gab-1 associated PI 3-kinase activity was decreased by 61% upon insulin (Fig. 4A, lane 3), and by 70% upon osmotic shock stimulation (Fig. 4B, lane 3). Effect of osmotic shock on actin filament rearrangement in insulin resistant 3T3-L1 adipocytes

Because membrane ruffling is a PI 3-kinase dependent phenomenon (9, 14 –16), we have investigated whether chronic insulin treatment affects this event stimulated by osmotic shock or insulin. In control cells, sorbitol and insulin stimulation for 15 min caused the appearance of membrane ruffles in 35% and 75% of the cells counted, respectively (Fig. 5, open bars). In contrast, in insulin-resistant cells, the appearance of membrane ruffles was decreased by 42 ⫾ 2% in sorbitol-stimulated cells and by 67 ⫾ 2% in insulin-stimulated cells (Fig. 5, solid bars). Discussion

It is well established that insulin stimulates the translocation of the GLUT4 glucose transporters from intracellular vesicular storage sites to the plasma membrane in both mus-

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FIG. 3. Effect of insulin resistance on the osmotic shock-induced association of Gab-1 with the p85 subunit of PI 3-kinase. Insulin resistant and nonresistant 3T3-L1 adipocytes were stimulated with 16.6 nM insulin or 600 mM sorbitol for 20 min. Lysates were immunoprecipitated with anti-Gab-1 antibodies. A, Immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and blotted with anti-p85 antibodies. Representative authoradiograph is shown. Identical amounts of proteins were analyzed as described in Materials and Methods. B, Graphical representation of Gab-1-p85 association densitometry data are the mean ⫾ SEM of three independent experiments represented in (A) and expressed in arbitrary units.

cle and adipose cells. There are also several insulinomimetic agents that can stimulate glucose transport and GLUT4 translocation (3). Recent studies have observed that, in addition to insulin, osmotic shock also stimulates glucose transport activity and GLUT4 translocation though a novel tyrosine kinase-dependent pathway (6, 7). An interesting observation was that chronic exposure to osmotic shock rendered cells unable to respond to subsequent insulin stimulation, creating a state of cellular insulin resistance (7). In our current study, we have taken adventage of the fact that chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes, to study the effect of insulin resistance on the mechanisms controlling osmotic shock-induced glucose transport. The major findings of these experiments are that chronic insulin treatment causes resistance to osmotic shockinduced glucose transport and GLUT4 translocation. With respect to mechanisms, we found that insulin resistance attenuates osmotic shock-induced Gab-1 tyrosine phosphorylation and Gab-1 associated PI 3-kinase activity, events that mediate shock-stimulated glucose transport (8). In skeletal muscle and in adipocytes, insulin resistance may be caused by defects in insulin-stimulated glucose transport, which results from impairment in the translocation, fusion, or activation of GLUT4 glucose transporters (3, 17). Chronic insulin treatment of 3T3-L1 adipocytes produces a persistent defect in the cells’ ability to respond to subsequent acute insulin stimulation with an increase in GLUT4 translocation and glucose transport activity. In addition, chronic

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hyperinsulinemia can also affect the expression of GLUT4 protein. Previously published chronic insulin treatment conditions dramatically decreased the total GLUT4 amount in 3T3-L1 adipocytes (13) and adipocytes from obese and type 2 diabetic patients also exhibit reduced levels of GLUT4 protein (18, 19). In the current series of experiments, we wanted to produce a state of cellular insulin resistance without a loss of GLUT4 protein content, so that any defects would be attributable to signaling abnormalities. Therefore, we used a reduced insulin concentration of 10 nm and, importantly, a reduced insulin treatment time of 10 h, and found that these conditions caused cellular insulin resistance with no loss of total cellular GLUT4 protein content. With this approach, we studied the effects of 10 h insulin treatment on the subsequent ability of cells to respond to an osmotic shock stimulus and found that, independent of changes in GLUT4 content, the insulin-resistant cells were also resistant to the stimulatory effects of osmotic shock on glucose transport and GLUT4 translocation to the plasma membrane. Although insulin- and osmotic shock-induced GLUT4 translocation to the plasma membrane seems to be completely blocked in resistant cells, there remains a residual amount of glucose transport following either acute stimulus. Kozka et al. (13) have shown that chronic insulin treatment, in addition to causing GLUT4 down-regulation, causes an increase in cell surface GLUT1 that is correlated with an increase in basal glucose uptake. Thus, it is possible that the residual glucose uptake we observe in resistant, acutely stimulated cells is due to transport via the GLUT1 transporter, although we did not measure GLUT1 levels. Additionally, enhanced intrinsic activity of either transporter may be responsible for the residual glucose transport we observe in resistant, acutely stimulated cells (20). Our findings provide data on the mechanisms of this effect. Because shock does not lead to activation of the insulin receptor or IRS-1 (7), we focused our attention on other potential signaling molecules. We have previously observed that osmotic shock leads to extensive tyrosine phosphorylation of a new member of the IRS family called Gab-1, which then associates with the p85 subunit of PI 3-kinase with subsequent activation of the enzyme (8). Our earlier studies also showed that Gab-1 is a necessary signaling element of shock-induced GLUT4 translocation (8). The current results show that chronic-insulin exposure attenuates the tyrosine phosphorylation of Gab-1 induced by both osmotic shock and insulin. This attenuation presumably results from a chronic insulin-induced modification of Gab-1 protein or inhibition of a Gab-1 kinase. Additionally, we have found that a decrease in both insulin- and shock-stimulated Gab-1 phosphorylation led to a marked impairment in the ability of Gab-1 to associate with the p85 regulatory subunit of PI 3-kinase following acute insulin or osmotic shock stimulation. The chronic insulin-induced inhibition of Gab-1 associated PI 3-kinase activity correlates with the reduction in Gab-1 tyrosine phosphorylation and Gab-1-p85 association upon shock or insulin stimulation, indicating that the phosphotyrosines involved in PI 3-kinase association were inhibited to a similar extent as overall Gab-1 phosphorylation. Given our previous results showing the importance of Gab-1 in the shock signaling pathway (8), it is reasonable to

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FIG. 4. Gab-1-associated PI 3-kinase activity induced by osmotic shock is decreased in insulin-resistant 3T3-L1 adipocytes. Insulin-resistant and nonresistant 3T3-L1 adipocytes were either untreated or stimulated with 16.6 nM insulin (panel A) or 600 mM sorbitol (panel B) for 20 min after pretreatment with wortmannin (20 min, 100 nM) as indicated. Gab-1-associated PI 3-kinase activity was determined by incorporation of 32 P into phosphatidylinositol and separation of phospholipids by TLC as described in Materials and Methods. The position of the PI 3-kinase product is indicated (PI-3P). Quantitative results were obtained using NIH Image. Representative experiments are shown and quantitative graphs shown on right are the mean ⫾ SEM of three experiments, expressed in arbitrary units. R, Resistant, N, nonresistant.

FIG. 5. Effect of osmotic shock on actin filament rearrangement in insulin resistant 3T3-L1 adipocytes. 3T3-L1 adipocytes were untreated (open bars) or incubated with 10 nM insulin for 10 h at 37 C (solid bars). Cells were then either stimulated with 16.6 nM insulin or 600 mM sorbitol for 15 min, fixed, stained, and analyzed for actin filament rearrangement (membrane ruffling) as described in Materials and Methods. Results are expressed as the percent of positive cells. Error bars represent the mean ⫾ SEM for three independent experiments. *, P ⬍ 0.01 (insulin-resistant vs. nonresistant); **, P ⬍ 0.05 (sorbitol-resistant vs. nonresistant).

conclude that defective Gab-1 phosphorylation is an important contributor to the osmotic shock resistance we have observed.

It is interesting to compare our current study with that of Chen et al. (7), who have shown that pretreatment of cells with hyperosmotic shock induces cellular insulin resistance. In their study, cells exposed to hyperosmotic conditions and subsequently acutely stimulated with insulin responded normally with respect to insulin receptor and IRS1 tyrosine phosphorylation, as well as PY20-associated PI 3-kinase activity; however, insulin-stimulated Akt/PKB phosphorylation was defective in these cells. The defect in Akt/PKB phosphorylation in their study was apparently due to the osmotic shock-induced activation of an Akt/PKB-directed phosphatase. Thus, the target of osmotic shock-induced insulin resistance is downstream of PI 3-kinase activation. In contrast, our study indicates that the target of insulininduced osmotic shock resistance is upstream of PI 3-kinase, presumably at the level of Gab-1 tyrosine phosphorylation. Akt/PKB phosphorylation does not occur in response to osmotic shock (7),and we also find no Akt/PKB phosphorylation in insulin-resistant cells treated with acute osmotic shock (Janez, A., D. S. Worrall, and J. M. Olefsky, unpublished observation). We have previously shown that insulin has a robust effect on stimulation of membrane ruffling in a PI 3-kinase-dependent manner (9, 14 –16). We have also observed that sorbitol stimulation is capable of inducing membrane ruffling, but only up to 50% of the insulin effect ( Ref. 8 and this study).

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Data from our present study demonstrate that insulin resistance inhibits both insulin- and sorbitol-induced membrane ruffling. Thus, measuring two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling, we found that both effects are decreased in chronically insulin-treated cells. The studies described above clearly demonstrate that chronic insulin exposure leads to desensitization of the osmotic shock stimulated pathway and induces a state of cellular osmotic shock resistance for glucose transport and GLUT4 translocation. In addition, chronic insulin treatment decreases Gab-1 phosphorylation and Gab-1 associated PI 3-kinase activity, events that are necessary for full stimulation of osmotic shock-induced glucose transport. It is possible that these findings may underlie the effect of insulin resistance on other insulinomimetic agent-regulated pathways leading to GLUT4 translocation and glucose transport.

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10. 11. 12.

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Acknowledgments We thank Donna Reichart for maintaining the 3T3-L1 adipocytes.

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