Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes.

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)-kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser307. The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.

In 3T3-L1 adipocytes, hyperosmotic stress was found to inhibit insulin signaling, leading to an insulin-resistant state. We show here that, despite normal activation of insulin receptor, hyperosmotic stress inhibits both tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated phosphoinositide 3 (PI 3)kinase activity in response to physiological insulin concentrations. Insulin-induced membrane ruffling, which is dependent on PI 3-kinase activation, was also markedly reduced. These inhibitory effects were associated with an increase in IRS-1 Ser 307 phosphorylation. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the osmotic shock-induced phosphorylation of IRS-1 on Ser 307 . The inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. In addition, prolonged osmotic stress enhanced the degradation of IRS proteins through a rapamycin-insensitive pathway and a proteasome-independent process. These data support evidence of new mechanisms involved in osmotic stress-induced cellular insulin resistance. Short-term osmotic stress induces the phosphorylation of IRS-1 on Ser 307 by an mTOR-dependent pathway. This, in turn, leads to a decrease in early proximal signaling events induced by physiological insulin concentrations. On the other hand, prolonged osmotic stress alters IRS-1 function by inducing its degradation, which could contribute to the down-regulation of insulin action.
Insulin regulates blood glucose levels through multiple regulatory mechanisms such as suppression of endogenous glucose production in liver and stimulation of glucose uptake into muscle and adipocytes (1). Glucose transport in muscle and adipose tissues is caused by the translocation of the glucose transporter Glut 4 from an intracellular pool to the plasma membrane. These biological responses require tyrosine phosphorylation of IRS-1, 1 which, in turn, binds and activates PI 3-kinase. Downstream effectors of PI 3-kinase such as protein kinase B (PKB) or atypical PKC could be involved in Glut 4 translocation (2,3). Further, it has been recently shown that insulin-induced Glut 4 translocation also requires the activation of the adaptor protein containing PH and SH2 domain (APS)/Cbl-associated protein (CAP)/Cbl/Crk-II/TC10 pathway independent of PI 3-kinase activation (4 -6). Other stimuli, such as osmotic shock, can promote Glut 4 translocation. However, osmotic shock only partly mimics the insulin effect on Glut 4 translocation and glucose uptake. The effect induced by osmotic stress requires the tyrosine phosphorylation of the adaptor protein Grb2-Associated binder-1 and is independent of PI 3-kinase/PKB activation (7)(8)(9)(10). We have recently shown that both osmotic shock and insulin share the Crk-II/TC10 pathway to stimulate Glut 4 translocation (10). However, like several other insulinomimetic agents, hyperosmolarity not only partly activates several insulin-specific biological responses but also induces a state of insulin resistance. Indeed, in rat epididymal fat cells, hyperosmotic stress markedly reduces insulin-induced glucose transport (11). In perfused rat liver, hyperosmolarity impairs insulin-mediated cell swelling and reverses the proteolysis inhibition induced by insulin (12). In 3T3-L1 adipocytes, pretreatment with sorbitol strongly decreases the ability of insulin to stimulate glucose uptake, lipogenesis, and glycogen synthesis (13). The molecular mechanism by which hyperosmotic stress antagonizes insulin-mediated responses has not yet been fully elucidated. A recent report has shown that hyperosmolarity prevents insulin-induced activation of PKB. This could be mediated by the stimulation of calyculin A-or okadaic acidsensitive protein phosphatases acting at the level of PKB in response to hyperosmotic stress (13). Thus, hyperosmolarityinduced insulin resistance could partially result from the stimulation of a PKB phosphatase that maintains PKB in an inactive state. However, because the role of PKB in insulinstimulated glucose transport is still controversial (14 -17), other mechanisms, in addition to dephosphorylation of PKB, could be involved in hyperosmolarity-induced insulin resistance. Indeed, it has recently been shown that insulin resistance caused by short-and long-term stress could be caused by an increase in the serine/threonine phosphorylation of IRS-1, with a subsequent inhibition of IRS-1 tyrosine phosphorylation. For instance, the inflammatory cytokine tumor necrosis factor-␣ induces the phosphorylation of IRS-1 on its Ser 307 (18). This serine residue is located near the phosphotyrosine binding (PTB) domain of IRS-1, and an interaction between this domain and the activated insulin receptor is required for the tyrosine phosphorylation of IRS-1 (2). IRS-1 phosphorylation on Ser 307 prevents this interaction and thus inhibits tyrosine phosphorylation of IRS-1 providing a potential mechanism to explain, at least in part, the insulin resistance induced by cellular stress (19). Whereas serine phosphorylation could be a short-term mechanism involved in the negative regulation of IRS-1 function, regulated degradation of IRS-1 might also promote longterm insulin resistance (2).
In this report, we provide evidences of new mechanisms, apart from PKB inactivation, that contribute to osmotic stressinduced insulin resistance. We show that short-term osmotic stress inhibits IRS-1 function by an mTOR-dependent phosphorylation of IRS-1 on Ser 307 , whereas prolonged osmotic stress promotes the degradation of IRS proteins by an mTOR-and proteasome-independent mechanism.
Cell Culture-3T3-L1 fibroblasts were grown in 35-or 100-mm dishes in DMEM, 25 mM glucose, and 10% calf serum and induced to differentiate in adipocytes as described previously (10,20). Differentiation medium was removed after 2 days and replaced with DMEM, 25 mM glucose, and 10% fetal calf serum supplemented with insulin for 2 more days. The cells were fed every 2 days with DMEM, 25 mM glucose, and 10% fetal calf serum. 3T3-L1 adipocytes were used 8 -15 days after the beginning of the differentiation protocol.
Immunoprecipitation Assays-3T3-L1 adipocytes were serumstarved overnight in DMEM/0.5% bovine serum albumin. 3T3-L1 adipocytes were incubated in serum-free medium supplemented or not with 600 mM sorbitol and subsequently treated with or without insulin, as indicated in figure legends. To study the effect of pharmacological inhibitors, the cells were pretreated for 30 min with various inhibitors in serum-free medium followed by incubation in the serum-free medium with or without 600 mM sorbitol and pharmacological inhibitors. Cells were subsequently washed with ice-cold buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 150 mM NaF, and 2 mM sodium orthovanadate) before solubilization for 30 min at 4°C in the lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 150 mM NaF, 2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, protease inhibitors, 100 nM okadaic acid, and 1% Triton X-100). Lysates obtained after centrifugation (15 min at 15,000 ϫ g at 4°C) were incubated for 3 h at 4°C with appropriate antibodies pre-adsorbed on protein-G-Sepharose (4 g of antibodies/sample). After washes with lysis buffer, immune pellets were resuspended in Laemmli buffer and proteins were separated by SDS-PAGE using a 7.5% resolving gel and transferred to polyvinylidene difluoride membrane. The membrane was blocked with saline buffer (10 mM Tris, pH 7.4, and 140 mM NaCl) containing 5% (w/v) bovine serum albumin for 2 h at room temperature and blotted overnight at 4°C with commercial antibodies at the dilution indicated by the manufacturer's instructions and at 0.5 g/ml for the anti-pS 307 -IRS1 antibody. After incubation with horseradish peroxidase-conjugated secondary antibodies, proteins were detected by enhanced chemiluminescence. In some cases, the membrane was stripped for 30 min at 50°C in 62 mM Tris, pH 7.6, 100 mM 2-mercaptoethanol, and 2% SDS, and reprobed with the indicated antibodies.
PI 3-Kinase Assay-3T3-L1 adipocytes were treated in the presence or absence of rapamycin for 30 min in serum-free medium. The cells were then incubated in serum-free medium supplemented with or without 600 mM sorbitol and with or without rapamycin for 40 min before 5 min of insulin stimulation (0.2 nM) as indicated in the figure legends. Cell lysates were immunoprecipitated with anti-IRS-1 antibodies. Thereafter, immune pellets were washed twice with each of the following buffers: 1) PBS containing 1% Nonidet P-40 and 200 M Na 3 VO 4 ; 2) 100 mM Tris, pH 7.4, 0.5 M LiCl, and 200 M Na 3 VO 4 ; and 3) 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, and 200 M Na 3 VO 4 . PI 3-kinase activity was measured on the immune pellets as described previously (21).
Immunofluorescence and Image Analysis-3T3-L1 adipocytes were grown and differentiated on glass coverslips. After overnight serum starvation, cells were treated with or without rapamycin for 30 min in the serum-free medium. Then, the medium was exchanged for serumfree medium supplemented with or without 600 mM sorbitol and with or without rapamycin for 40 min before 20 min of insulin stimulation (0.5 nM). Cells were washed twice with ice-cold PBS and fixed with 4% paraformaldehyde for 20 min on ice. After two washes with ice-cold PBS, the cells were incubated with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin for 30 min at room temperature. After three washes with ice-cold PBS, cells were incubated with Texas redphalloidin (Molecular Probes, Inc., Eugene, OR) in PBS/0.1% Triton X-100/1% bovine serum albumin for 30 min at room temperature. Cells were then washed twice with ice-cold PBS, and coverslips were mounted onto glass slides. Cells were examined using a Leica confocal microscope equipped with a Leica confocal laser scanning imaging. Cells were studied at a magnification of 40ϫ using a 1.0 -0.50 oilimmersion objective. Series of images were collected along the z-axis and examined. In each condition, 200 cells in random fields were examined by two different persons blind to the images' origin. Cells that showed clustering of actin staining at the periphery were scored as positive for membrane ruffles. The experiment was repeated three times.
Statistical Analysis-Statistical analysis was performed by Student's t test. Statistical significance was assessed at p Ͻ 0.05

Hyperosmotic Stress Impairs IRS-1 Tyrosine Phosphorylation, Subsequent PI 3-Kinase Activation, and Membrane Ruf-
fling Induced by Insulin-Insulin effect on glucose uptake requires the tyrosine phosphorylation of IRS-1 and the activation of PI 3-kinase (3). Because hyperosmotic stress antagonizes insulin-mediated Glut 4 translocation and glucose uptake (13), we investigated the effect of hyperosmotic stress on insulinmediated tyrosine phosphorylation of IRS-1. IRS-1 was immunoprecipitated from 3T3-L1 adipocytes pre-treated or not with 600 mM sorbitol for 40 min before insulin stimulation using a physiological or a supraphysiological insulin concentration (0.2 and 100 nM, respectively). IRS-1 tyrosine phosphorylation was analyzed by immunoblotting with phosphotyrosine antibodies. As shown in Fig. 1, sorbitol alone did not induce IRS-1 tyrosine phosphorylation. Sorbitol treatment inhibited insulin-induced tyrosine phosphorylation of IRS-1 by 50% at physiological insulin concentration (0.2 nM), but did not modify IRS-1 tyrosine phosphorylation induced by 100 nM insulin. We then investigated whether this decrease in IRS-1 tyrosine phosphorylation also impairs its ability to recruit the p85 subunit of the PI 3-kinase and to activate the enzyme. IRS-1 was immunoprecipitated from 3T3-L1 adipocytes pretreated with or without 600 mM sorbitol for 40 min before 0.2 nM insulin stimulation. The level of p85 subunit of the PI 3-kinase coimmunoprecipitated with IRS-1 was detected with an anti-p85 antibody. As shown in Fig. 2A, treatment of cells with sorbitol induced a 57% inhibition in the amount of p85 associated with IRS-1 in response to a physiological insulin concentration. This was correlated with a reduction in PI 3-kinase activity (Fig.  2B). Indeed, in absence of sorbitol, PI 3-kinase activity associated to IRS-1 was increased 14-fold by insulin, and sorbitol induced a 50% inhibition of insulin effect. This indicates that osmotic stress inhibits both the tyrosine phosphorylation of IRS-1 and subsequent PI 3-kinase activation.
Because the activation of PI 3-kinase (22)(23)(24) and not PKB (25) plays an important role in insulin-induced membrane ruffling, we investigated the effect of hyperosmotic stress on this insulin effect. 3T3-L1 adipocytes were incubated with or without sorbitol before insulin stimulation. Cells were then fixed and permeabilized, and F-actin structures were visualized using Texas red coupled-phalloidin by confocal fluorescence microscopy as described under "Experimental Procedures." Representative fields obtained in each condition are shown in Fig.  3A. Quantification of several experiments (Fig. 3B) indicates that sorbitol induced an increase of nearly 2-fold in the number of cells with membrane ruffles (12% in unstimulated cells versus 22% in sorbitol-treated cells). Insulin stimulation promoted a 4-fold increase in the number of cells harboring membrane ruffles (12% in unstimulated cells versus 47% in insulin-stimulated cells), an effect that was totally abolished by sorbitol treatment. This indicates that sorbitol pretreatment markedly inhibits the insulin-induced membrane ruffling, suggesting that inhibition of IRS-1-associated PI3-kinase activity by hyperosmotic stress appears sufficient to alter this insulin effect.
Hyperosmotic Stress Does Not Alter the Activation of Insulin Receptor-The marked reduction in insulin-induced IRS-1 phosphorylation after pretreatment with sorbitol could result from an alteration in insulin receptor kinase activity. To test this hypothesis, we determined the tyrosine phosphorylation level of insulin receptor from cells preincubated with or without sorbitol before insulin stimulation (0.2 nM). As shown in Fig. 4, hyperosmotic stress did not impair the ability of insulin to stimulate the autophosphorylation of its receptor. Thus, in sorbitol-treated cells, the decrease in insulin-stimulated IRS-1 function did not result from an alteration in insulin-mediated receptor activation.
Hyperosmotic Stress Promotes the Phosphorylation of IRS-1 on Serine 307-A potential mechanism involved in the decrease in insulin-induced IRS-1 tyrosine phosphorylation is an increase in its serine/threonine phosphorylation (2,26,27). Several recent reports have pointed out the role of the Ser 307 phosphorylation in IRS-1 as an inhibitory mechanism to trigger the decrease in insulin-induced IRS-1 tyrosine phosphorylation (18,19,28). To determine whether Ser 307 is phosphorylated after sorbitol stimulation, 3T3-L1 adipocytes were treated with or without 600 mM sorbitol and IRS-1 was immunopurified and immunoblotted with a phosphospecific antibody against Ser 307 (␣-pS 307 ). As shown in Fig. 5A, when IRS-1 was immunoblotted with an anti-IRS-1 antibody, we observed a reduction in the electrophoretic mobility of IRS-1 in response to sorbitol, which could result from an increase in its serine/threonine phosphorylation. Immunoblotting with ␣-pS 307 antibody indicates that the phosphorylation of this serine residue was very low in unstimulated cells but markedly increased after sorbitol treatment. Taken together, these results indicate that hyperosmotic stress increases the serine phosphorylation of IRS-1 and that one of the phosphorylated sites is the Ser 307 .
Phosphorylation of Ser 307 in Response to Hyperosmotic Stress Is Prevented by Inhibition of mTOR-IRS-1 could be phosphorylated by several serine/threonine kinases (2). We wanted to determine the signaling pathway involved in the phosphorylation of IRS-1 during sorbitol treatment. 3T3-L1 adipocytes were pretreated with a MEK1 inhibitor (40 M PD-98059), a PI 3-kinase inhibitor (100 nM wortmannin), or an mTOR inhibitor (40 nM rapamycin) before sorbitol treatment. IRS-1 was immunoprecipitated and immunoblotted with ␣-pS 307 antibody or anti-IRS-1 antibody. As shown in Fig. 5A, inhibition of extracellular signal-regulated kinase by PD-98059 did not alter sorbitol-induced phosphorylation of Ser 307 . Inhibition of PI 3-kinase activation by wortmannin induced a slight increase in the electrophoretic mobility of IRS-1 but did not significantly impair the Ser 307 phosphorylation. This could indicate that a PI 3-kinase-dependent pathway could phosphorylate Ser/Thr res-idues other than Ser 307 . In contrast, cell treatment with rapamycin before sorbitol stimulation not only prevented the decrease in the electrophoretic mobility of IRS-1 but also abrogated its phosphorylation on Ser 307 (Fig. 5A), indicating that a mTOR signaling pathway was the major pathway involved in sorbitol-induced phosphorylation of IRS-1. IRS-1 has been shown to be a potential substrate of both mTOR and its effectors, the p70 S6-kinases (29). Using a phosphospecific antibody, we did not detect the phosphorylation of threonine 389 in p70 S6-kinases in sorbitol-treated cells (Fig. 5B). Because phosphorylation of this site in p70 S6-kinases is correlated with their activation (30), it was unlikely that these kinases were involved in the phosphorylation of IRS-1 during sorbitol treatment. This suggests that mTOR could be the kinase responsible for Ser 307 phosphorylation of IRS-1 after osmotic stress.

Inactivation of mTOR by Rapamycin Prevents the Inhibitory Effect of Hyperosmotic Stress on Insulin-mediated IRS-1 Tyrosine Phosphorylation and PI 3-Kinase Activation-Because
mTOR seems to be involved in the serine phosphorylation of IRS-1 in response to sorbitol, we determined whether inhibition of mTOR could prevent the inhibitory effect of sorbitol on insulin signaling. 3T3-L1 adipocytes were incubated with or without rapamycin before sorbitol and insulin stimulation. Both the IRS-1 tyrosine phosphorylation level (Fig. 6A) and PI 3-kinase activity associated to IRS-1 (Fig. 6B) were measured as described under "Experimental Procedures." As previously shown, sorbitol treatment reduced the insulin-induced IRS-1 tyrosine phosphorylation and its associated PI 3-kinase activ- ity. More importantly, inactivation of mTOR by rapamycin prevented these inhibitory effects of sorbitol (Fig. 6). As shown in Fig. 7, mTOR inhibition by rapamycin did not reverse the inhibitory effect of sorbitol on insulin-induced membrane ruffling. Taken together, these results show that mTOR plays a crucial role in the serine phosphorylation of IRS-1 and, thus, in the negative regulation of IRS-1 function. However, other mechanisms are also involved in the inhibition of insulin-induced membrane ruffling by hyperosmotic stress.
Long-term Hyperosmotic Stress Enhances the Degradation of Both IRS-1 and IRS-2-It has been reported recently that long-term insulin stimulation could trigger IRS-1 degradation by proteasome in 3T3-L1 adipocytes. Moreover, it has been suggested that a rapamycin-sensitive IRS-1 phosphorylation on serine allows for this degradation (31)(32)(33)(34). Because hyperosmolarity also promotes the serine phosphorylation of IRS-1 by a rapamycin-dependent pathway, we investigated the effect of long-term hyperosmolarity on IRS protein expression.
3T3-L1 adipocytes were treated with either 600 mM sorbitol (30 min or 4 h) or insulin for 4 h. The amounts of IRS-1, IRS-2, and p85 were analyzed by immunoblotting proteins of cell lysates. After 4 h, both hyperosmotic stress and insulin dramatically reduced the level of IRS-1 without altering p85 amount. In contrast, only sorbitol treatment reduced the amount of IRS-2 (Fig. 8A).
Long-term insulin treatment induces the degradation of IRS-1 through both a rapamycin-sensitive pathway and a proteasome-dependent process (32)(33)(34). Proteasome-induced protein degradation appears as the major determinant in the with 600 mM sorbitol for 20 min at 37°C. Cell lysates were then immunoprecipitated (IP) using anti-IRS-1 (␣-IRS1) antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE and blotted (IB) using anti-pSer 307 IRS-1 (␣-pS 307 ) antibodies. The membrane was then stripped and probed with anti-IRS-1 antibodies. Top, representative autoradiographs are shown. Bottom, phosphorylation of Ser 307 was quantified by densitometry scanning analysis and normalized for the total IRS-1 amounts. Data are expressed as percentage of insulin effect and presented as the means Ϯ S.E. of three independent experiments. *, p Ͻ 0.05 compared with sorbitol alone. B, after serum starvation, 3T3-L1 adipocytes were incubated with either sorbitol (600 mM) or insulin (100 nM) for 30 min. Total cell lysates (50 g of protein) were blotted using indicated antibodies. ␣p-S6K, anti-phospho-S6 kinase; p70 S6K p-T389 , phospho-p70 S6 kinase (Thr 389 ).

FIG. 6. Inactivation of mTOR prevents the inhibitory effect of hyperosmotic stress on insulin-mediated IRS-1 tyrosine phosphorylation and subsequent PI 3-kinase activity.
After serum starvation, 3T3-L1 adipocytes were incubated with vehicle or rapamycin (40 nM) for 30 min. The cells were left untreated (empty bars) or were stimulated (gray bars) with 600 mM sorbitol for 40 min before a 5-min insulin stimulation (0.2 nM). Cell lysates were immunoprecipitated using anti-IRS-1 (␣-IRS1) antibodies. A, immunoprecipitated (IP) proteins were resolved by SDS-PAGE and blotted using indicated antibodies. Top, representative autoradiographs are shown. IB, immunoblots; ␣-pY, anti-phosphotyrosine. Bottom, Tyr-phosphorylation of IRS1 was quantified by densitometry scanning analysis and normalized for the total IRS-1 amounts. Data are expressed as percentage of insulin effect and presented as the means Ϯ S.E. of four independent experiments. *, p Ͻ 0.01 compared with insulin B, IRS-1-associated PI 3-kinase activity was measured, as described under "Experimental Procedures." Means ϩ S.E. of three independent experiments are shown. *, p Ͻ 0.05 compared with insulin growth arrest caused by hyperosmotic stress (35). We examined the effect of mTOR and proteasome inhibitors on IRSs expression levels. 3T3-L1 adipocytes were preincubated with a proteasome inhibitor (20 M lactacystin) or mTOR inhibitor (40 nM rapamycin) before stimulation with either sorbitol (600 mM) or insulin (100 nM) for 4 h. As shown in Fig. 8B, cell treatment with rapamycin or lactacystin largely prevented the insulininduced IRS-1 degradation as described previously (31)(32)(33)(34). In contrast, both inhibitors were without effect on hyperosmotic stress-induced IRS-1 or IRS-2 degradation. This indicates that although both long-term insulin treatment and hyperosmotic stress induce the degradation of IRS-1, both agents mediate this response through different processes. Long-term insulin treatment promotes the serine/threonine phosphorylation of IRS-1, which is correlated with IRS-1 degradation via a proteasome-dependent process (31)(32)(33)(34). In contrast, after a longlasting hyperosmotic stress, the degradation of both IRS-1 and IRS-2 occurs through an mTOR-and proteasome-independent process.

DISCUSSION
The preincubation of 3T3-L1 adipocytes with sorbitol decreases the ability of insulin to stimulate glucose uptake, lipogenesis, and glycogen synthesis (13). The molecular mechanisms by which hyperosmolarity induces cellular insulin resistance have not yet been fully elucidated. These alterations could result, in part, from the stimulation of phosphatases that maintain PKB in an inactive state (13).
In the present study, we show that hyperosmotic stress antagonizes the effect of physiological insulin concentrations on IRS-1 tyrosine phosphorylation, the subsequent PI 3-kinase activation and membrane ruffling. This is associated with an increase in the phosphorylation of IRS-1 on Ser 307 in sorbitoltreated cells. The Ser 307 residue is located close to the PTB domain. It has been proposed that the phosphorylation of the Ser 307 prevents the association between IRS-1 and the insulin receptor, leading to a decrease in IRS-1 tyrosine phosphorylation (19). Several stimuli, such as fatty acids (36), tumor necrosis factor-␣ (18, 28), or long-term administration of insulin or IGF-1 (18), induce the phosphorylation of Ser 307 leading to the reduction in both IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. The importance of the Ser 307 in the negative regulation of IRS-1 seems to be a general mechanism. However, although hyperosmotic stress promotes the phosphorylation of Ser 307 , it inhibits the tyrosine phosphorylation of IRS-1 and the subsequent PI 3-kinase activation only when physiological insulin concentrations were used. Osmotic stress did not inhibit insulin-induced IRS-1 tyrosine phosphorylation when a supraphysiological concentration of insulin (100 versus 0.2 nM) was used ( Fig. 1 and Ref. 13). Because high insulin concentrations activate IGF-1 receptors, the lack of sorbitol inhibitory effect could indicate that phosphorylation of Ser 307 in IRS-1 induced by hyperosmotic stress does not prevent its tyrosine phosphorylation by IGF-1 receptors. However, this explanation seems unlikely because 3T3-L1 adipocytes contain only low amounts of IGF-1 receptors compared with insulin receptors (10,000 versus 200,000) (37). Moreover, at 13 nM of IGF-1, the tyrosine phosphorylation of IRS-1 is similar to the phosphorylation observed at the 0.2 nM insulin concentration (data not shown). When MCF-7 cells were treated with an activator of the c-Jun NH 2 -terminal kinase pathway, which strongly phosphorylates IRS-1 on Ser 307 , IGF-1-induced PI 3-kinase-dependent PKB activation and cell survival responses were largely reduced (38). Furthermore, treatment of 3T3-L1 adipocytes with 13 nM IGF-1 also led to the phosphorylation of IRS-1 on Ser 307 (18). This indicates that phosphorylation of IRS-1 on Ser 307 inhibits both insulin and IGF-1 signaling. Thus, activation of IGF-1 receptors by a high insulin concentration cannot explain this observation ( Fig. 1 and Ref. 13).
A more conceivable explanation could be that the inhibitory effect of the phosphorylation of the Ser 307 in IRS-1 could depend on the number of activated insulin receptors. Both PH and PTB domains of IRS-1 participate in the efficient tyrosine phosphorylation of IRS-1 by the insulin receptor. In cells expressing a high level of receptors, either the PH or the PTB domain is sufficient to promote IRS-1 tyrosine phosphorylation. In cells expressing a low level of receptors, both domains are required for efficient tyrosine phosphorylation of IRS-1 (39,40). It is thus possible that at high insulin concentration, the PH domain of IRS-1 could be sufficient to compensate for the inhibitory effect of the phosphorylation of the Ser 307 on the PTB domain function (19). In contrast, at low insulin concentration, when fewer insulin receptors are activated, the interaction of IRS-1 with insulin receptor would require both PH and PTB domains. In this case, hyperosmotic stress-induced phosphorylation of Ser 307 in IRS-1 by inhibiting the function of the PTB domain could reduce the coupling between IRS-1 and the insulin receptor leading to a partial inhibition of the tyrosine phosphorylation of IRS-1. A delicate balance between positive IRS-1 tyrosine phosphorylation versus negative IRS-1 serine phosphorylation could regulate IRS-1 function depending on the number of activated receptors.
Ser 307 can be phosphorylated by different kinases, such as c-Jun NH 2 -terminal kinase (28), MEK-dependent kinase (18), inhibitor B kinase (41), or PI 3-kinase-dependent pathway (18). Inactivation of extracellular signal-regulated kinases (by MEK1 inhibitor), PI 3-kinase (Fig. 5A), or c-Jun NH 2 -terminal kinase (data not shown) by specific inhibitors did not prevent hyperosmotic stress-mediated Ser 307 phosphorylation. In contrast, inhibition of mTOR by rapamycin completely abrogated the phosphorylation of Ser 307 in response to sorbitol. Furthermore, mTOR-induced serine phosphorylation of IRS-1 seems to play a major role in the inhibitory effect of osmotic stress on insulin signaling. Indeed, rapamycin prevents the shift in the apparent molecular weight of IRS-1 in sorbitol-treated cells, and this was concomitant with the suppression of the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation. Because phosphorylation of Ser 307 is sensitive to rapamycin, this indicates that mTOR and/or downstream kinases such as p70 S6kinases could be responsible for IRS-1 phosphorylation. It is unlikely that p70 S6-kinases are involved in Ser 307 phosphorylation. Indeed, as shown in Fig. 5B, sorbitol fails to activate the p70 S6-kinases and instead may lead to the stimulation of phosphatases, which maintain these kinases in an inactive state (42). Insulin treatment is also able to induce the phosphorylation of IRS-1 on Ser 307 by an mTOR-dependent pathway (43,44) but independent of p70 S6-kinase (43). We can hypothesize that Ser 307 is directly phosphorylated by mTOR. In favor of such an hypothesis, it has been shown that mTOR and IRS-1 were constitutively associated (45). Moreover, mTOR catalyzes the phosphorylation of a set of Ser/Thr-Pro sites that have a proline in the ϩ1 position, as is the case for Ser 307 in IRS-1 (46). Thus, we demonstrate that the mTOR-signaling pathway is involved in the phosphorylation of Ser 307 . Moreover, our results strengthen the observation that distinct kinases mediated by different stimuli might converge at Ser 307 to inhibit insulin response. Although we have identified Ser 307 in IRS-1 as a site phosphorylated in response to osmotic stress, we cannot exclude the possibility that other serine or threonine residues are also phosphorylated in sorbitol-treated cells. In agreement with this, it has been reported that mTOR is able to phosphorylate IRS-1 511-772 in vitro, leading to the inhibition of JAK-1-dependent IRS-1 tyrosine phosphorylation induced by interferon-␣ (29). Furthermore, tumor necrosis factor-␣ is also able to induce the phosphorylation of IRS-1 on Ser 636/639 by a mTOR-dependent pathway (45).
Whereas inhibition of mTOR completely reversed the inhibitory effect of hyperosmotic stress on insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase activation, cell treatment with rapamycin did not prevent the inhibition of insulininduced membrane ruffling by sorbitol. This could indicate that other mechanisms, in addition to inhibition of IRS-1 function, are involved in the inhibitory mechanism of insulin-induced response by hyperosmolarity. Insulin-induced membrane ruffling may involve the activation of the PI 3-kinase/Rac/Protein kinase N pathway (22)(23)(24)47). Activation of protein kinase N in response to insulin is also dependent of the PI 3-kinase/PDK1 pathway (47). In contrast, sorbitol promotes Rac activation and membrane ruffling through a PI 3-kinase-independent manner (9,48). Osmotic stress negatively regulates PKB and p70-S6kinase by activating phosphatases (13,42). An attractive explanation could be that osmotic stress also inhibits the Protein kinase N activity by the same mechanism.
Although serine phosphorylation is usually considered a short-term inhibitory mechanism, regulated degradation of IRS proteins may also promote long-term insulin-resistance. As described previously, prolonged insulin treatment reduces the level of IRS-1 through a proteasome-dependent process. Moreover, it has been proposed that the mTOR-dependent IRS-1 phosphorylation on serine could allow its degradation by the proteasome (31-34, 44). As described previously (33,34,49), we did not detect a significant reduction of IRS-2 amount after long-term exposure of 3T3-L1 adipocytes to insulin, although Rui et al. (50) reported its degradation. We also show that long-term hyperosmotic stress stimulates the degradation not only of IRS-1 but also of IRS-2, as described in Fao cells (50). This effect was completely insensitive to mTOR and proteasome inhibitors, suggesting that hyperosmotic stress induces IRS-1 degradation through a lysosomal process in 3T3-L1 adipocytes. On the other hand, in Fao cells, IRS-2 degradation induced by osmotic stress seems to require proteasome activity (50). Thus, depending on cell types, mechanisms induced by osmotic stress to promote IRS-2 degradation could be different. Although both insulin and osmotic stress induced the serine phosphorylation of IRS-1 through an mTOR-dependent pathway, other events activated specifically by insulin treatment could be required to trigger the IRS-1 degradation by the proteasome. Because the N-terminal region of IRS-1 contains a structural element that is crucial for the specificity of ubiquitination and proteasome degradation in response to insulin (51), another region of this protein could be required for its osmotic stress-induced degradation.
In summary, short-term osmotic stress can inhibit insulin action by distinct mechanisms including the activation of a phosphatase, which dephosphorylates PKB (13) and p70 S6 kinases (13,42). We provide first evidence for another mechanism demonstrating that the hyperosmotic stress inhibits IRS-1 function by increasing its serine phosphorylation. mTOR could play a role in both mechanisms. Indeed, mTOR is partly associated with mitochondria and senses osmotic stress via mitochondrial dysfunction resulting in an activation of phosphatases (52). On the other hand, mTOR kinase activity could phosphorylate IRS-1 on Ser 307 , inducing its decoupling with insulin receptor. Both pathways could allow the cells to integrate a variety of stress signals and to adapt their metabolism to the environmental modifications. A long-term hyperosmotic stress reduces the IRS-1 expression level by stimulating a degradation process.