Hyperosmotic Stress Inhibits Insulin Receptor Substrate-1 Function by Distinct Mechanisms in 3T3-L1 Adipocytes*
Philippe Gual
,
Teresa Gonzalez,
Thierry Grémeaux,
Romain Barrés,
Yannick Le Marchand-Brustel and
Jean-François Tanti
From the
INSERM U 568 and l'Institut Fédératif de Recherches 50,
Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 02,
France
Received for publication, December 3, 2002
, and in revised form, April 30, 2003.
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ABSTRACT
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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.
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INTRODUCTION
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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
(46).
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
(710).
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 acid-sensitive protein phosphatases
acting at the level of PKB in response to hyperosmotic stress
(13). Thus,
hyperosmolarity-induced 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 insulin-stimulated glucose transport is
still controversial
(1417),
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 Ser307
(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
Ser307 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 stress-induced insulin resistance. We
show that short-term osmotic stress inhibits IRS-1 function by an
mTOR-dependent phosphorylation of IRS-1 on Ser307, whereas
prolonged osmotic stress promotes the degradation of IRS proteins by an mTOR-
and proteasome-independent mechanism.
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EXPERIMENTAL PROCEDURES
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MaterialsDulbecco's modified Eagle's medium (DMEM), fetal
calf serum, and calf serum were obtained from Invitrogen. PD-98059, rapamycin,
lactacystin, and okadaic acid were from Calbiochem. Wortmaninn was purchased
from Sigma. [
-32P]ATP was purchased from Amersham
Biosciences. Polyvinylidene difluoride membranes were purchased from
Millipore. BCA reagent was obtained from Pierce. Protease inhibitors mixture
was obtained from Roche Diagnostics. All other chemical reagents were
purchased from Sigma. Polyclonal anti-phospho-IRS-1 (Ser307)
antibody (
-pS307) was raised in rabbit against a synthetic
peptide (ESITATpS307PASMVGGK) flanking Ser307 that is
conserved among mouse, rat, and human (Eurogentec, Seraing, Belgium)
(18). Antibodies against
phosphotyrosine (clone p-Tyr-100) and phospho-p70 S6-kinase
(Thr389) were purchased from Cell Signaling. Antibodies against
IRS-1, IRS-2, and the p85 subunit of PI 3-kinase were purchased from Upstate
Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated secondary
antibodies were obtained from Jackson Immunoresearch Laboratories, Inc. (West
Grove, PA). Enhanced chemiluminescence reagent was purchased from PerkinElmer
Life Sciences.
Cell Culture3T3-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 815 days after the beginning of the
differentiation protocol.
Immunoprecipitation Assays3T3-L1 adipocytes were
serum-starved 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 x g at 4 °C) were
incubated for 3 h at 4 °C with appropriate antibodies pre-adsorbed on
protein-G-Sepharose (4 µg 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 µg/ml for the
anti-pS307-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 Assay3T3-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 µM
Na3VO4; 2) 100 mM Tris, pH 7.4, 0.5
M LiCl, and 200 µM Na3VO4; and
3) 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM
EDTA, and 200 µM Na3VO4. PI 3-kinase
activity was measured on the immune pellets as described previously
(21).
Immunofluorescence and Image Analysis3T3-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 serum-free 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 red-phalloidin (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 40x using a 1.00.50 oil-immersion
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 AnalysisStatistical analysis was performed by
Student's t test. Statistical significance was assessed at p
< 0.05
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RESULTS
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Hyperosmotic Stress Impairs IRS-1 Tyrosine Phosphorylation, Subsequent
PI 3-Kinase Activation, and Membrane Ruffling Induced by
InsulinInsulin 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 insulin-mediated 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 co-immunoprecipitated
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
(2224)
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.

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FIG. 3. Hyperosmotic stress strongly inhibits insulin-induced membrane
ruffling. After serum starvation, 3T3-L1 adipocytes were either left
untreated (empty bars) or stimulated (gray bars) with 600
mM sorbitol for 40 min before 20 min of insulin stimulation (0.5
nM). The cells were then fixed and subjected to confocal
fluorescent microscopy using Texas red-phalloidin. A, representative
fields of cells are shown. B, results are expressed as the percentage
of positive cells. Bars represent the means ± S.E. of three
independent experiments in which at least 200 cells were scored in each
condition (see "Experimental Procedures"). *, p < 0.05
compared with basal; , p < 0.01 compared with insulin
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Hyperosmotic Stress Does Not Alter the Activation of Insulin
ReceptorThe 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
307A 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 Ser307 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
Ser307 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
Ser307 (
-pS307). 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
-pS307 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 Ser307.

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FIG. 5. Hyperosmotic stress triggers the phosphorylation of IRS-1 on serine 307
via a rapamycin-sensitive pathway. A, 3T3-L1 adipocytes were
incubated with vehicle, PD-98059 (10 µM, PD), rapamycin
(40 nM, Rapa), or wortmannin (100 nM, W) for 30 min. The
cells were either left untreated (empty bars) or stimulated (gray
bars) 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-pSer307IRS-1
( -pS307) antibodies. The membrane was then stripped
and probed with anti-IRS-1 antibodies. Top, representative
autoradiographs are shown. Bottom, phosphorylation of
Ser307 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 ± S.E. of three independent
experiments. *, p < 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 µg of protein) were blotted using indicated antibodies.
p-S6K, anti-phospho-S6 kinase; p70
S6Kp-T389, phospho-p70 S6 kinase (Thr389).
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Phosphorylation of Ser307 in Response to Hyperosmotic Stress
Is Prevented by Inhibition of mTORIRS-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
µM 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
-pS307 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 Ser307. 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
Ser307 phosphorylation. This could indicate that a PI
3-kinase-dependent pathway could phosphorylate Ser/Thr residues other than
Ser307. 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 Ser307
(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
Ser307 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 ActivationBecause 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
activity. 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.

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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 ±
S.E. of four independent experiments. *, p < 0.01 compared with
insulin B, IRS-1-associated PI 3-kinase activity was measured, as
described under "Experimental Procedures." Means + S.E. of three
independent experiments are shown. *, p < 0.05 compared with
insulin
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FIG. 7. Inactivation of mTOR does not prevent the inhibitory effect of
hyperosmotic stress on insulin-induced membrane ruffling. 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 20 min of insulin stimulation (0.5 nM). The cells were
then fixed and subjected to confocal fluorescent microscopy using Texas
red-phalloidin and analyzed as described in the legend to
Fig. 3. Results are expressed
as percentage of insulin effect. Bars represent the means ±
S.E. of three independent experiments in which at least 200 cells were scored
in each condition (see "Experimental Procedures"). *, p
< 0.01 compared with insulin alone and is not significantly modified by
rapamycin.
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Long-term Hyperosmotic Stress Enhances the Degradation of Both IRS-1
and IRS-2It 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
(3134).
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).

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FIG. 8. Degradation of both IRS-1 and IRS-2 is enhanced by chronic hyperosmotic
stress by a mechanism which is mTOR and proteasome independent.
A, 3T3-L1 adipocytes were incubated with either sorbitol (600
mM) for 0.5 or 4 h, as indicated, or with insulin (Ins)
for 4 h. Total cell lysates (50 µg of protein) were blotted using indicated
antibodies. IB, immunoblots. B, 3T3-L1 adipocytes were
incubated with vehicle, lactacystin (Lact) (10 µM), or
rapamycin (Rapa) (40 nM) for 30 min. The cells were left
untreated or were stimulated with either 600 mM sorbitol or 100
nM insulin for 4 h. Total cell lysates (50 µg of protein) were
blotted using indicated antibodies. Typical autoradiographs representative of
three to four experiments are shown.
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Long-term insulin treatment induces the degradation of IRS-1 through both a
rapamycin-sensitive pathway and a proteasome-dependent process
(3234).
Proteasome-induced protein degradation appears as the major determinant in the
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 µM
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 insulin-induced IRS-1 degradation as described previously
(3134).
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
(3134).
In contrast, after a long-lasting hyperosmotic stress, the degradation of both
IRS-1 and IRS-2 occurs through an mTOR- and proteasome-independent
process.
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DISCUSSION
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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
Ser307 in sorbitol-treated cells. The Ser307 residue is
located close to the PTB domain. It has been proposed that the phosphorylation
of the Ser307 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 Ser307 leading to the reduction in both IRS-1
tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity. The
importance of the Ser307 in the negative regulation of IRS-1 seems
to be a general mechanism. However, although hyperosmotic stress promotes the
phosphorylation of Ser307, 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 Ser307 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
NH2-terminal kinase pathway, which strongly phosphorylates IRS-1 on
Ser307, 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 Ser307
(18). This indicates that
phosphorylation of IRS-1 on Ser307 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 Ser307 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
Ser307 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
Ser307 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.
Ser307 can be phosphorylated by different kinases, such as c-Jun
NH2-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
NH2-terminal kinase (data not shown) by specific inhibitors did not
prevent hyperosmotic stress-mediated Ser307 phosphorylation. In
contrast, inhibition of mTOR by rapamycin completely abrogated the
phosphorylation of Ser307 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
Ser307 is sensitive to rapamycin, this indicates that mTOR and/or
downstream kinases such as p70 S6-kinases could be responsible for IRS-1
phosphorylation. It is unlikely that p70 S6-kinases are involved in
Ser307 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 Ser307 by an
mTOR-dependent pathway (43,
44) but independent of p70
S6-kinase (43). We can
hypothesize that Ser307 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
+1 position, as is the case for Ser307 in IRS-1
(46). Thus, we demonstrate
that the mTOR-signaling pathway is involved in the phosphorylation of
Ser307. Moreover, our results strengthen the observation that
distinct kinases mediated by different stimuli might converge at
Ser307 to inhibit insulin response. Although we have identified
Ser307 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-1511772 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
Ser636/639 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 insulin-induced 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
(2224,
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-S6-kinase 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
(3134,
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 Ser307, 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.
 |
FOOTNOTES
|
---|
* This work was supported by grants from INSERM (France), the University of
Nice, the Fondation Bettencourt-Schueller, the Fondation pour la Recherche
Médicale, the Région Provence-Alpes Côte d'Azur, the
Conseil Général des Alpes Maritimes, and the Association pour la
Recherche Contre le Cancer Grant 7449. This work was also supported by a grant
from ALFEDIAM-Takeda Laboratories (Puteaux, France) (to J. F. T.). The costs
of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Supported by fellowships from La Ligue Contre le Cancer and ALFEDIAM
(France). 
To whom correspondence should be addressed. Tel.: 33-4-93-37-77-99; Fax:
33-4-93-37-77-01; E-mail:
tanti{at}unice.fr.
1 The abbreviations used are: IRS-1, insulin receptor substrate-1; PI
3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PTB,
phosphotyrosine binding; mTOR, mammalian target of rapamycin; DMEM, Dulbecco's
modified Eagle's medium;
-pS307, polyclonal
anti-phosphoserine 307-IRS-1 antibody; PBS, phosphate-buffered saline; PH,
Pleckstrin homology; MEK, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Mireille Cormont for helpful discussion and assistance with
immunofluorescence experiments and confocal microscopy and F. Bost, B.
Binetruy, and J. Vukmirica for critical reading of the manuscript.
 |
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