 |
INTRODUCTION |
The information regarding the biological actions of reactive
oxygen and nitrogen species has increased considerably in recent years,
revealing diverse functions (1, 2). Exposure of tissues to free
radicals in a variety of experimental systems leads to apoptosis and to
cell damage (2). Paradoxically, reactive oxygen species have been
demonstrated to participate in normal cellular responses including in
signal transduction pathways and in gene regulation (1, 3, 4).
H2O2, for example, has been shown to be
produced intracellularly following stimulation with platelet-derived growth factor and to mediate the normal response to this growth factor
(5). Similarly, insulin was reported to activate an adipocyte membrane
bound NADPH oxidase (6, 7), further supporting a potential role for
reactive oxygen species as second messengers. In agreement with this
concept, direct exposure of cells to H2O2 has
been demonstrated to result in insulinomimetic effects, as demonstrated
both by mimicking the metabolic response to insulin as well as by
activating components of its signal transduction machinery (8-11).
However, prolonged exposure of 3T3-L1 adipocytes to micromolar
concentrations of H2O2 resulted in impaired
insulin-stimulated lipogenesis, activation of glycogen synthase,
glucose transport, and GLUT4 translocation to the plasma membrane
(PM)1 (12, 13).
Insulin-stimulated GLUT4 translocation has been suggested to depend
upon the activation of phosphatidylinositol 3-kinase (PI 3-kinase),
which in fat cells occurs in various cellular fractions (14, 15).
Recently, the concept that the activation of PI 3-kinase in the low
density microsomal fraction (LDM) or in GLUT4 containing vesicles is
necessary for the specific ability of insulin to promote GLUT4
translocation has been suggested (16-19). In addition, overexpression
of a constitutively active p110 (the catalytic subunit of PI 3-kinase),
which resulted in increased total and LDM PI 3-kinase activity in
primary adipocytes or in 3T3-L1 adipocytes, dramatically stimulated
glucose uptake and GLUT4 translocation (18, 20). In a recent study we
observed that impaired insulin-stimulated GLUT4 translocation following
oxidation could not be attributed to defects in activation of PI
3-kinase as detected in total cell lysate (13). Thus, impaired
compartment-specific activation of PI 3-kinase by insulin may represent
a putative cellular mechanism for oxidation induced insulin resistance.
The events leading to the specific activation of PI 3-kinase in the LDM
are not clear. Recently, insulin-induced redistribution of insulin
receptor substrates (IRS) was suggested to play a role in the
compartment-specific activation of PI 3-kinase in 3T3-L1 adipocytes
(21). Insulin stimulation was found to induce a reduction in the amount
of IRS1/2 in the LDM, while IRS tyrosine phosphorylation was elevated.
The tyrosine-phosphorylated IRS in the LDM was suggested to serve as a
docking molecule for PI 3-kinase, leading to a rapid translocation of
the p85 subunit from the cytosol to the LDM, resulting in increased
PI 3-kinase activity in this fraction (21, 22).
Although the insulin signaling steps toward GLUT4 translocation distal
to PI 3-kinase activation are currently not fully understood, a number
of downstream targets for PI 3-kinase have been identified (23, 24).
Among them, a serine/threonine kinase of 60 kDa (24, 25) termed protein
kinase B (PKB) also known as RAC protein kinase or Akt (26-28). PI
3-kinase is both necessary and sufficient for
insulin-dependent phosphorylation and activation of PKB,
although the exact mode of activation has not been fully elucidated
(reviewed in Ref. 29). It was suggested that binding of
phosphatidylinositol 3,4,5-trisphosphate or phosphatidylinositol
3,4-bisphosphate to the pleckstrin homology domain of PKB and its
translocation to the membranes are necessary for PKB phosphorylation on
Thr308 and Ser473 by its upstream kinases PDK1
and PDK2, respectively. In the search for its relevant biological
activity, PKB was found to mediate some of insulin's effects, such as
the inhibition of glycogen synthase kinase-3 (30, 31), the stimulation
of glucose and amino acids uptake (23), and protein synthesis (32).
Several lines of evidence strongly support a crucial role for PKB in
mediating GLUT4 translocation. In agreement with this notion, insulin
activation of PKB precedes the hormonal effect on glucose transport
(33). Moreover, overexpression of a constitutively active form of PKB resulted in enhanced glucose transport and GLUT4 translocation (32-36), while inhibition of PKB activity by transfecting rat
adipocytes with a dominant PKB-inactive mutant, significantly inhibited
insulin-stimulated GLUT4 translocation (34). The respective role of the
different isoforms of PKB in mediating insulin-stimulated GLUT4
translocation in various cell types is as yet unclear.
Although the involvement of IRS, PI 3-kinase, and PKB cellular
redistribution and activation in the normal response to insulin is
increasingly recognized, their relevance for the understanding of the
cellular mechanisms leading to insulin resistance is largely unclear.
In this study we report that impaired insulin-stimulated GLUT4
translocation induced by oxidative stress is associated with disruption
of insulin-induced IRS-1 and PI 3-kinase intracellular trafficking and
with inhibition of PKB
and PKB
activation. This represents a
novel cellular mechanism for the understanding of insulin resistance in
response to a change in the extracellular environment.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture medium, serum, and antibiotic
solutions were obtained from Biological Industries (Beit-Haeemek,
Israel). Recombinant human insulin was from Novo Nordisk (Bagsvaerd,
Denmark). Anti-GLUT4 antibodies were from Chemicon International Inc.
(Temecula, CA). Crosstide, anti-sheep IgG, anti-IRS-1, anti-p85,
anti-phosphotyrosine (4G10), anti-PKB
(pleckstrin homology domain)
antibodies were from Upstate Biotechnology (Lake Placid, NY).
Anti-phosphospecific PKB (Ser473) antibodies were from New
England Biolabs Inc. (Beverly, MA). Anti-PKB
and -PKB
were a kind
gift from Dr. D. Alessi (University of Dundee, Dundee, United Kingdom)
and were used as described previously (37). Anti-PKB (C-terminal)
antibodies were kindly provided by Dr. R. Seger (Weizmann Institute,
Rehovot, Israel). Peroxidase-conjugated anti-rabbit IgG, anti-mouse
IgG, and [
-32P]ATP were from Amersham Life Sciences
(Buckingham, United Kingdom). Protein G-Sepharose and protein
A-Sepharose were from Pharmacia Biotech (Uppsala, Sweden).
2-Deoxy-[3H]glucose was purchased from Nuclear Research
Center-Negev (Dimona, Israel). All other chemicals were obtained from Sigma.
Cell Culture--
3T3-L1 pre-adipocytes (American Type Culture
Collection) were grown to confluence in Dulbecco's modified Eagle's
medium containing 25 mM glucose, as described previously
(12). 48 h following confluence cells were induced to
differentiate to adipocytes by changing the medium to Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 5 µg/ml recombinant human insulin, 0.5 mM
3-isobutylmethylxanthine and 0.25 µM dexamethasone for 48-72 h. Cells were used 9-10 days following differentiation
induction when exhibiting >90% adipocyte phenotype.
H2O2 was generated by adding 100 milliunits/ml
glucose oxidase (type II from Aspergillus niger, 20,000 units/g solid in non-oxygen-saturated conditions, Sigma) to serum-free
Dulbecco's modified Eagle's medium supplemented with 0.5%
radioimmunoassay grade bovine serum albumin. The addition of 100 milliunits/ml glucose oxidase resulted in medium
H2O2 concentration that achieved a steady state
of 27.4 ± 0.3 µM after 15 min. Following a 2-h
incubation, medium glucose concentrations determined with hexokinase
and glucose-6-phosphate dehydrogenase (38) were 18.2 ± 2.1 and
17.4 ± 1.3 mM for control and glucose oxidase-treated cells, respectively.
Cellular Membrane Preparations--
Following treatment with or
without glucose oxidase, cells were rinsed three times with
phosphate-buffered saline (PBS) and incubated for 7 or 20 min with or
without 100 nM insulin in freshly prepared medium
supplemented with 0.5% bovine serum albumin (radioimmunoassay grade).
Membrane preparations (4 × 10-cm plates per condition) were
performed following the procedure described by Clancy and Czech (39).
The activity of the PM marker 5'-nucleotidase (EC 3.1.3.5) was
determined as described previously (39). The specific activity
(nmol/min/mg protein) of PM 5'-nucleotidase for control and glucose
oxidase-treated cells was 28.1 ± 3.3 and 27.9 ± 1.9, respectively, which reflected purification of more then 10-fold
versus homogenate. Protein recovery of the different membrane fractions was not altered by glucose oxidase treatment. The
various cellular compartments were resuspended in the original fractionation buffer (255 mM sucrose, 20 mM
HEPES, pH 7.4, 1 mM EDTA, 0.2 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µM aprotonin, 1 µM leupeptin, 1 µM pepstatin A), with no addition of detergent, as
described (13, 39).
Plasma Membrane Lawn Preparation--
Plasma membrane sheets
were prepared as described by Chen (40). Differentiated 3T3-L1
adipocytes were washed with PBS and incubated for 1 min with 0.5 mg/ml
poly-D-lysine followed by three washes with hypotonic
buffer (23 mM KCl, 10 mM HEPES, pH 7.5, 1.7 mM MgCl2, 1 mM EGTA). The cells
were then covered with sonication buffer (3 × hypotonic buffer
containing 1 mM dithiothreitol and 0.1 mM
phenylmethylsulfonyl fluoride) and sonicated with a probe membrane
disrupter. Following sonication, the plasma membrane sheets were washed
three times with sonication buffer and were used for immunoblotting as
described below.
Cell Lysates and Western Blots--
Cells (1 × 10-cm plate
per condition) were rinsed three times with PBS and incubated in the
absence or presence of insulin for 7 min. Cells were then scraped into
0.6 ml of ice-cold lysis buffer (50 mM Tris-HCl, 1%
Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium vanadate, 1 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µM
aprotonin, 1 µM leupeptin, 1 µM pepstatin
A). Lysates were gently shaken for 15 min at 4 °C, centrifuged
(12,000 × g, 15 min at 4 °C), supernatant
collected, and protein content determined (BCA method, Pierce). Laemmli
buffer was added and samples boiled for 10 min. Protein samples were
resolved on 7.5-10% SDS-polyacrylamide gel electrophoresis and
subjected to Western blot, followed by quantitation using video
densitometry analysis, as described (12).
Immunoprecipitation and PI 3-Kinase Assay--
Cells were lysed
in ice-cold deoxycholate based buffer, as described above. For
immunoprecipitation, either 0.5 mg protein of cell lysate and cytosols
or 75 µg of protein of LDM were used and assayed as described (13).
PI 3-kinase assay was performed following the protocol described by
Hadari et al. (10), using phosphatidylinositol and
[
-32P]ATP as substrates. The phosphorylated
phosphatidylinositols were separated using thin layer chromatography
and quantified by video densitometry.
PKB Kinase Assay--
Cell lysates were prepared as described
above using PKB lysis buffer (50 mM Tris-HCl pH 7.5, 0.1%
(w/v) Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM sodium
-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium vanadate,
0.1% (v/v) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl
fluoride, 1 µM aprotonin, 1 µM leupeptin, 1 µM pepstatin A).
Protein content was determined using the Bio-Rad Bradford procedure
(41). Lysates were subjected to immunoprecipitation with the different
PKB antibodies and were assayed for kinase activity using Crosstide
(GRPRTSSFAEG) as a substrate according to the manufacturer's
instructions as described previously (31).
Hexose Transport Determinations--
2-Deoxyglucose uptake
measurements were performed as described previously (12). Assays were
performed for 10 min using 50 µM 2-deoxy-[3H]glucose (1 µCi/ml). Nonspecific uptake (less than 10% of the total) was
determined in the presence of cytochalasin B (50 µM) and was
subtracted from the total uptake.
Statistical Analysis--
Data are expressed as mean ± S.E. Each treatment was compared with control, and statistical
significance between two groups was evaluated using the Student's
t test. The criterion for significance was set at
p < 0.01.
 |
RESULTS |
Oxidative Stress Impairs the Compartment-specific Activation
of PI 3-Kinase by Insulin--
In order to study the effect of
oxidative stress on the ability of insulin to activate PI 3-kinase in
various cellular fractions, fully differentiated 3T3-L1 adipocytes were
exposed for 2 h to 27.4 ± 0.8 µM
H2O2 continuously generated by addition of 100 milliunits/ml glucose oxidase to the culture medium. In agreement with
recent reports (16, 21), insulin induced a 2.8-fold increase in p85 content in the LDM (Fig. 1A),
which was associated with a 50% reduction in its abundance in the
cytosolic fraction (Fig. 1C). PI 3-kinase activity measured
in p85 immunoprecipitates revealed an approximately 7-fold increase by
insulin in the LDM and a 50% reduction in the cytosolic fraction (Fig.
1, B and D, respectively). These results indicate
that insulin-induced activation of PI 3-kinase in the LDM can be
attributed to both translocation of this enzyme from the cytosol, as
well as to its activation in the LDM fraction. Oxidative stress induced
a 3-fold increase in PI 3-kinase activity in the LDM in the absence of
insulin, which was not associated with increased LDM p85 content (Fig.
1, B and A). This may suggest that oxidative
stress increased LDM PI 3-kinase activity primarily by inducing
activation rather than translocation of this enzyme in the LDM. Yet,
oxidative stress resulted in a significant reduction in the ability of
insulin to increase p85 content in the LDM (Fig. 1A) or to
reduce its cytosolic content (Fig. 1C). This was associated with inhibition of insulin-stimulated PI 3-kinase activation in the LDM
(Fig. 1B), as well as in impaired insulin induced reduction in its cytosolic activity (Fig. 1D). In total cell lysates,
both p85 content as well as the activation of p85-associated PI
3-kinase activity by insulin were not affected following oxidation
(Fig. 1, E and F, respectively). Taken together
these data suggest that both the insulin-stimulated translocation of PI
3-kinase to the LDM and its activation in this fraction are impaired
following oxidative stress and are associated with retention of this
enzyme in its cytosolic pool.

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Fig. 1.
Insulin-induced PI 3-kinase redistribution
and activation in cellular fractions of control and glucose
oxidase-treated cells. Differentiated 3T3-L1 adipocytes were
serum-starved for 16 h, followed by 2-h treatment without
(Control) or with 100 milliunits/ml glucose oxidase
(GO). Cells were then rinsed three times with PBS followed
by a further 7-min incubation with (+) or without ( ) 100 nM insulin. Cells were then subjected to subcellular
fractionation, and the LDM and cytosol fractions were collected, as
described under "Experimental Procedures." Each panel contains a
representative blot of at least three independent experiments, under
which the results of densitometric analysis is presented. Values are
the mean ± S.E. of the densitometry values expressed in arbitrary
units as compared with values obtained in unstimulated control cells.
A, 40 µg of LDM protein were subjected to polyacrylamide
gel electrophoresis and immunoblotted with antibody against the p85
regulatory subunit of PI 3-kinase. B, 75 µg of protein
from LDM were subjected to immunoprecipitation with an antibody against
p85 and assayed for PI 3-kinase activity using phosphatidylinositol as
a substrate, as detailed under "Experimental Procedures".
C, 500 µg of protein from the cytosolic fractions were
immunoprecipitated using anti-p85 antibody followed by Western blot
analysis for the amount of p85 or assayed for PI 3-kinase activity
(D). E, 50 µg of protein of whole cell lysate
was assayed for total p85 content by Western blot analysis.
F, 500 µg of protein of total cell lysate were subjected
to immunoprecipitation using anti-p85 antibody, after which PI 3-kinase
activity was assessed. *, p < 0.01 versus
unstimulated control cells; **, p < 0.01 versus insulin-stimulated control cells.
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Oxidative Stress Alters the Pattern of Insulin-stimulated IRS-1
Redistribution--
In 3T3-L1 adipocytes most of PI 3-kinase
activation by insulin occurs via its interaction with
tyrosine-phosphorylated IRS-1 (21). In order to assess whether altered
IRS-1 cellular compartmentalization may account for the impaired PI
3-kinase activation in the LDM, IRS-1 content and interaction with PI
3-kinase were evaluated in the LDM and cytosolic fractions. In control
cells, an approximately 50% reduction in IRS-1 LDM content following
7-min insulin stimulation was observed (Fig.
2A). In oxidized cells, IRS-1
content in the LDM was significantly reduced by approximately 35%,
with no further reduction following insulin stimulation. In control
cells, insulin induced an inhibition in IRS-1 gel mobility (Fig.
2A), which could be attributed to increased serine/threonine
phosphorylation, since incubation of LDM with alkaline phosphatase
diminished this change in gel mobility (data not shown). In addition,
despite the reduction in LDM IRS-1 content (Fig. 2A),
insulin induced a marked elevation in IRS tyrosine phosphorylation in
this fraction (Fig. 2B). In LDM prepared from oxidized
cells, insulin stimulation failed to induce the change in IRS gel
mobility (Fig. 2A), suggesting inhibition of its insulin
stimulated serine/threonine phosphorylation. Moreover, insulin-induced
tyrosine phosphorylation of IRS-1 was significantly impaired following
oxidation (Fig. 2B). In accordance with this finding, the
16-fold increase in IRS-1-associated PI 3-kinase activity observed in
the LDM of control cells was markedly impaired by oxidative stress
(Fig. 2C).

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Fig. 2.
Insulin-induced IRS-1 redistribution in
control and glucose oxidase-treated cells. Cell treatment and
cellular fraction isolation were identical to those described in the
legend for Fig. 1. A, total IRS-1 content in the LDM was
measured by Western blot analysis. B, tyrosine
phosphorylation of LDM proteins was evaluated using
anti-phosphotyrosine antibodies (4G10). C, 75 µg of LDM
were immunoprecipitated using anti-IRS-1 antibody, and the activity of
PI 3-kinase co-precipitated with IRS-1 was assayed. D, 500 µg of cytosol were subjected to immunoprecipitation using anti-IRS-1
antibody and assessed for IRS-1 content, as well as for PI 3-kinase
activity using phosphatidylinositol as substrate (E). Shown
are representative blots of at least three independent experiments.
Values of densitometry analysis presented as mean ± S.E. are
expressed in arbitrary units as compared with values obtained in
unstimulated control cells. *, p < 0.01 as compared
with values of unstimulated control cells; **, p < 0.01 versus insulin-stimulated control cells.
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In the cytosolic fraction, insulin stimulation of either control or
oxidized cells did not result in changes in IRS-1 gel mobility, as in
the LDM fraction (Fig. 2D). Yet, both the increase in
cytosolic IRS-1 content induced by insulin (Fig. 2D), as well as the
stimulation of IRS-1-associated PI 3-kinase activity in this fraction
(Fig. 2E), were significantly impaired by oxidative stress. Taken
together these results suggest that oxidation disrupted IRS-1 cellular
redistribution and tyrosine phosphorylation in the LDM. This in turn
resulted in the reduced ability of IRS-1 to serve as a LDM docking
protein for PI 3-kinase.
Insulin-stimulated Activation of PKB
and PKB
Is Impaired
following Oxidative Stress--
To assess whether the defect in PI
3-kinase redistribution is associated with impairment in the activation
of PKB by insulin, PKB phosphorylation and activation were evaluated
following oxidative stress. Immunoblot analysis of total cell lysates
using anti-phospho-Ser473 PKB antibody demonstrated that
oxidative stress induced a 3.47 ± 0.85-fold increase in PKB
phosphorylation, which was wortmannin-sensitive (Fig.
3A). Exposure to insulin led
to a rapid, time-dependent stimulation of PKB
phosphorylation reaching maximal stimulation by 10 min in control
cells, while in oxidized cells PKB phosphorylation was dramatically
reduced (Fig. 3B). To correlate changes in PKB phosphorylation with its activity, total cell lysates were subjected to
immunoprecipitation using anti PKB antibodies raised against its
C-terminal tail. The efficiency of the immunoprecipitation protocol was
not affected by oxidation, as assessed by immuno- blotting the
supernatant above the immunoprecipitate (data not shown). Fig.
3C demonstrates a 12.2 ± 2.3-fold increase in PKB activity following 7-min exposure to insulin. In accordance with the
serine phosphorylation pattern, oxidative stress induced a 3.6 ± 1.7-fold increase in PKB activity, while the net insulin effect on PKB
activity was markedly reduced from 24.10 ± 2.7 to 4.65 ± 0.9 milliunits/mg of protein/min (p < 0.001). Both
basal activation of PKB induced by oxidative stress, as well as its stimulation by insulin, were completely inhibited by wortmannin, indicating the involvement of PI 3-kinase in both processes.

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Fig. 3.
PKB phosphorylation and activation by insulin
in control and oxidized cells. A, cells were treated
without or with 250 nM wortmannin for 15 min before and
during the 2-h incubation without (C) or with glucose
oxidase (GO). Subsequently, cells were lysed in PKB lysis
buffer, and 50 µg of protein were separated on SDS-polyacrylamide gel
electrophoresis and immunoblotted with phosphoserine 473-specific PKB
antibody, as described under "Experimental Procedures."
B, control and oxidized cells were incubated without or with
insulin for the indicated times, and the phosphorylation pattern was
studied by Western blot analysis using anti-phospho-Ser473
antibody. C, 200 µg of cell lysate were subjected to
immunoprecipitation with anti PKB (C-terminal) antibody and assayed for
PKB activity using Crosstide as a substrate. Wortmannin treatment was
as described above and also during the 7-min insulin stimulation. *,
p < 0.01 as compared with unstimulated control cells;
**, p < 0.01 versus insulin-stimulated
control cells.
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To further characterize the effect of oxidative stress on basal and
insulin stimulated PKB activity, the various isoforms of PKB were
studied using isoform specific antibodies. In 3T3-L1 adipocytes, all
known PKB isoforms were found to be expressed, and their total cellular
content was not significantly altered by oxidation (Fig.
4A). PKB activity was measured
in immunoprecipitates of the different PKB isoforms from total cell
lysates of control and oxidized cells before and after exposure to 100 nM insulin for 7 min. Fig. 4B demonstrates that
in 3T3-L1 adipocytes the basal activity of PKB
, PKB
, and PKB
was 1.87 ± 0.25, 2.74 ± 0.42, and 6.02 ± 0.96 milliunits/mg of protein/min, respectively. Insulin increased the
activity of PKB
and PKB
by approximately 13- and
6-fold, respectively, while exerting only a minor effect on PKB
activity. Exposure of the cells to oxidative stress resulted in a
significant increase in basal PKB
activity. The ability of insulin
to further activate PKB
was completely inhibited, while a
significant reduction in insulin stimulated PKB
activity was
observed (from 32.3 ± 4.9 to 15.6 ± 2.6 milliunits/mg of
protein/min, p < 0.01). These data demonstrate the
role of PKB
in the induction of basal PKB activity observed in
oxidized cells, while the reduction in insulin-stimulated PKB activity
could be mainly related to PKB
and PKB
isoforms.

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Fig. 4.
The effect of oxidation on the PKB isoforms
expression and regulation by insulin. A, control or
glucose oxidase (GO)-treated 3T3-L1 adipocytes were lysed
using PKB lysis buffer, and 50 µg of protein were subjected to
SDS-polyacrylamide gel electrophoresis followed by immunoblotting with
anti-PKB , anti-PKB , and anti-PKB antibodies. Each blot is a
representative of at least three independent experiments. B,
200 µg of protein from control and GO-treated cells, stimulated
without or with 100 nM insulin for 7 min, were lysed and
subjected to immunoprecipitation using the specific PKB isoform
antibodies. PKB activity was assayed on the immunoprecipitates using
Crosstide as a substrate, as further detailed under "Experimental
Procedures." Values are expressed as milliunits of activity/mg of
protein and are the mean ± S.E. of three independent experiments.
*, p < 0.01 as compared with unstimulated control
cells; **, p < 0.01 versus
insulin-stimulated control cells.
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To assess whether oxidative stress also impairs the ability of PKB to
be activated through non-PI 3-kinase-mediated pathways, the
phosphorylation and activation of PKB by heat shock were studied in
oxidized cells. In control cells, exposure of 3T3-L1 adipocytes for 20 min to 44 °C resulted in a wortmannin-insensitive increase in both
PKB serine phosphorylation (Fig.
5A) and in PKB activity (Fig.
5B). Cells treated for 2 h with glucose oxidase, and
subsequently exposed to heat shock treatment, exhibited increased PKB
phosphorylation and activation comparable with control cells. Taken
together, these results suggest that oxidative stress does not
interfere with activation of PKB through wortmannin-insensitive
pathway(s).

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Fig. 5.
The effect of oxidative stress on heat
shock-induced PKB activation. Control and glucose oxidase
(GO)-treated 3T3-L1 adipocytes were further incubated in
fresh medium for 20 min at either 37 or 44 °C (Heat
shock). Heat shock-exposed cells were co-treated without or with
250 nM wortmannin as described in the legend for Fig. 3.
A, 50 µg of protein of whole cell lysate were subjected to
Western blot analysis using anti-phospho-specific PKB antibody (raised
against phospho-Ser473). B, 200 µg of cell
lysate were subjected to immunoprecipitation with anti PKB (C-terminal)
antibody and assayed for PKB activity. Values are expressed as
milliunits of activity/mg of protein and represent the mean ± S.E. of two independent experiments. *, p < 0.01 as
compared with unstimulated control cells.
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Oxidative Stress-impaired Insulin-stimulated GLUT4 Translocation
and Glucose Transport Activity--
The metabolic relevance of the
impaired insulin stimulation of PI 3-kinase redistribution and PKB
activation was evaluated by assessing the effects of oxidative stress
on glucose transport and GLUT4 translocation. Fully differentiated
3T3-L1 adipocytes exhibited an approximately 12-fold increase in
glucose transport activity following 20-min stimulation with 100 nM insulin (Fig. 6A). When cells were exposed
for 2 h to glucose oxidase, a marked reduction in
insulin-stimulated glucose transport activity was observed. This was
associated with a significant 2.1 ± 0.15-fold increase in basal
glucose transport, resulting in a reduction in the net insulin effect
on glucose transport activity above basal from 510 ± 17 to
77 ± 13 pmol/mg of protein/min. As observed with the activation
of PKB (Fig. 3C), both the basal activation as well as the
insulin stimulation of glucose transport were completely inhibited by
wortmannin. Since insulin acutely activates glucose transport mainly by
promoting the translocation of GLUT4 from internal membrane pools to
the plasma membrane, GLUT4 translocation was assessed. PMs were
prepared using the plasma membrane lawn technique, as described (40),
and subjected to Western blot analysis (Fig. 6B, left
side). Using this method, insulin induced a 4.87 ± 1.43-fold
increase in PM GLUT4 content in control cells, while only a 1.6 ± 0.3-fold increase was observed in cells treated with glucose oxidase
prior to insulin stimulation. To further established whether oxidative
stress inhibits translocation of GLUT4 from LDM to the PM, GLUT4
content was evaluated in the subcellular fractions using the sucrose
cushion methods (39). As can be evaluated, insulin induces 2.55 ± 0.35-fold increase in PM GLUT4 content in control cells (Fig.
6B, right side). This was associated with a
55 ± 1.2% reduction in its abundance in the LDM, indicating insulin-stimulated GLUT4 translocation. In glucose oxidase-treated cells, no significant increase in PM GLUT4 content was observed with
insulin, while a nonsignificant 15% reduction in LDM GLUT4 was
detected. Taken together these results indicate that the changes in
normal insulin-induced PI 3-kinase redistribution and PKB activation by
oxidative stress correlate with impairment in insulin-stimulated GLUT4
translocation and activation of glucose transport activity.

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Fig. 6.
The effect of oxidative stress on
insulin-stimulated 2-deoxyglucose uptake and GLUT4 translocation.
Control or glucose oxidase (GO)-treated cells were incubated
without or with 100 nM insulin for 20 min. Wortmannin (250 nM) was added for 15 min prior to and during the 2-h
incubation without or with glucose oxidase and again for the 20-min
incubation without or with insulin. A, 2-Deoxyglucose uptake
was measured as described under "Experimental Procedures." Values
are mean ± S.E. of five independent experiments performed at
least in duplicates. B, plasma membrane (PM)
lawns (left side) were prepared as described under
"Experimental Procedures." Alternatively, PM and low density
microsomes (LDM) were prepared by subcellular fractionation
(right side). GLUT4 protein content in control and glucose
oxidase-treated cells was assessed by Western blot analysis using anti
GLUT4 antibody. Blots are representatives of four independent
experiments for subcellular fractionation and of three independent
experiments for the PM lawn procedure. Densitometry analysis of PM
GLUT4 content is presented as mean ± S.E. *, p < 0.01 as compared with unstimulated control cells; **, p < 0.01 versus insulin-stimulated control cells.
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 |
DISCUSSION |
This study was aimed at investigating the cellular mechanisms by
which oxidative stress disrupts insulin action in 3T3-L1 adipocytes.
The data presented demonstrate that while certain insulinomimetic
effects of micromolar H2O2 concentrations could be observed, oxidative stress impaired the compartment-specific activation of PI 3-kinase, IRS-1 redistribution, and PKB activation induced by insulin. These provide a putative mechanism for impaired insulin-stimulated GLUT4 translocation and glucose transport activity following oxidative stress.
The concept of signal transduction events taking place in specific
cellular compartments has been suggested as a plausible explanation for
understanding the specificity of hormones and growth factors action. In
isolated adipocytes and in 3T3-L1 adipocytes, the activation of PI
3-kinase in the LDM has been suggested as necessary for the specific
ability of insulin to induce GLUT4 translocation to the PM, resulting
in enhanced glucose transport activity (16-19, 22, 42). Using the
subcellular fractionation protocol on sucrose cushion, the LDM fraction
has been shown to contain intracellular membranes comprising recycling
endosomes, the Golgi apparatus, intracellular GLUT4 storage vesicles
(43-45), as well as cytoskeleton components (22). Our findings of
insulin-stimulated IRS-1 redistribution from the LDM to the cytosol,
and of increased PI 3-kinase and tyrosyl-phosphorylated IRS-1 in this
fraction following insulin, are in close agreement with previous
reports (21, 22, 46). Whether the IRS-1-PI 3-kinase complex in the LDM
directly interacts with the GLUT4 vesicles during the normal insulin
signaling toward GLUT4 translocation is currently a subject of debate.
Heller-Harrison et al. (19) reported a direct interaction that was demonstrated by insulin-stimulated IRS-1-associated PI 3-kinase activity in isolated GLUT4 vesicles. Yet, other reports challenged this view (15, 22). Clark et al. (22) suggested that IRS-1 and PI 3-kinase could be recovered in cytoskeleton components that were nonsoluble by nonionic detergents, as opposed to
the GLUT4 protein. The role of such interaction in the normal response
to insulin is suggested by the demonstration that PI 3-kinase is
necessary for insulin-induced actin rearrangement (47, 48) and by the
observation that disruption of the actin filament network by
cytochalasin D or by latrunculin B impairs insulin-stimulated GLUT4
translocation and glucose transport activity (49). Whether the impaired
insulin-induced PI 3-kinase and IRS-1 redistribution in 3T3-L1
adipocytes exposed to oxidative stress (Figs. 1 and 2, respectively)
represent a defect in their interaction with GLUT4 vesicles or
cytoskeleton elements could not be determined by this study. Yet, these
observations demonstrate that insulin stimulated PI 3-kinase
translocation to and activation in the LDM, possibly through its
interaction with tyrosyl phosphorylated IRS-1, are oxidation sensitive
steps that may be essential for GLUT4 translocation. To the best of our
knowledge, this is the first demonstration that changes in the extra
cellular environment induce insulin resistance by impairing IRS-1
and/or PI 3-kinase trafficking, without affecting proximal insulin
signaling events as detected in total cell lysates.
Insulin is known to induce both the tyrosine phosphorylation of IRS-1,
as well as its phosphorylation on serine/threonine residues. The role
and interrelation of these two processes is not fully understood. While
the propagation of early steps in the insulin signal appears to depend
on tyrosine phosphorylation (50), serine/threonine phosphorylation may
be involved in its termination (51, 52). Several serine/threonine
kinases which are normally activated in response to insulin stimulation
have been suggested to phosphorylate IRS-1, potentially representing an
inherent shut-down mechanism for the insulin signal. These include ERK
1/2 MAP kinases (53), GSK3 (54), PKB (55), and certain PKC isoforms
(56). Moreover, activation of insulin stimulated serine/threonine
kinase(s) was suggested to play a role in the translocation of IRS-1
from the LDM fraction to the cytosol (21). In this study we demonstrate
that the activation of the serine/threonine kinase PKB was dramatically
inhibited by oxidative stress (Figs. 3 and 4). Insulin induced
activation of the ERK1/2 MAP kinase was also impaired (unpublished
results). Interestingly, insulin induced serine/threonine
phosphorylation of IRS-1 in the LDM was markedly reduced by oxidation
(Fig. 2A), and was associated with inhibition of its translocation from
the LDM to the cytosol. These results are consistent with the notion
that serine/threonine phosphorylation of LDM IRS-1 may be needed for
its normal cellular redistribution. Nevertheless, insulin stimulated
tyrosine phosphorylation of IRS-1 was also impaired in the LDM
following oxidation (Fig. 2B), and was associated with reduced PI
3-kinase activity in this fraction (Fig. 1 and 2). Thus, the
oxidation-induced reduction in tyrosine phosphorylated IRS-1 in the LDM
may represent the cause rather than the effect of alterations in
serine/threonine phosphorylation of LDM IRS-1. Moreover, reduced
tyrosine-phosphorylated IRS-1 in the LDM appears to represent the
limiting factor for the translocation and activation of PI 3-kinase in
this fraction.
It is well established that PKB activation by insulin is dependent on
PI 3-kinase (29). Yet whether insulin stimulation of PI 3-kinase in the
LDM is required for the activation of the various known isoforms of PKB
is largely unknown. Thus, the effect of oxidative stress on the basal
and insulin-stimulated activity of PKB
, PKB
, and PKB
was
evaluated. In 3T3-L1 adipocytes, similar to observations in L6 myotubes
(23), most of the insulin-stimulated PKB activation could be attributed
to PKB
and PKB
(Fig. 4). Surprisingly, insulin barely affected
PKB
activity, which was demonstrated as the major PKB isoform
regulated by insulin in primary rat adipocytes (57). Oxidative stress
profoundly impaired both insulin-stimulated PKB
and PKB
activities (Fig. 4), resulting in an overall reduction of total
cellular PKB activity. The striking similarity between the effect of
oxidation on PKB (Figs. 3C and 4) and on glucose uptake (Fig.
6A) suggests a potential cause and effect relationship
between the impairment in both insulin stimulated PKB activation and
GLUT4 translocation.
The ability to activate PKB through wortmannin-insensitive pathway(s)
following oxidative stress was evaluated to exclude a direct inhibitory
effect of oxidation on either PKB or its immediate upstream kinases
PDK1 and PDK2 (29, 58). As demonstrated in COS-7 and NIH 3T3 cells
(59), heat shock treatment of 3T3-L1 adipocytes induced phosphorylation
and activation of PKB through wortmannin-insensitive mechanisms (Fig.
5, A and B, respectively). Following oxidative
stress, this activation of PKB remained intact, supporting the notion
that the impaired insulin-stimulated PKB activation induced by
oxidation may be a consequence of the reduced ability of insulin to
activate PI 3-kinase in the LDM. This, in turn, may offer the
possibility that activation of PKB is also an insulin signaling event
dependent on normal cellular compartmentalization of PI 3-kinase. In
support of this concept is the observation that membrane-localized p110
was found to be sufficient to activate PKB in COS-7 cells (60).
This paper presents evidence that while micromolar concentrations of
H2O2 inhibits acute metabolic response to
insulin, they also induce a certain insulinomimetic effect. Exposure to
H2O2 for 2 h resulted in
wortmannin-sensitive increase in basal glucose transport and in PKB
activities, as well as in basal PI 3-kinase activity in the LDM
(Figs. 6A, 3C, and 1B, respectively).
These seemingly opposing effects suggest several cellular targets for H2O2 along the multistep insulin signaling
network. While one or more targets may be activated by
H2O2, other can eventually become rate-limiting
for additional insulin stimulation.
The potential relevance of findings presented herein may be in the
understanding of the cellular mechanisms leading to peripheral insulin
resistance in various conditions. Increased oxidative stress has been
suggested by various mechanisms to occur in diabetic or prediabetic
individuals (61-63) and to contribute to the pathogenesis of diabetic
late complications (64), as well as to peripheral insulin resistance
(65, 66). Reduced GLUT4 expression and impaired translocation in
response to insulin stimulation were reported in adipocytes of
insulin-resistant individuals (67). The impaired insulin response was
suggested to represent both receptor and postreceptor mechanisms. This
study offers a potential mechanism by which oxidative stress can
contribute to the development of impaired insulin-stimulated GLUT4
translocation in adipocytes of diabetic subjects.