Tumor Necrosis Factor
-Mediated Insulin Resistance, but Not Dedifferentiation, Is Abrogated by MEK1/2 Inhibitors in 3T3-L1 Adipocytes
Jeffrey A. Engelman,
Anders H. Berg,
Renée Y. Lewis,
Michael P. Lisanti and
Philipp E. Scherer
Department of Cell Biology and Diabetes Research and Training
Center (A.H.B., R.Y.L., P.E.S.) Department of Molecular
Pharmacology and Diabetes Research and Training Center (J.A.E.,
M.P.L.) Albert Einstein College of Medicine Bronx, New York
10461
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ABSTRACT
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Tumor necrosis factor-
(TNF
) has been
implicated as a contributing mediator of insulin resistance observed in
pathophysiological conditions such as obesity, cancer-induced cachexia,
and bacterial infections. Previous studies have demonstrated that
TNF
confers insulin resistance by promoting phosphorylation of
serine residues on insulin receptor substrate 1 (IRS-1), thereby
diminishing subsequent insulin-induced tyrosine phosphorylation of
IRS-1. However, little is known about which signaling molecules are
involved in this process in adipocytes and about the temporal sequence
of events that ultimately leads to TNF
-stimulated IRS-1 serine
phosphorylation. In this study, we demonstrate that specific inhibitors
of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein
(MAP) kinase pathway restore insulin signaling to normal levels
despite the presence of TNF
. Additional experiments show that
MEK1/2 activity is required for TNF
-induced IRS-1 serine
phosphorylation, thereby suggesting a mechanism by which these
inhibitors restore insulin signaling.
We observe that TNF
requires 2.54 h to markedly reduce
insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1
adipocytes. Although TNF
activates p42/44 MAP kinase, maximal
stimulation is observed within 1030 min. To our surprise, p42/44
activity returns to basal levels well before IRS-1 serine
phosphorylation and insulin resistance are observed. These activation
kinetics suggest a mechanism of p42/44 action more complicated than a
direct phosphorylation of IRS-1 triggered by the early spike of
TNF
-induced p42/44 activity.
Chronic TNF
treatment (>> 72 h) causes adipocyte
dedifferentiation, as evidenced by the loss of triglycerides and
down-regulation of adipocyte-specific markers. We observe that this
longer term TNF
-mediated dedifferentiation effect utilizes
alternative, p42/44 MAP kinase-independent intracellular pathways.
This study suggests that TNF
-mediated insulin resistance, but not
adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP
kinase pathway.
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INTRODUCTION
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Insulin induces receptor dimerization and triggers the receptors
intrinsic tyrosine kinase activity. This results in the tyrosine
phosphorylation of a number of different intracellular substrates.
Insulin receptor substrates (IRSs) 14 are the major targets for the
activated insulin receptor (1). Once tyrosine-phosphorylated by
activated insulin receptor, they propagate intracellular signaling by
binding to a variety of SH2 domain-containing proteins. In
particular, the binding of Grb2 and the regulatory p85 subunit of
phosphatidylinositol-3-kinase (PI3 kinase) to specific
tyrosine-phosphorylated residues on IRS-1 has been well documented (2, 3). Binding of the p85 subunit to IRS-1 stimulates PI3-kinase activity,
resulting in the activation of the downstream signaling molecules (4).
In the adipocyte, insulin stimulates GLUT4 translocation,
triglyceride synthesis (3), and a number of other cellular
processes.
Under a variety of conditions, signaling from the insulin receptor is
impaired. This resistance to insulin is strongly implicated in the
pathogenesis of type II diabetes mellitus. In recent years, it has been
amply demonstrated that adipocytes with impaired insulin signaling are
highly enriched in serine-phosphorylated IRS-1. Elevated serine
phosphorylation of IRS-1 and IRS-2 inhibits their binding to the
juxtamembrane region of the insulin receptor and impairs their ability
to undergo insulin-induced tyrosine phosphorylation (5). Additionally,
there is evidence that immunoprecipitated IRS-1, which has been serine
phosphorylated in response to tumor necrosis factor-
(TNF
), is a
direct inhibitor of insulin receptor tyrosine kinase activity (6).
IRS-1 serine phosphorylation can be induced by treating cells with a
variety of agents such as serine phosphatase inhibitors
(e.g. okadaic acid), activators of protein kinase C, or with
different cytokines such as TNF
and platelet-derived growth factor
(PDGF) (6, 7, 8, 9, 10, 11).
In particular, TNF
-induced insulin resistance has received
much recent attention. TNF
levels are elevated in a variety of
disease states associated with insulin resistance in peripheral
tissues. There is increasing evidence that implicates TNF
as one of
the key factors involved in obesity-induced insulin resistance (6, 12, 13). TNF
levels are elevated in obese patients due, at least in
part, to increased secretion of TNF
from adipose tissue (13).
Adipocytes, in turn, express TNF
receptors and are highly
susceptible to the effects of TNF
with respect to insulin signaling
(reviewed in Ref. 14). Mice carrying deletions of the TNF
receptor
are more resistant to the development of diabetes (12), and
neutralization of TNF
in rodent models of obesity increases insulin
sensitivity (13, 15). On the other hand, a clinical study by Ofei and
colleagues demonstrated that TNF
neutralization (using a
recombinant-engineered human TNF
-neutralizing antibody) over a
period of 4 weeks had no effect on insulin sensitivity in obese
non-insulin-dependent diabetes mellitus (NIDDM) subjects (16). The most
likely explanation for these seemingly contradictory observations is
that TNF
acts primarily through a local, paracrine effect and much
less through a systemic effect, such that systemic inhibition of TNF
is not expected to have an impact on insulin sensitivity.
Although TNF
-induced IRS-1 serine hyperphosphorylation was
demonstrated in adipocytes, the components of the various signal
transduction pathways that mediate this phosphorylation event have not
been described. In this study, we find that both the TNF
-mediated
serine phosphorylation of IRS-1 and concomitant reduction of insulin
signaling are completely abolished by PD98059, a widely used and highly
specific inhibitor of MAP kinase kinase (MEK)1/2 (11, 17, 18, 19, 20), the
upstream activator of p42/44 MAP kinase. Kinetic analysis reveals that
TNF
-induced IRS-1 phosphorylation and insulin resistance require
2.54 h. Interestingly, TNF
-induced p42/44 activity occurs much
more rapidly with activity returning to baseline within 90 min. These
activation kinetics suggest a mechanism more complex than the direct
serine phosphorylation of IRS-1 by TNF
-activated p42/44.
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RESULTS
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The Effects of Insulin and TNF
on the Activation State of MAP
Kinase Pathways in 3T3-L1 Adipocytes
TNF
and insulin have distinct physiological effects on
adipocytes. In other cell types, both ligands signal, in part, through
different MAP kinases. Because MAP kinases have many significant roles
in cellular physiology, we chose to investigate their potential roles
in TNF
-mediated effects in adipocytes. However, it remains largely
unknown which MAP kinase pathways are activated by these hormones in
the mature adipocyte. We used antibodies that specifically recognize
the phosphorylated, activated forms of the classical MAP kinases
[p42/44, p38 and c-jun N-terminal kinase (JNK)] to determine which
pathways are primarily stimulated by either insulin or TNF
in 3T3-L1
adipocytes. As shown in Fig. 1
, insulin
induced a robust activation of p42/44 MAP kinase after both 2 and 5
min. Insulin also activated JNK but did not significantly activate p38
MAP kinase. In contrast, TNF
activated both p42/44 and p38 MAP
kinase, but did not lead to a significant induction of JNK. After a
5-min treatment, at the concentrations used in this experiment, insulin
is more potent than TNF
in activating p42/44. Jain and colleagues
(21) have also observed that treatment of 3T3-L1 adipocytes with TNF
does not lead to activation of JNK. On the other hand, Font de Mora
et al. (19) used an indirect kinase assay and observed
TNF
-induced JNK activation. However, the availability of
phospho-specific anti-MAP kinase antibodies may allow a more direct
assessment of the activation state of the respective kinase than
in vitro kinase assays.
TNF
-Induced Insulin Resistance Is Blocked by Treatment with
Inhibitors of MEK1/2
TNF
inhibits insulin signaling in adipocytes and muscle
(22, 23). TNF
has been implicated as an important factor responsible
for insulin resistance in NIDDM (24). However, the proteins involved in
mediating the signal from the TNF
receptor that ultimately trigger
serine phosphorylation of IRS-1 have not been identified. To determine
whether the p42/44 or p38 MAP kinase pathways are necessary for this
effect, serum-starved 3T3-L1 adipocytes were treated with TNF
(10
ng/ml) for 6 h with or without the addition of PD98059 (a specific
MEK1/2 inhibitor) or SB203580 (a specific p38 inhibitor). The
adipocytes were then incubated with insulin for 5 min followed by
immediate lysis in immunoprecipitation buffer. The lysates were
immunoprecipitated with anti-IRS-1 antibodies, and the precipitates
were probed with antiphosphotyrosine antibodies. As shown in Fig. 2A
, insulin stimulated tyrosine
phosphorylation of IRS-1. As expected, TNF
pretreatment resulted in
a 2.5-fold reduction of tyrosine-phosphorylated IRS-1, a phenomenon
observed by many other investigators (6, 8, 25). Treatment of the cells
with PD98059 completely abolished the TNF
-induced reduction of IRS-1
tyrosine phosphorylation. SB203580 (the p38 MAP kinase inhibitor) did
not effect the TNF
-mediated reduction of IRS-1 tyrosine
phosphorylation. As controls, TNF
alone has no effect on IRS-1
tyrosine phosphorylation, and PD98059 does not alter insulin-induced
IRS-1 tyrosine phosphorylation (Fig. 2B
). In agreement with previously
reported findings (25), at the low concentrations of TNF
used (10
ng/ml), we do not observe a significant reduction of the tyrosine
phosphorylation state of the insulin receptor (Fig. 2C
). Peraldi and
colleagues (25) observed significant effects on insulin receptor
phosphorylation only at higher TNF
concentrations, whereas effects
on IRS-1 could be seen even at the lowest doses studied.
TNF
-Induced IRS-1 Serine Phosphorylation Is Blocked by
PD98059
Several studies demonstrated that TNF
stimulates serine
phosphorylation of IRS-1, which in turn blocks insulin-induced IRS-1
tyrosine phosphorylation (6, 8). Thus, it is possible that PD98059
inhibits TNF
-induced insulin resistance by abrogating
TNF
-induced IRS-1 serine phosphorylation. To test this hypothesis,
3T3-L1 adipocytes were labeled with
[32P]orthophosphate and treated with TNF
+/- PD98059 for 6 h. The cells were lysed in IP buffer, and the
lysates were immunoprecipitated with antibodies to IRS-1. As shown in
Fig. 3A
, TNF
promoted an approximately
2.5-fold increase in IRS-1 phosphorylation. Cotreatment with PD98059
completely abolished the TNF
-induced IRS-1 phosphorylation.
Treatment with PD98059 alone did not have a significant effect (not
shown). The results were quantitated and are displayed as a graph
in Fig. 3B
. Additionally, phosphoamino acid analysis was performed on
the immunoprecipitated IRS-1. As shown in Fig. 3C
, PD98059 treatment
indeed reduced the TNF
-induced serine phosphorylation of IRS-1.
Therefore, TNF
promotes IRS-1 serine phosphorylation in a
MEK1/2-dependent manner.
To test whether TNF
s inhibitory action requires
transcription, 3T3-L1 adipocytes were treated with TNF
either in the
presence or the absence of the RNA polymerase inhibitor
-amanitin
for 6 h. Additionally, to further corroborate the observations
above obtained with the MEK1/2 inhibitor, PD98059, we used another,
chemically distinct MEK1/2 inhibitor, U0216 (26), in the same
experiment. At the end of the incubation period, insulin was added for
5 min, and extracts were prepared and separated on a 515% gradient
SDS-PAGE followed by Western blotting with anti-IRS-1 antibodies (Fig. 4
). Note that IRS-1 isolated from
unstimulated (lane1), insulin-stimulated (lane 2), and TNF
+
insulin-stimulated (lane 3) adipocytes possesses distinct
electrophoretic mobility shifts due to differential phosphorylation, as
previously demonstrated by others (10, 11). The two independent
MEK 1/2 inhibitors, PD98059 and U2106 (lanes 6 and 7), completely
abrogated the TNF
-induced electrophoretic shift of IRS-1, confirming
the requirement of the MEK1/2 pathway in TNF
-induced IRS-1 serine
phosphorylation as depicted in the previous experiment (Fig. 3A
).
However,
-amanitin treatment (lane 4) did not have an effect,
suggesting that TNF
-induced serine phosphorylation of IRS-1 does not
require a transcriptional event.
TNF
-Induced Signaling Resistance Requires Several Hours of
TNF
Treatment
Many studies have been conducted investigating TNF
s
inhibition of insulin signaling. It has been shown that while long-term
(>>72 h) TNF
treatment results in the down-regulation of IRS-1
(27), the short-term effects (
6 h) are mediated by IRS-1
serine phosphorylation. To determine the kinetics of TNF
-induced
p42/44 mitogen-activated protein kinase (MAPK) activation, 3T3-L1
adipocytes were incubated in the presence of TNF
for various lengths
of time up to 6 h. Cells were lysed in boiling sample buffer, and
active p42/44 was assessed by Western blot analysis using antibodies
that specifically bind to the activated, phosphorylated form of p42/44
(Fig. 5A
). Maximal p42/44 activation
occurred between 10 and 30 min after TNF
treatment. However, long
overexposures reveal that there is basal p42/44 activity present even
at 0 min, 180 min, and 360 min time points (data not shown). To
determine the kinetics of TNF
-induced serine phosphorylation on
IRS-1, a similar experiment was performed as in Fig. 5A
. Extracts were
prepared and analyzed with anti-IRS-1 antibodies. Note that the samples
were not treated with insulin at the end of the incubation to visualize
the TNF-induced mobility shift. Figure 5B
shows a distinct shift toward
decreased electrophoretic mobility between 60 and 180 min, suggesting
that serine phosphorylation occurs between these two time points.
In a separate experiment, the kinetics of the TNF
-induced reduction
of insulin signaling was determined. 3T3-L1 adipocytes were treated
with TNF
for 0, 90, 150, 240, and 420 min. Cells were then
stimulated with insulin for 5 min. Extracts were prepared,
immunoprecipitated with anti-IRS-1 antibodies, and probed with
antiphosphotyrosine antibodies (Fig. 5C
). The TNF
-induced decrease
in IRS-1 phosphorylation occurs within 150240 min. The Western blot
of IRS-1 (lower panel) also reveals a mobility shift at 150
min. Thus, the TNF
-induced decrease in IRS-1s electrophoretic
mobility caused by serine phosphorylation (Fig. 5
, B and C) coincides
with TNF
s inhibition of insulin signaling (Fig. 5C
). Therefore,
although TNF
-stimulated p42/44 activity is required for its
antiinsulin actions, maximal p42/44 activity is achieved within 30 min
but is back to basal levels well before the antiinsulin effect is
observed.
Epidermal Growth Factor (EGF) Promotes Insulin Resistance in a
PD98059-Dependent Manner
To determine whether insulin desensitization in the adipocyte
could be accomplished by activating the p42/44 MAPK via ligands other
than TNF
, 3T3-L1 adipocytes were incubated with EGF before insulin
stimulation. While both PDGF and EGF activate p42 MAP kinase in 3T3-L1
cells, EGF does not activate PI3-kinase activity (28). However, EGF
pretreatment did inhibit insulin-induced IRS-1 phosphorylation in a
p42/44-dependent manner, indicating that other growth factors that
stimulate the p42/44 MAP kinase pathway can mimic the inhibitory action
of TNF
on insulin signal transduction (Fig. 6A
). As a control, extracts were analyzed
for the effective inhibition of MEK1/2 with PD98059 by Western blotting
with anti-phospho42/44-specific antibodies (Fig. 6B
). The presence of
PD98059 effectively reduces the levels of active p42/44.

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Figure 6. EGF Induces Insulin Resistance in a
PD98059-Dependent Manner
3T3-L1 adipocytes were serum starved for 3648 h. Cells were
then treated with vehicle alone or EGF (50 ng/ml) for 1.5 h.
PD98059 (50 µM) was included in these incubations where
indicated. The cells were then stimulated with insulin for 5 min where
indicated. Cells were lysed in IP buffer. Equal amounts of precipitate
were analyzed by Western blot analysis using antiphosphotyrosine
antibodies (upper) or anti-IRS-1 antibodies. Note that
EGF treatment decreased tyrosine phosphorylation of IRS-1 in response
to insulin. B, Extracts from the experiment shown in panel A as well as
cells that have been treated for 6 h with TNF followed by
stimulation with insulin for 5 min in the presence or absence of
PD98059 were probed by Western blot analysis for activated p42/44.
PD98059 effectively reduced the activation of p42/44 in all cases.
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Anisomycin Treatment of 3T3-L1 Cells Leads to Reduced IRS-1
Tyrosine Phosphorylation through a p42/44-Independent Mechanism
An elegant study by White and colleagues (29) recently
demonstrated that JNK associates with IRS-1 in Chinese hamster ovary
cells. Anisomycin, a strong activator of JNK in these cells, stimulates
the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated
tyrosine phosphorylation of IRS-1. Even though we do not observe
significant induction of JNK activity by TNF
in 3T3-L1 cells, we
wanted to test whether anisomycin treatment of 3T3-L1 adipocytes has an
effect on the phosphorylation state of IRS-1, and whether such an
effect would mechanistically resemble the pathway used by TNF
.
Figure 7A
shows that anisomycin leads to
the activation of both p38 and p42/44 MAP kinase. However, we were
unable to observe activation of JNK by anisomycin under conditions that
readily detected JNK activation by insulin. Using conditions very
similar to the ones used by Aguirre et al. (29), we also
observe that a 30-min pretreatment of 3T3-L1 adipocytes with anisomycin
leads to a significant reduction of insulin-induced tyrosine
phosphorylation on IRS-1 (Fig. 7B
). However, this effect could not be
prevented by pretreatment of cells with MEK1/2 inhibitor. This suggests
that, even though both TNF
and anisomycin treatment result in
decreased insulin-induced IRS-1 tyrosine phosphorylation, the two
processes are mechanistically quite different, as judged by their
differential susceptibility to MEK1/2 inhibition as well as the rather
different kinetics with which the effects on IRS-1 are exerted.
Furthermore, anisomycin did not result in an electrophoretic mobility
shift of IRS-1 similar to the one observed for TNF
treatment.

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Figure 7. Anisomycin Induces Reduced Insulin-Stimulated
Phosphorylation of IRS-1 in 3T3-L1 Adipocytes but Does Not Activate JNK
A, Differentiated 3T3-L1 adipocytes were incubated in the presence of 5
µg/ml anisomycin for the indicated amount of time. As a positive
control for JNK activation, one sample was also treated for 5 min with
160 ng/ml insulin. Cells were lysed and analyzed by Western blot
analysis for total as well as activated forms of the indicated MAP
kinases. B, Cells were treated as indicated, lysed, and
immunoprecipitated with anti-IRS-1 antibodies. Immunoprecipitates were
analyzed by Western blot analysis with either antiphosphotyrosine
antibodies (top panel) or anti-IRS-1 antibodies
(bottom panel).
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TNF
-Induced Insulin Resistance Is Not Mediated by SOCS-3
(Suppressor of Cytokine Signaling-3) Induction
Emanuelli and colleagues (30) have recently reported that SOCS-3
is an insulin-induced negative regulator of insulin signaling. SOCS is
a family of proteins initially characterized by their ability to
negatively regulate cytokine signaling. We wanted to test whether
TNF
would induce the expression of SOCS-3 in 3T3-L1 adipocytes and
potentially mediate the effects we observe. Even though TNF
indeed
induces expression of SOCS-3 in adipocytes (Fig. 8
), its induction was not prevented by
the presence of MEK1/2 inhibitor. Since SOCS-3 activity is primarily
controlled at the transcriptional level, this is also consistent with
our observation that the p42/44-mediated TNF
effect is not
critically dependent on a transcriptional event (as shown in Fig. 4
by
pretreatment with
-amanitin). SOCS-3 is therefore an unlikely
mediator of p42/44-mediated IRS-1 serine phosphorylation.
TNF
-Induced 3T3-L1 Adipocyte Dedifferentiation Is Not Mediated
by the p42/44 Pathway
Upon prolonged (>> 72 h) exposure to TNF
, mature
adipocytes lose their terminally differentiated phenotype. We have
shown above that the p42/44 MAP kinase pathway mediates the acute (<4
h) antiinsulin effects of TNF
. To determine whether the p42/44 MAP
kinase pathway also mediates the long-term dedifferentiation effects of
TNF
in adipocytes, 3T3-L1 adipocytes were treated with TNF
(10
ng/ml) for 7 days with or without the addition of the MEK1/2 inhibitor,
PD98059, or the p38 inhibitor, SB203580. As shown in Fig. 9A
, TNF
promoted dedifferentiation of
adipocytes as detected by loss of expression of adipocyte-specific
markers, fatty-acyl CoA synthase, caveolin-1, and IRS-1.
TNF
-mediated dedifferentiation was not blocked by inhibition of the
p42/44 (PD98059) or p38 (SB203580) pathways. Additionally, when these
cells were viewed microscopically, the TNF
-treated cells (with or
without MAP kinase inhibitors) no longer appeared as lipid-laden fat
cells, but instead had a more fibroblastic morphology with decreased
lipids. Staining the cells with Oil Red O demonstrates the reduced
overall lipid content of the cells treated with TNF
in the presence
or absence of the MAP kinase inhibitors (Fig. 9B
).

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Figure 9. TNF -Induced Dedifferentiation of 3T3-L1
Adipocytes Is Not Inhibited by PD98059 or SB203580
Differentiated 3T3-L1 adipocytes were incubated in the presence or
absence of TNF for 7 days. Cells treated with TNF were incubated
in the presence or absence of SB203580, PD98059, or both inhibitors to
assess the role, if any, that the p42/44 and p38 MAP kinase pathways
play in this dedifferentiation process. Media were replaced every 2
days. A, After 7 days of treatment, cells were lysed, and 50 µg of
extracts were probed with antibodies specific for FACS, caveolin-1,
IRS-1, and GDI. Cells were treated as in panel A and then fixed and
stained with Oil Red O (Materials and Methods). Note
that there is decreased staining in TNF regardless of the presence
of MAP kinase inhibitors.
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DISCUSSION
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Peripheral adipocyte and muscle resistance to insulin is
believed to be a critical factor in the development of type II diabetes
mellitus. Although much is known about the pathogenesis of this
disease, the intracellular mechanisms that promote resistance to
insulin remain largely undefined.
TNF
is a cytokine primarily produced in macrophages. It is,
however, also secreted from adipocytes (13). Even though predominantly
an inflammatory cytokine (31), it has been implicated in conferring
insulin resistance in peripheral tissues in a number of different
disease states associated with elevated systemic TNF
levels, such as
obesity, cancer, and sepsis (13, 32, 33). Additionally, mice carrying
deletions of the TNF
receptor are resistant to the development of
diabetes (12), and neutralization of TNF
in rodent models of obesity
increases insulin sensitivity (13, 15). Treatment of cultured murine
adipocytes with TNF
was shown to induce serine phosphorylation of
insulin receptor substrate 1 (IRS-1) and convert IRS-1 into an
inhibitor of the IR tyrosine kinase activity in vitro
(6).
In this study, we have focused on the process of TNF
-induced
insulin resistance in 3T3-L1 adipocytes. In light of the accumulating
data linking TNF
to insulin resistance in vivo, this
appears to be a critical and physiologically relevant paradigm to
address. We attempted to identify which, if any, MAP kinase pathways
are involved in the process. No detailed studies had been performed
with respect to the temporal sequence of events and signal transduction
pathways that lead to the acute TNF
-induced reduction of
insulin-induced IRS-1 tyrosine phosphorylation. Guo and Donner (34)
found that TNF
treatment for 30 min leads to enhanced IRS-1 tyrosine
phosphorylation and association with the p85 PI 3-kinase subunit in
3T3-L1 adipocytes. In marked contrast, Liu and colleagues (35) report
that TNF
treatment for 15 min results in 6070% reduction of
insulin signaling in human adipocytes isolated from mammary tissue. We
have also investigated earlier time points of TNF
treatment in
3T3-L1 adipocytes. However, we do not observe a reduction or
enhancement of insulin-stimulated IRS-1 phosphorylation after 15 or 60
min of TNF
treatment (data not shown). In agreement with our
findings, others have used a 6-h pretreatment of 3T3-L1 adipocytes with
TNF
to observe reduced tyrosine phosphorylation of IRS-1 in response
to insulin (15, 25).
As stated earlier, it is quite apparent that the peak of p42/44
activity in response to TNF
stimulation occurs much more rapidly
than its induction of IRS-1 serine phosphorylation and insulin
resistance. There are many potential explanations for the apparent
temporal discrepancy between TNF
-induced p42/44 activation and IRS-1
serine phosphorylation. We cannot exclude either a p42/44-dependent
activation of another serine kinase or inhibition of a serine
phosphatase that eventually leads to serine phosphorylation of IRS-1.
Alternatively, it may be that MEK1/2 directly serine phosphorylates
IRS-1 or activates a kinase distinct from p42/44 leading to IRS-1
serine phosphorylation. Yet another possibility is that TNF
treatment may result in a change in either the conformation or
microenvironment of IRS-1 that allows basal level p42/44 activity to
phosphorylate it after 150 min.
Recently, several groups have investigated the process of
inhibition of insulin signaling by other growth factors. Recently, Li
et al. (36) demonstrated that, in 3T3-L1 fibroblasts, PDGF
treatment for 20 min inhibits insulin-induced IRS-1 association with
PI-3 kinase. They found that PDGFs effect was blocked by inhibitors
to PI-3 kinase, but not to p42/44. Their data implicate a serine kinase
(possibly mTOR) that is activated through the PI-3 kinase-Akt pathway
in promoting this effect. In another study, Staubs et al.
(11) found that, in 3T3-L1 adipocytes, the PDGF treatment requires
approximately 6090 min to result in a significant reduction of IRS-1
tyrosine phosphorylation. In agreement with the previous study, they
also found that PDGFs effect was mediated by PI-3 kinase, but not the
p42/44 pathway. Ricort et al. (10) observed a reduction of
IRS-1-associated phosphotyrosine within 515 min of PDGF treatment
with levels returning to baseline values by 60 min. In the same study,
PDGFs antiinsulin effects were also blocked by treatment with
wortmannin. In our studies, we did not observe that TNF
s
antiinsulin effect was abrogated by the PI-3 kinase inhibitor LY29004
(data not shown). Instead, we observed that TNF
-induced insulin
resistance is mediated by MEK1/2, implicating the p42/44 MAP kinase
pathway. Additionally, EGF, a growth factor that does not activate PI3
kinase in 3T3-L1 adipocytes (28), also leads to reduced tyrosine IRS-1
phosphorylation via a p42/44-dependent pathway.
Phorbol ester-induced IRS-1 serine phosphorylation and resulting
insulin resistance were investigated by De Fea and Roth (20). They have
suggested that p44/42 MAP kinase is directly involved in 12,13-phorbol
myristate acetate (PMA)-induced serine phosphorylation of IRS-1
in 293 cells stably expressing recombinant IRS-1. Additionally, they
showed that p42 MAP kinase phosphorylates IRS-1 in vitro on
Ser612, which is part of a MAP kinase consensus phosphorylation site
and has been implicated in protein kinase C-mediated IRS-1
phosphorylation. Interestingly, the study by Liu et al. (17)
suggests that PDGF, which inhibits insulin signaling via a
p42/44-independent pathway, does not exert its effects via
phosphorylation of Ser612.
Thus, there appear to be several distinct mechanisms to inhibit
insulin signaling in adipocytes. One pathway, induced by PDGF, utilizes
PI-3 kinase and is not significantly affected by MEK1/2 inhibitors
(36). Another pathway, induced by TNF
and EGF, requires the p42/44
MAP kinase pathway. Similarly, anisomycin treatment of 3T3-L1 cells
results in rapid reduction of insulin-induced tyrosine phosphorylation,
which is mechanistically distinct from the TNF
/p42/44-mediated
pathway, but which may or may not use components of the PDGF-mediated
cascade that leads to serine phosphorylation. Finally, a pathway that
is mechanistically distinct from all the other pathways but also
results in the down-regulation of insulin signaling is mediated by
SOCS-3 (30). Although TNF
induces SOCS-3 expression, inhibition of
the p42/44 pathway does not prevent its expression.
TNF
activates many different signaling pathways in the course of
adipogenesis and in the fully differentiated adipocyte. Font de Mora
et al. (19) have determined that the inhibitory activity
that TNF
exerts on adipocyte differentiation process can be
abrogated by PD98059, suggesting that TNF
blocks differentiation via
a p42/44 MAP kinase- mediated pathway. Long-term TNF
treatment of
mature adipocytes causes a step-wise reduction of lipid accumulation
and a concomitant decrease of adipocyte-specific marker expression. Our
studies show that, in contrast to the differentiation process, the
TNF
-induced dedifferentiation process does not require the p42/44
(nor the p38) MAP kinase pathways, since inhibitors to these pathways
do not effectively block the dedifferentiation process.
In contrast to TNF
-induced adipocyte dedifferentiation,
TNF
-induced insulin resistance requires active MEK1/2. Currently,
the true physiological role of TNF
in the development of insulin
resistance in obese patients with type II diabetes mellitus remains
undefined. However, this study raises the distinct possibility that the
MEK1/2-p42/44 MAP kinase pathway may mediate insulin resistance in the
adipocyte. In light of the strong activation of p42/44 MAP kinase in
response to insulin, this pathway may also contribute to adipocyte
insulin insensitivity during hyperinsulinemia. Additionally, other, as
yet undefined, factors that promote insulin resistance in adipocytes
may utilize this pathway as well.
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MATERIALS AND METHODS
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Materials
DMEM was purchased from Cellgro Inc.;
[32P]orthophosphate was purchased from NEN Life Science Products (Boston, MA) at a specific activity of
9,000Ci/mmol. DMEM lacking methionine, cysteine, and glutamate was
purchased from ICN Biochemicals, Inc. (Costa Mesa,
CA), and DMEM lacking phosphate and pyruvate was purchased from
Specialty Media, Inc. (Lavallette, NJ). SB203580 and PD98059 were
purchased from Calbiochem (San Diego, CA) and dissolved in
dimethylsulfoxide (DMSO) at a concentration of 10 mM and 50
mM, respectively, and used at a final concentration of 10
µM and 50 µM. U2106 was purchased from
Calbiochem and used at a final concentration of 10
µM. Murine TNF
was purchased from
PharMingen (San Diego, CA) and used at a final
concentration of 10 ng/ml.
-Amanitin and anisomycin were purchased
from Sigma (St. Louis, MO) and used at a final
concentration of 2.5 µg/ml and 5 µg/ml. Insulin was purchased from
Sigma and used at 100 nM. EGF was purchased
from Promega Corp. (Madison, WI) and used at 50 ng/ml. All
other chemicals were purchased from Fisher Scientific
(Pittsburgh, PA).
Cell Culture
3T3-L1 murine fibroblasts (a generous gift of Dr. Charles Rubin,
Department of Molecular Pharmacology, Albert Einstein College of
Medicine) were propagated and differentiated according to the protocol
described previously (18). In brief, the cells were propagated in FCS
[DMEM containing 10% FCS (JRH Biosciences, Lenexa, KS)
and penicillin/streptomycin (100U/ml each)] and allowed to reach
confluence (day -2). After 2 days (day 0), the medium was changed to
DM1 (containing FCS and 160 nM insulin, 250
µM dexamethasone, and 0.5 mM
3-isobutyl-1-methylxanthine). Two days later (day 2), the medium was
switched to DM2 (FCS containing 160 nM insulin). After
another 2 days, the cells were switched back to FCS.
Antibodies
The antibodies to fatty acyl CoA-synthase (FACS) and GDP
dissociation inhibitor (GDI) were generous gifts from Drs. Jean
Schaffer and Perry Bickel (Washington University, St. Louis, MO).
Antibodies to the following proteins were purchased from the indicated
sources: phospho-p38, p42/44, phospho-p42/44, phospho-JNK from
New England Biolabs, Inc.; caveolin-1, phosphotyrosine and
insulin receptor antibodies from Transduction Laboratories, Inc. (Lexington, KY); IRS-1 from Upstate Biotechnology, Inc. (Lake Placid, NY); and SOCS-3 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Immunoprecipitations
For immunoprecipitations of IRS-1, cells were lysed in 50
mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton
X-100, 10% glycerol, 4 mM ortho-vanadate, 17
mM Na-pyrophosphate, 100 mM NaF, 0.1 µg/ml
okadaic acid, and protease inhibitors. Lysates were precleared by
addition of 50 µl of a 1:1 slurry of Protein A Sepharose
(Amersham Pharmacia Biotech, Arlington Heights, IL) in
TNET buffer (1% Triton X-100, 150 mM NaCl, 2
mM EDTA, 20 mM Tris, pH 8.0) containing 1 mg/ml
BSA. After 30 min at 4 C, samples were centrifuged for 5 sec at
15,000 x g, the supernatant was transferred to a fresh
tube, and 50 µl Protein A Sepharose were added together with the
corresponding antiserum. Samples were then incubated for 3 h at 4
C. Immunoprecipitates were washed six times in IP buffer lacking
okadaic acid and analyzed by SDS-PAGE.
Oil Red O staining
Staining was performed as described previously (37).
Immunoblotting
After SDS-PAGE, proteins were transferred to BA83 nitrocellulose
(Schleicher & Schuell, Inc., Keene, NH). Nitrocellulose
membranes were blocked in PBS or TBS with 0.1% Tween-20 and 5% nonfat
dry milk. Primary and secondary antibodies were diluted in PBS or TBS
with 0.1% Tween-20 and 1% BSA. Bound antibodies were detected by
enhanced chemiluminescence according to the manufacturers
instructions (NEN Life Science Products). Immunoblots
probed with antiphosphotyrosine antibodies were blocked with 1% BSA in
TBST.
In Vivo Phosphorylation Experiments
Cells were washed twice in DMEM lacking phosphate and incubated
for 6 h in DMEM lacking phosphate supplemented with 1 mCi
[32P]orthophosphate per 10-cm dish in the
presence or absence of TNF
and 50 µM PD98059. Control
cells were treated with an equal volume of DMSO. This medium was then
removed, and cells were washed in ice-cold PBS and subsequently lysed
in IP buffer.
Phosphoamino Acid Analysis
Phosphoamino acid analysis was performed as described previously
(38, 39).
Other Methods
Separation of proteins by SDS-PAGE, fluorography, and
immunoblotting was performed as described previously (40).
 |
ACKNOWLEDGMENTS
|
---|
We thank the members of the Scherer and Lisanti laboratories and
Dr. Jonathan Backer for helpful discussions, Drs. Perry Bickel and Jean
Schaffer for donating antibodies, and Dr. Joseph Glavy for assistance
in phosphoamino acid analysis.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Philipp E. Scherer, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: scherer{at}aecom.yu.edu
This work was supported by NIH Medical Scientist Training Grant
T32-GM07288 (J.A.E.), the Training Program in Cellular & Molecular
Biology & Genetics T32-GM07491 (A.H.B.), a NIH grant from the NIDDK
(1R01-DK55758; to P.E.S.), a grant from the American Diabetes
Association (P.E.S.), by the G. Harold and Leila Y. Mathers foundation
(M.P.L. and P.E.S.), a NIH grant from the National Cancer Institute
(R01-CA-80250; to M.P.L.) and grants from the Charles E. Culpeper
Foundation (M.P.L.) and the Sidney Kimmel Foundation for Cancer
Research (M.P.L.).
Received for publication December 18, 1999.
Revision received June 19, 2000.
Accepted for publication July 11, 2000.
 |
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