From the Department of Molecular Endocrinology-Diabetes, Merck Research Laboratories, Rahway, New Jersey 07065
Received for publication, June 5, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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Salicylates, including aspirin, have been shown
to improve insulin sensitivity both in human and animal models.
Although it has been suggested that salicylates sensitize insulin
action by inhibiting I I Salicylates, including aspirin, have been shown to be antidiabetic in
both animal models and man in both long term (i.e. 3-4 week) and short term (i.e. overnight) treatments (3-5).
Several lines of evidence suggest that the antidiabetic effects of
salicylates are mediated via direct inhibition of IKK Serine phosphorylation of insulin receptor substrate proteins (IRSs)
such as IRS1 has been strongly implicated as a mechanism of insulin
resistance (10-13). Upon serine phosphorylation, IRS1 proteins have a
reduced ability to interact with the insulin receptor, to be tyrosine
phosphorylated by the insulin receptor, and to bind
phosphatidylinositol 3-kinase (PI3K) (10, 14-17). Such negative phosphorylation in IRS1 has been mapped to several serine residues (13,
14, 18-21). It was reported recently that IRS1 Ser-307 (refers
to the rat IRS1 protein or Ser-312 in human IRS1) is a key regulatory
site (17, 22). Moreover, phosphorylation at this site can be mediated
by the c-Jun N-terminal kinase (JNK) and may be an important
contributor to the inhibitory effects of tumor necrosis factor Salicylates inhibit the kinase activity of recombinant IKK The current study was initiated to investigate the effects of salicylic
acid on phorbol 12-myristate-13-acetate (PMA)- and TNF Materials--
Insulin, TNF Cell Culture, Compound Treatment, and Protein
Extraction--
HEK293 cells stably expressing recombinant human IRS1
(HEK293.IRS1 cells) were a gift from Dr. Richard Roth. The cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C in 10% CO2. For the
experiments, the cells were first incubated in serum-free Dulbecco's
modified Eagle's medium overnight. The cells were then treated with
vehicle, salicylic acid, or SP600125 for 2 h at 37 °C. PMA or
TNF Western Blot Analysis--
Cell lysates were resuspended in
SDS-loading buffer (Invitrogen) and separated in precast 4-20%
gradient NuPAGE SDS-PAGE gels (Invitrogen). The proteins were then
transferred to polyvinylidene difluoride membrane and probed with
primary antibodies. Detection was performed with ECF Western
blotting Kit (Amersham Biosciences) by scanning with a Storm® gel and
blot imaging system (Amersham Biosciences) per the
manufacturer's recommendation.
Salicylic Acid Reverses PMA and TNF Salicylic Acid Reverses PMA and TNF Salicylic Acid Does Not Affect PMA-induced Activation of Multiple
Isoforms of PKCs in the HEK293.IRS1 Cells--
PMA is known to
activate multiple isoforms of classical and novel PKCs (31, 32). We
performed experiments to identify the PKC isoforms that were activated
by PMA in the HEK293.IRS1 cells and determine whether the activation of
any of the PKC isoforms was reversed by salicylic acid. To this end,
Western blots were performed using antibodies against PKC isoforms that
were phosphorylated at their activation loops, autophosphorylation
sites, or at hydrophobic sites within the C terminus of the enzyme.
Such phosphorylation is known to correlate to the activation and
catalytic activity of the PKC isoforms (33, 34). As expected, PMA
treatment did not activate PKC TNF
As shown in Fig. 4, PMA treatment led to
phosphorylation (and activation) of both IKK JNK Is Activated by PMA and TNF
Next we assessed whether JNK is critical or required for PMA and
TNF
It was reported that, although SP600125 potently inhibits the kinase
activity of recombinant JNK enzymes (IC50 of 40-90
nM), it is rather inactive against the kinase activity of
recombinant p42/44, p38, IKK
Finally, we determined whether JNK enzymes are important in mediating
PMA- and TNF Salicylic Acid Inhibits PMA and TNF SP600125 Inhibits Insulin-stimulated Tyrosine Phosphorylation of
Akt, IRS1, and Insulin Receptor In the current study, several observations have been made using a
cellular model system for insulin signaling, i.e. a HEK293 cell line stably expressing recombinant human IRS1. First, both PMA and
TNF The goal of this study was to further elucidate the role of IKK The results from the current study indicate that PMA activates multiple
isoforms of PKC and IKK enzymes in the HEK293.IRS1 cells (Fig. 3). In
addition to activating IKK How does salicylic acid reverse insulin resistance in these cells if
not via inhibition of IKK To elucidate the role of JNK in PMA- and TNF Fig. 7 summarizes the working hypothesis
on the effects of salicylic acid on PMA- and TNFB kinase
(IKK
), the detailed mechanisms
remain unclear. Protein kinase C isoforms and tumor necrosis
factor
(TNF
) signaling pathways are well described mediators of
insulin resistance; they are implicated in the activation of IKK
and the subsequent inhibition of proximal insulin signaling via insulin receptor substrate 1 (IRS1) and Akt. This study investigated the effect
of salicylic acid on phorbol 12-myristate 13-acetate (PMA)- and
TNF
-induced insulin resistance in a human embryonic kidney 293 (HEK293) cell line stably expressing recombinant human IRS1. The
results showed that both PMA and TNF
inhibited insulin-induced Akt
phosphorylation and promoted IRS1 phosphorylation on Ser-307. Salicylic acid pretreatment completely reversed the effects of PMA and
TNF
on both Akt and IRS1. Whereas PMA activated protein kinase C
isoforms and IKK
, TNF
activated neither. On the other hand, both
PMA and TNF
activated the c-Jun N-terminal kinase (JNK), which has
been reported to directly phosphorylate IRS1 Ser-307. SP600125, a JNK
inhibitor, prevented PMA and TNF
-induced IRS1 Ser-307
phosphorylation. Finally, salicylic acid inhibited JNK activation
induced by both PMA and TNF
. Taken together, these observations
suggest that salicylic acid can reverse the inhibitory effects of
TNF
on insulin signaling via an IKK
-independent mechanism(s), potentially involving the inhibition of JNK activation. The role of JNK
in salicylic acid-mediated insulin sensitization, however, requires
further validation because the JNK inhibitor SP600125 appears to have
other nonspecific activity in addition to inhibiting JNK activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B kinase
(IKK
),1 a serine/threonine
kinase, is a component of the larger IKK signalosome, which also
consists of IKK
and IKK
. Whereas IKK
is also a
serine/threonine kinase and is highly homologous to IKK
, IKK
is a
scaffold protein with no enzymatic activity. Upon exposure to stimuli
including stress or cytokines, both IKK
and IKK
undergo
phosphorylation and activation. The activated IKK kinases phosphorylate
serine residues in I
B, which subsequently becomes degraded via the
proteasome pathway. Prior to its degradation, I
B binds to NF
B and
prevents its translocation into the nucleus. Thus, IKKs potentiate
NF
B-mediated gene transcription (as occurs in the inflammatory
states). Interestingly, IKK
but not IKK
deficiency appears to
result in immunodeficiency in mice, suggesting that IKK
but not
IKK
plays a dominant role in immunoresponses (1, 2).
. First,
salicylates were reported to specifically inhibit the kinase activity
of recombinant IKK
but not IKK
proteins in vitro and
to down-regulate NF
B-mediated transcription regulation in cultured
cells (6-8). Second, IKK
heterozygous mice have improved insulin
sensitivity and reduced plasma glucose levels (9). Third, IKK
heterozygosity in ob/ob background also improves
insulin sensitivity in the mice (9). Finally, IKK
heterozygosity and
salicylate treatment prevents lipid-induced insulin resistance in
animal models (5).
(TNF
) on insulin signaling. It was further proposed that salicylate
treatment or IKK
deficiency can improve insulin sensitivity by
reducing the serine phosphorylation of IRS proteins (i.e.
IRS1) (5, 9).
in
vitro and the biological function of endogenous IKK
in cultured cells with an EC50 of 50-100 µM (6). On the
other hand, in in vivo experiments in which antidiabetic
effects were demonstrated, salicylates reached millimolar
concentrations in the plasma (5). Such a discrepancy between in
vitro and in vivo potency may be ascribed to, for
example, potentially lower salicylate levels in the intracellular
environment than in the plasma. On the other hand, it is also possible
that at such a high concentration in vivo, salicylates may
manifest antidiabetic effects by regulating other targets in addition
to or other than IKK
. In fact, at high concentrations salicylates
have been shown to have additional effects such as inhibition of
mitochondria function (23, 24) and inhibition of phosphorylation and
nuclear translocation of signal transducers and activators of
transcription (STATs) (25). Furthermore, it was reported that treatment
of intact cells with salicylate inhibited TNF
-induced but not
interleukin-1-induced IKK activity and that this inhibition was
prevented by a p38 MAPK inhibitor, suggesting that salicylate does not
directly bind to and inhibit IKK
in intact cells (26). These
observations suggest that the antidiabetic effects of salicylates may
not be mediated exclusively by the inhibition of IKK
.
-induced
insulin resistance in a HEK293 cell line stably expressing recombinant
IRS1. Results from this study suggest the following. 1) PMA and TNF
inhibit insulin-stimulated Akt phosphorylation and promote inhibitory
IRS1 serine 307 phosphorylation. 2) Salicylic acid reverses the effects
of PMA and TNF
on Akt and IRS1 phosphorylation. 3) Salicylic acid
attenuates the inhibitory effects of TNF
on insulin signaling
through an IKK
-independent mechanism. 4) JNK kinase is activated by
and may be important for both PMA and TNF
-induced insulin resistance
in these cells. 5) Salicylic acid partially inhibits PMA and
TNF
-induced JNK activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PMA, and salicylic acid were
purchased from Sigma. Salicylic acid was prepared as 1 M
stock solution in 1 M Tris-HCl, pH7.5, aliquoted, and
stored at
20 °C. Rabbit polyclonal antibodies against total
insulin receptor
subunit, total IRS1, IRS1 phosphorylated at
Ser-307 (IRS1-pS307), and total PKC
were purchased from
Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal antibody
(4G10) against tyrosine-phosphorylated proteins was also purchased from
Upstate Biotechnology. Rabbit polyclonal antibodies against total Akt,
Akt phosphorylated at threonine 308 (Akt-pT308), total IKK
, total
IKK
, both IKK
phosphorylated at serine 180 and IKK
phosphorylated at serine 181, phosphorylated PKC isoforms (PKC-pan),
PKC
/
II phosphorylated at threonine 638 and 641, PKC
phosphorylated at threonine 505, PKC
phosphorylated at serine 643, PKCµ phosphorylated at serine 744/748, PKCµ phosphorylated at
serine 916, PKC
phosphorylated at threonine 538, PKC
/
phosphorylated at threonine 410 and 403, total PKCµ, total c-Jun, and
c-Jun phosphorylated at serine 73 were purchased from Cell Signaling
(Beverly, MA). Rabbit polyclonal antibodies against total p38 MAP
kinase, p38 MAPK phosphorylated at threonine 180 and tyrosine 182, total p42/44 MAP kinase, p42/44 MAP kinase phosphorylated at threonine
202 and tyrosine 204, total JNK, and JNK phosphorylated at Thr-183, and
Tyr-185 were purchased from New England Biolabs (Beverly, MA). Mouse
monoclonal antibody against total PKC
was purchased from Santa Cruz
Biotechnology. SP600125, a JNK inhibitor, was purchased from Biomol
(Plymouth Meeting, PA).
were then added to the media. After incubation for another 30 min, the cells were exposed to insulin for 15 min, rinsed with ice-cold
phosphate-buffered saline, and then lysed in ice-cold lysis buffer. The
lysis buffer contains 20 mM HEPES, pH7.4, 1% Triton X-100,
20 mM
-glycerophosphate, 150 mM sodium
chloride, 1 mM sodium orthovanadate, 10 mM
sodium fluoride, and 1× concentration of a protease inhibitor mixture (Roche Diagnostics). Cell lysates were cleared by centrifugation. Protein concentrations were determined using Bradford reagent (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mediated Inhibition on
Insulin-stimulated Akt Phosphorylation in the HEK293.IRS1
Cells--
Both PMA (19, 27, 28) and TNF
(14, 18, 29) have been
shown to inhibit insulin signaling in cultured cells. Akt phosphorylation (and activation) is a key event in insulin signaling and has been commonly used as a surrogate marker for assessing the
level of activation of the proximal insulin signaling pathway (30). We
therefore determined whether PMA and/or TNF
inhibit insulin-stimulated Akt phosphorylation in the HEK293.IRS1 cells. If so,
we sought to see if such inhibition could be reversed by salicylic
acid. As shown in Fig. 1A,
insulin stimulation resulted in an apparent increase in Akt
phosphorylation at threonine 308 (Akt-pT308) in the HEK293.IRS1 cells
(Fig. 1A, lane 2 versus 1). PMA treatment reduced insulin-stimulated Akt phosphorylation (Fig. 1A, lane 3 versus 2).
Although salicylic acid pre-treatment by itself did not affect
insulin-stimulated Akt phosphorylation (Fig. 1A, lane
5 versus 2), it reversed the negative
effects by PMA (Fig. 1A, lane 4 versus
3). Similar observations were made on cells treated with
TNF
(Fig. 1B). Taken together, these results suggest that
PMA and TNF
negatively affect insulin signaling in HEK293.IRS1 cells
and that such negative regulation can be reversed by salicylic
acid.
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Fig. 1.
Salicylic acid reverses PMA and
TNF -induced inhibition of insulin-stimulated Akt
phosphorylation in the HEK293.IRS1 cells. Cells were serum-starved
overnight and treated with vehicle (V), 5 mM
salicylic acid (SA), 2 µM PMA, 6 nM TNF
, and 100 nM insulin (Ins)
as described under "Experimental Procedures." Equivalent amounts of
crude proteins were loaded in each of the lanes. Western blots were
performed using antibodies against total Akt (Akt-t) or Akt
phosphorylated at threonine 308 (Akt-pT308). Similar results
were obtained in at least three experiments for both A and
B. Similar abbreviations are used in all figures.
-induced IRS1 Ser-307
Phosphorylation in HEK293.IRS1 Cells--
Both PMA and TNF
have
been shown to inhibit insulin signaling by promoting IRS1
phosphorylation at multiple serine residues (13, 14, 18-21).
Furthermore, it was reported recently that IRS1 Ser-307 phosphorylation
mediates the inhibitory effects of TNF
on insulin signaling (17,
22). We therefore investigated whether PMA and/or TNF
induce IRS
Ser-307 phosphorylation in the HEK293.IRS1 cells. We further determined
whether salicylic acid could attenuate the effect of PMA or TNF
on
IRS Ser-307 phosphorylation. As shown in Fig.
2A, PMA treatment resulted in ~4-fold increase in IRS1 Ser-307 phosphorylation. Such
phosphorylation was reversed by pre-treatment with salicylic acid.
Similar observations were made using cells treated with TNF
(Fig.
2B). Taken together, these results suggest that PMA and
TNF
induce IRS1 Ser-307 phosphorylation and that such
phosphorylation can be effectively blocked by salicylic acid treatment
in the HEK293.IRS1 cells.
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Fig. 2.
Salicylic acid reverses PMA and
TNF -induced IRS1 phosphorylation at Ser-307 in the HEK293.IRS1
cells. Cells were maintained and treated as described in the Fig.
1 legend. Western blots were performed using antibody against total
IRS1 (IRS1-t) or IRS1 phosphorylated at Ser-307
(IRS1-pS307). Quantification was done using phosphorimaging.
Similar results were obtained in at least three experiments for both
A and B. V, vehicle; SA,
salicylic acid.
/
, the atypical PKC isoforms, in
the HEK293.IRS1 cells (Fig. 3,
bottom panel). Although PMA did not appear to activate classical PKC
/
II (Fig. 3, second panel from
top), it activated novel PKCµ, PKC
, and PKC
(Fig.
3, third through ninth
panels from top). Pre-treatment with
salicylic acid did not appear to affect PMA-mediated activation of any
of these PKC isoforms. Taken together, these results suggest that
salicylic acid did not affect PMA-mediated PKC activation.
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Fig. 3.
Salicylic acid does not affect PMA-mediated
activation of multiple isoforms of PKCs in the HEK293.IRS1 cells.
Cells were maintained and treated as described in the Fig. 1 legend.
Western blots were performed using antibodies against total
(t) or phosphorylated (p) PKC isoforms. It was
noticed that PMA treatment increased both total and phosphorylated
PKC . Similar results were obtained in at least three experiments.
V, vehicle.
Does Not Activate PKC Isoforms, IKK
, or IKK
in the
HEK293.IRS1 Cells--
Some of the PMA-responsive PKC isoforms
(i.e. PKC
and PKC
) have been implicated in the
activation of IKK
(35-37). It has also been reported that TNF
activates IKK
(2). We decided to investigate whether PMA and/or
TNF
activate IKK
in the HEK293.IRS1 cells and, if so, whether
such activation could be reversed by salicylic acid. To this end, we
determined the activation of IKK
and IKK
using an antibody that
specifically recognizes both IKK
phosphorylated at serine 180 and
IKK
phosphorylated at serine 181. Phosphorylation of serine 176 and
180 in IKK
(177 and 181 in IKK
) are required for activation of
the kinases (38). In fact, for IKK
there is full correspondence
between its kinase activity and its Ser-177 and Ser-181 phosphorylation
(39).
and IKK
(Fig. 4,
top three panels, lane 3 versus 1). In contrast, TNF
stimulation did
not result in the phosphorylation of either IKK
or IKK
(Fig. 4,
top three panels, lane 2 versus 1). In addition, TNF
treatment did not activate PKC isoforms based on Western blot using an antibody against
phosphorylated pan-PKC (Fig. 4, bottom panel,
lane 2 versus 1) or antibodies against
phosphorylated individual PKC isoforms (data not shown). The effect of
PMA to induce IKK
and IKK
phosphorylation implies that
these two IKKs are functional in these cells. Because the same
concentration of TNF
that was sufficient to impair insulin signaling
did not activate either IKK
or IKK
, it appears that IKK
is not
involved in TNF
-mediated intracellular signaling in these cells.
Furthermore, these results exclude the possibility that the salicylic
acid-elicited blockade of TNF
-induced insulin resistance is mediated
via IKK
in these cells.
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Fig. 4.
PMA but not TNF treatments activate
IKK
in the HEK293.IRS1 cells. Cells were
maintained and treated as described in Fig. 1. Western blots were
performed using antibodies against total IKK
(IKK
-t),
total IKK
(IKK
-t), both IKK
and IKK
that were
serine phosphorylated (IKK
/
-p), and phosphorylated PKC
isoforms (pan-PKC-p). Similar results were obtained in at
least three independent experiments. V, vehicle.
and May Be Important for PMA and
TNF
-induced IRS1 Ser-307 Phosphorylation in the HEK293.IRS1
Cells--
JNK has been reported to phosphorylate IRS1 at serine 307 and inhibit insulin signaling (17, 22). We performed experiments to
address whether JNK was activated by PMA and/or TNF
in the HEK293.IRS1 cells. As shown in Fig.
5A, PMA treatment leads to phosphorylation (and activation) of the 54- and 46-kDa isoforms of JNK.
A similar observation was made about TNF
-treated cells (Fig.
5B). Taken together, these results demonstrated that both PMA and TNF
treatments in the HEK293.IRS1 cells activate JNK.
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Fig. 5.
Salicylic acid reverses PMA and
TNF -induced JNK activation in the HEK293.IRS1 cells. Cells were
maintained and treated as described in the Fig. 1 legend. Western blots
were performed using antibodies against total JNK (JNK-t)
and phosphorylated and activated JNK (JNK-p) (A)
and total p42/44 MAPK kinase (p42/44-t) and phosphorylated
and activated p42/44 (p42/44-p) (B).
Quantification was done using phosphorimaging. Similar results were
obtained in two experiments. V, vehicle; SA,
salicylic acid.
-mediated effects on insulin signaling. To address this question,
we determined the effect of SP600125, a JNK small molecular inhibitor
(40), on PMA- and TNF
-induced IRS1 Ser-307 phosphorylation. It was
reported that, at 5µM, SP600125 inhibits JNK activity in intact hemopoietic cells (40). We first determined whether SP600125 inhibits JNK activity in the HEK293.IRS1 cells. As shown in Fig. 6, SP600125 did not affect JNK
phosphorylation (and activation) upon both TNF
(Fig. 6B,
third panel from top,
lanes 5 and 6 versus 3 and
4) and PMA treatment (data not shown). On the other hand, SP600125 inhibited phosphorylation of the transcription factor c-Jun, a
physiological substrate of JNK, upon PMA treatment (Fig. 6A,
third panel from top, lanes 5 and 6 versus 3 and 4). We had difficulty
detecting TNF
-induced c-Jun phosphorylation (data not shown). Taken
together, these results confirm that SP600125 indeed inhibits the
kinase activity but not the activation process of JNK in the
HEK293.IRS1 cells.
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Fig. 6.
SP600125, a JNK inhibitor, reverses PMA and
TNF -induced phosphorylation of IRS1 at serine 307 in the
HEK293.IRS1cell. A and B, cells were
maintained and treated with vehicle (V), 5 µM
SP600125 (SP), PMA, and TNF
as described in the Fig. 1
legend. Western blots were performed using antibodies against total
and/or phosphorylated IRS1, JNK, c-Jun, p42/44 MAPK, p38 MAPK, IKK
,
and IKK
. The level of total c-Jun, p42/44, p38 MAPK, IKK
, and
IKK
were similar across all treatments (data not shown). The levels
of total c-Jun, p42/44, p38 MAPK, IKK
, and IKK
were similar
across all treatments (data not shown). C, cells were
treated with vehicle (V) alone (left lane), 100 nM insulin alone (center lane), or pretreated
with 5 µM SP600125 (SP) and then stimulated
with 100 mM insulin (right lane). Western blots
were performed using antibodies against total IRS-1, total Akt, and
phosphorylated Akt as described in the Figs. 1 and 2 legends. Western
blots were also performed to determine total insulin receptor
(IR) using a rabbit polyclonal antibody against the insulin
receptor
subunit and determine the tyrosine-phosphorylated insulin
receptor (IR-pTyr) and IRS1 (IRS1-pTyr) using the
mouse monoclonal antibody 4G10. The level of total Akt, IRS1, and
insulin receptor were similar across all treatments (data not shown).
Similar results were obtained in at least two experiments for
A, B, and C.
, IKK
, and PKC isoforms (including
PKC
) (IC50 of more than 10 µM), indicating
a high degree of JNK selectivity for SP600125 (40). Furthermore, it was
also reported that at 5 µM or less the effect of SP600125
on JNK activity is specific because SP600125 does not affect the
activity or activation of other serine/threonine kinases in the intact
hemopoietic cells (40). We decided to confirm such specificity in the
HEK293.IRS1 cells. Indeed, SP600125 did not appear to affect
PMA-induced activating phosphorylation of IKK
and IKK
(Fig.
6A, bottom panel, lanes 5 and 6 versus 3 and 4).
SP600125 treatment also did not affect PMA- (data not shown) and
TNF
-induced activation of both p42/44 MAPK kinases (Fig.
6B, fourth panel from top, lanes 5 and 6 versus 3 and 4) and p38
MAPK (Fig. 6B, bottom panel,
lanes 5 and 6 versus 3 and
4). Taken together, these results are consistent with the
notion that at 5 µM SP600125 specifically inhibits JNK activity in the HEK293.IRS1 cells (see more in Fig. 6C and
the corresponding discussion for additional nonspecific activity).
-induced IRS1 Ser-307 phosphorylation. The
results indicate that SP600125 treatment prevented IRS1 Ser-307 phosphorylation induced by both PMA (Fig. 6A, top
two panels, lanes 5 and 6 versus 3 and 4) and TNF
(Fig.
6B, top two panels, lanes 5 and 6 versus 3 and
4). Taken together, these results suggest that JNK is
activated by PMA and TNF
and may be important for PMA and
TNF
-induced IRS1 Ser-307 phosphorylation.
--
mediated JNK
Activation
The above results suggest that JNK was required for PMA-
and TNF
-mediated IRS1 Ser-307 phosphorylation and insulin
resistance. A key question was whether the effects of salicylic acid on
insulin signaling, including IRS1 Ser-307 phosphorylation, were
mediated by the inhibition of JNK. Thus, we determined whether
salicylic acid affected JNK activation by PMA and TNF
. As shown in
Fig. 5, salicylic acid significantly inhibited the activating
phosphorylation of both the 54- and 46-kDa JNK induced by both PMA
(Fig. 5A) and TNF
(Fig. 5B). Furthermore, the
inhibition of JNK phosphorylation by salicylic acid appeared to be
specific because salicylic acid did not inhibit the activating phosphorylation of p42/44 MAP kinases induced by both PMA (Fig. 5A, bottom two panels,
lanes 5 and 6 versus 3 and
4) and TNF
(data not shown). Salicylate acid also did not
affect the activating phosphorylation of the p38 MAPK kinases by PMA
and TNF
(data not shown). Taken together, these results suggest that
salicylic acid specifically inhibits JNK activation by PMA and
TNF
.
Subunit--
To further
demonstrate that JNK plays a negative role in insulin signaling and
that its activation is required for PMA and TNF
-mediated insulin
resistance, we decided to determine whether SP600125 can reverse PMA
and TNF
-mediated inhibition on insulin signaling. Rather
surprisingly, however, we found that SP600125 by itself completely
inhibited insulin-stimulated threonine phosphorylation of Akt (Fig.
6C, top panel) as well as tyrosine
phosphorylation of IRS-1 (middle panel) and the
insulin receptor
subunit (bottom panel).
These results suggest that SP600125 has some nonspecific activity that
has not been reported previously (40). Unfortunately, such nonspecific
activity excludes us from determining whether SP600126 treatments can
reverse PMA- and TNF
-mediated inhibition of insulin signaling.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
inhibited insulin-stimulated activating phosphorylation of Akt
and promoted IRS1 Ser-307 phosphorylation. Second, the effects of PMA
and TNF
on Akt phosphorylation and IRS1 serine phosphorylation were
reversed by salicylic acid pretreatment. Third, PMA treatment led to
activation of multiple serine/threonine kinases including PKC isoforms
and IKK
; however, such activation was not affected by
salicylic acid. Fourth, TNF
treatment did not active either PKC
isoforms or IKK
. Fifth, JNK was activated by both TNF
and PMA,
and its inhibition by a small molecule inhibitor prevented TNF
- and
PMA-induced IRS1 Ser-307 phosphorylation. The small molecular
inhibitor, however, has some additional nonspecific activity. Finally,
salicylic acid also inhibited TNF
- and PMA-induced activating
phosphorylation of JNK.
and
salicylic acid in inhibiting or augmenting insulin signaling, respectively. In our experimental system, PMA and TNF
were chosen to
induce insulin resistance for several reasons. First, the negative effects of PMA and TNF
on insulin signaling are well documented, at
least in cultured cells in vitro (11, 14, 18, 19, 27-29, 41). Second, IKK
is a well established downstream kinase for TNF
(2). It has also been shown that IKK
can be activated by PKC
isoforms (i.e. PKC
and PKC
) (35-37). In fact, it was
recently reported that PKC
deficiency in mice impaired IKK
activation (37).
(2), TNF
has also been shown to
activate PKC in multiple cell types (42-46). It was therefore rather
surprising that TNF
did not activate IKK
, IKK
, and the
multiple PKC isoforms examined in the HEK293.IRS1 cells (Fig. 4). Based
on these observations, we conclude that in the HEK293.IRS1 cells IKK
is intact but is not involved in TNF
signaling. Therefore, the lack
of IKK
activation by TNF
in the HEK293.IRS1 cells provided us
with a unique setting to determine whether IKK
activation and its
inhibition are involved and/or required for salicylate-mediated insulin
sensitization. It then follows that the effect of TNF
on insulin
signaling in the HEK293.IRS1 cells is independent of IKK
activation.
Because salicylate was readily able to reverse TNF
-induced effects
on insulin signaling, it also appears that these effects of the drug must be also IKK
-independent. In contrast, the current study does
not provide sufficient evidence to assess whether the action of
salicylic acid to reverse the effects of PMA on insulin action is
mediated via IKK
inhibition.
? We considered that salicylic acid might
improve insulin sensitivity by inhibiting kinase(s) other than IKK
,
which were responsive to both PMA and TNF
. To this end, we examined
the serine/threonine kinase JNK. JNK has been shown to be activated by
both PMA (47-49) and TNF
(50-52). Most interestingly, TNF
was
recently shown to inhibit insulin signaling by activating JNK, which
subsequently becomes associated with Ser-307 in IRS1 and phosphorylates
it (22).
-induced insulin
resistance and its reversal by salicylic acid, we performed a separate
series of experiments and made several interesting observations. First,
by demonstrating that both PMA and TNF
treatment induced activating
phosphorylation of JNK (Fig. 5), we confirmed that JNK is involved in
signaling in response to both PMA and TNF
in the HEK 293 cells.
Second, by showing that SP600125 inhibits PMA- and
TNF
-induced IRS1 Ser-307 phosphorylation, we propose that JNK activity may be important for the effects of PMA and TNF
on
insulin signaling in these cells (see more discussion later on the
nonspecific activity of SP600125). Finally, we showed that salicylic
acid inhibits PMA- and TNF
-induced JNK activating phosphorylation
(Fig. 6). Thus, these observations suggest that JNK is activated by
(and its activity may be important for) PMA- and TNF
-induced insulin
resistance. Most interestingly, the results also suggest that salicylic
acid improves insulin sensitivity potentially by inhibiting JNK activation.
-induced insulin
resistance in the HEK293.IRS1 cells. In short, we propose that JNK may
be directly responsible for IRS1 Ser-307 phosphorylation. IRS1
phosphorylated at Ser-307 has an impaired ability to undergo tyrosine
phosphorylation by insulin receptor, leading to insulin resistance as
reflected in reduced Akt phosphorylation (and activation). Salicylic
acid reversed the effects of PMA and TNF
in association with reduced JNK activation. Although the detailed mechanism by which salicylic acid
inhibits JNK activation in these cells remains unknown, it is most
likely to be IKK
-independent. Although both PMA and TNF
treatment
lead to JNK activation, the signaling pathways were apparently
different. PMA activates PKC isoforms as well IKK kinases. TNF
did
not activate either. Nevertheless, salicylic acid appears to inhibit
JNK activation by both PMA and TNF
.
View larger version (12K):
[in a new window]
Fig. 7.
Salicylic acid reverses
TNF -induced insulin resistance by an IKK
-independent
fashion, probably by inhibiting JNK activation. Shown is a
schematic that summarizes the results from the current experiments and
depicts our proposed mechanism(s) by which salicylic acid promotes
insulin signaling. IRS1-pY, tyrosine phosphorylated IRS1.
Solid arrow, direct interaction.
Dotted line, interaction with known or unknown
intermediate steps. The thickness of an arrow
represents the rate of signaling events.
In summary, the results from the current study suggest that IKK
inhibition is not necessarily critical to the mechanism(s) for
salicylate-mediated insulin sensitization in the TNF
treatment paradigm. One approach to further validate our results would be to
determine whether PMA and TNF
can induce IRS1 Ser-307
phosphorylation in IKK
-deficient cells and, if so, whether the
effects by PMA and TNF
can be reversed by salicylic acid. However,
it also remains to be established that the antidiabetic effects of
salicylate in vivo are directly tied to inhibition of
IKK
. Nevertheless, the apparent existence of IKK
-independent
alternative pathway(s) underscores the need for additional knowledge
regarding the mechanism(s) of salicylate-mediated insulin
sensitization
Finally, although the current study suggests that JNK activation plays
an important role in PMA and TNF-mediated insulin resistance and
that its inhibition may be responsible for salicylic acid-mediated
insulin sensitization, such a proposal needs further validation because
some of the results were derived from the use of the JNK inhibitor
SP600125. We found that although SP600125 does not affect PMA- or
TNF
-induced activation of several kinases (Fig. 6,
A and B), it inhibits insulin signaling (Fig. 6C), indicating that the inhibitor may not be as specific as
originally reported (40). One approach to further test the current
theory would be to determine whether PMA and TNF
induce IRS1 Ser-307 phosphorylation and inhibit insulin signaling in JNK-deficient cells.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed: Metabolic
Disorders-Diabetes, Merck Research Laboratories, RY80N-C31, P.O. Box
2000, Rahway, NJ 07065. Tel.: 732-594-2176; Fax: 732-594-3925; E-mail: guoqiang_jiang@merck.com or bei_zhang{at}merck.com.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M205565200
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ABBREVIATIONS |
---|
The abbreviations used are:
IKK, IB kinase;
IRS, insulin receptor substrate;
JNK, c-Jun N-terminal kinase;
TNF
, tumor necrosis factor
;
MAP, mitogen-activated protein;
MAPK, MAP
kinase;
PMA, phorbol 12-myristate 13-acetate;
HEK293, human embryonic
kidney 293;
PKC, protein kinase C.
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