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J. Biol. Chem., Vol. 277, Issue 50, 48565-48573, December 13, 2002
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From the Department of Pathology, Division of Molecular and
Cellular Pathology, University of Alabama,
Birmingham, Alabama 35294
Received for publication, August 1, 2002
Insulin regulates metabolic activity, gene
transcription, and cell growth by modulating the activity of several
intracellular signaling pathways. Insulin activation of one
mitogen-activated protein kinase cascade, the MEK/ERK kinase
cascade, is well described. However, the effect of insulin on the
parallel p38 pathway is less well understood. The present work examines
the effect of inhibiting the p38 signaling pathway by use of specific
inhibitors, either alone or in combination with insulin, on the
activation of ERK1/2 and on the regulation of gene transcription in rat
hepatoma cells. Activation of ERK1/2 was induced by insulin and was
dependent on the activation of MEK1, the kinase upstream of ERK in this pathway. Treatment of cells with p38 inhibitors also induced ERK1/2 activation/phosphorylation. The addition of p38 inhibitors followed by
insulin addition resulted in a greater than additive activation of
ERK1/2. The two genes studied, c-Fos and Pip92, are immediate-early genes that are dependent on the ERK1/2 pathway for insulin-regulated induction because the insulin effect was inhibited by pretreatment with
a MEK1 inhibitor. The addition of p38 inhibitors induced transcription
of both genes in a dose-dependent manner, and insulin stimulation of both genes was enhanced by prior treatment with p38
inhibitors. The ability of the p38 inhibitors to induce ERK1/2 and gene
transcription, both alone and in combination with insulin, was
abolished by prior inhibition of MEK1. These data suggest possible
cross-talk between the p38 and ERK1/2 signaling pathways and a
potential role of p38 in insulin signaling.
Insulin is a primary regulator of energy metabolism, is essential
for normal growth and development, and can directly or indirectly stimulate DNA synthesis (1). Upon binding of insulin, the insulin receptor becomes activated, resulting in its phosphorylation and the
phosphorylation of insulin receptor substrate (IRS) proteins. The growth factor binding protein 2 (Grb2)-son of sevenless complex (SOS) can then be recruited directly along with Shc or via insulin receptor substrate 1 or 2 (2). Usually, this leads to activation of
Ras, Raf, and the MEK1 kinase
cascade, resulting in phosphorylation and activation of ERK1/2 (3, 4).
A parallel MAPK signaling pathway is the mixed lineage kinase/apoptosis
signaling kinase, MAPK kinase 3/6, p38 kinase cascade. The effects of
insulin on the p38 pathway are inconsistent. In fetal neurons, insulin
can inhibit the p38 pathway as well as apoptosis (5). Conversely, in
cultured myoblasts, insulin can induce p38 activity and is required for
cytoskeletal rearrangement and myoblast differentiation (6, 7). Recent studies have indicated that insulin stimulates p38 activity in L6
myotubes and that this activation is important for insulin-induced glucose transport (8-11).
Following fasting, the liver is the major source of plasma glucose, and
hepatic glucose output is tightly regulated by insulin (12). Recently,
the significance of hepatic insulin signaling was elegantly
demonstrated in liver-specific insulin receptor knockout mice. These
animals demonstrated severe hyperglycemia in the presence of dramatic
hyperinsulinemia (13). The minimal deviation hepatoma cell line H35
(and its derivatives Fao as well as the H4IIE cells used in the present
studies) has long been used as a model for the effect of insulin on
intracellular signaling and hepatic gene expression (14, 15). These
cells express receptors that bind insulin with a similar affinity to
isolated rat hepatocytes and adipocytes, and insulin induces glycogen
synthesis at physiologic concentrations (16). Further, as in the liver, in these cell lines insulin regulates the transcription, mRNA levels, and activity of gluconeogenic enzymes such as
phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and ornithine
decarboxylase (17-24).
Aside from its metabolic role, another important action of insulin is
promoting growth. The induction of gene expression, including several
immediate-early genes, may be important in cell cycle progression (25,
26). The proto-oncogene c-Fos is an early response gene and a member of
a family of genes that, in combination with c-Jun and its family
members, binds to AP-1 sites. In addition to insulin, c-Fos is induced
by a variety of stimuli including a number of mitogens (26-28).
Pip92 is a proline-rich cytoplasmic protein that shares little homology
to any known proteins but is another regulated immediate-early gene.
Its mRNA is induced by serum in fibroblasts and pheochromocytoma cells (29, 30) and is increased in activated T-cells, and differentiated HL-60 cells (31, 32). We have previously shown that
insulin regulates Pip92 mRNA abundance in rat hepatoma cells (33).
Studies examining the mechanism of regulation of the Pip92 and c-Fos
genes found that their induction in response to stimuli is dependent
upon a serum-responsive element, in addition to other elements, within
each gene (34, 35).
Little is known about interactions between the p38 and ERK1/2 MAPK
signaling pathways. A few recent reports have suggested that some
cross-talk may occur but with variable results. The data published to
date indicate that any interplay between these two MAPK pathways may be
specific to the cell type and the particular stimulus. Even less is known about the effects of the p38 pathway on
insulin activation of ERK1/2. The present work describes cross-talk between the insulin-activated MEK/ERK pathway and the p38 pathway and
the effects of the cross-talk on expression of two insulin-regulated immediate-early genes, c-Fos and Pip92. We identify the MEK/ERK pathway
as a necessary component of insulin induction of these genes and
demonstrate the ability of p38 inhibitors to modulate MEK/ERK signaling
and insulin- and MEK/ERK-dependent gene expression.
Cell Culture--
Rat H4IIE hepatoma cells were obtained from
ATCC (Manassas, VA) and maintained at 37 °C and 5% CO2
in Swim's 77 medium (Sigma-Aldrich) supplemented with 2% fetal bovine
serum, 3% calf serum, 5% horse serum (Invitrogen), 100 units/ml
penicillin, and 100 µg/ml streptomycin (Mediatech, Herndon, VA).
Serum was withdrawn from subconfluent cultures 24-48 h prior to
experimental treatments. All of the insulin treatments (Sigma) were 10 nM. SB202190, SB202474, and SB203580 (Calbiochem, San
Diego, CA) were used at 2 and 10 µM. PD98059 (50 µM) was obtained from Cell Signaling Technology (Beverly, MA), as were phosphorylated (active) ERK1/2, total ERK, and secondary anti-rabbit horseradish peroxidase-conjugated antisera.
Gene Expression--
The transcription rates were assayed
by the nuclear run-on method (36). Briefly, transcriptionally active
nuclei were labeled with [32P]UTP (ICN, Costa Mesa, CA),
RNA-isolated, hybridized with cDNA spotted on nitrocellulose, and
autoradiographed. Densitometric data from autoradiographs were analyzed
using ZeroD Scan from Scanalytics. The rat Pip92 cDNA was cloned
from a library of insulin-induced genes as previously described (33),
and the complete murine c-Fos cDNA in pBR322 was obtained from the
ATCC (pc-fos3).
Total and Active ERK1/2 Measurements--
Serum-deprived
cultures were washed with 37 °C phosphate-buffered saline. Whole
cell (SDS) lysates were prepared as previously reported (37) and were
assayed for protein content using the Bio-Rad DC assay. The lysates (50 µg) were resolved by discontinuous polyacrylamide gel electrophoresis
with a 10% resolving gel (38) using the Bio-Rad MiniProtean II system
and transferred to S & S Protran BA85 membranes using the Mini
Transblot apparatus (39, 40). Total proteins or phosphoproteins were
visualized by ECL Plus (Amersham Biosciences) reagents and
autoradiography or digital image recording (41). Using the
AlphaInnotech Fluorchem FC imager, sequential images of the
chemiluminescent membranes were recorded. The range of emission
intensities between faint bands and bright bands detected provides a
linear data relationship with antigen concentration (42-46). Only
bands in the linear optical density (O.D.) range were analyzed, and
those beyond this range in each exposure were identified and rejected
by the onboard software. Comparison of experimental bands to control
bands of intermediate intensity allowed a broad range of signal
intensities to be analyzed. Densitometric data from three to six
experiments were averaged.
In Vitro Kinase Assays--
Nonradioactive kinase assay kits
were obtained from Cell Signaling Technology and used according to the
supplied protocols. Briefly, p38 and ERK kinases were purified by
immunoprecipitation with specific immobilized antiserum from 0.5-1.0
mg of soluble cell lysate by standard methods. The immune complex was
washed two times in lysis buffer (1% Triton X-100, 10% glycerol, 20 mM Tris, pH 7.5, 150 mM NaCl, 7.5 µg/ml
aprotonin, 5 mM benzamidine, 5 mM
phenylmethylsulfonyl fluoride, 50 mM NaF, and 0.125 mM NaVaO4) and then twice in kinase buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM Statistical Analysis--
Analysis of variance and Student's
t tests were performed using the Instat program (Graphpad
Software Inc., San Diego, CA). The values from the experimental
treatments were expressed as fold change compared with the
vehicle-treated controls within each experiment. The relative strength
of correlations was calculated using the Spearman rank correlation
coefficient (rs).
Insulin, MEK/ERK, and p38 Signaling Cross-talk--
To directly
characterize the intracellular signaling pathways activated by insulin
in rat hepatoma cells, Western blots were performed using antisera to
total and phosphorylated ERK1/2 (P-ERK1/2). As expected, treatment of
H4IIE cells with insulin caused a large and rapid increase in
phosphorylation of both ERK1 and ERK2 (Fig. 1A, upper panel).
Pretreatment of cells with the MEK 1 inhibitor (PD98059 (PD)) abolished
the insulin effect on ERK1/2 phosphorylation at all time points tested
(Fig. 1A).
Studies were performed to investigate the cross-talk between the
MEK/ERK and p38 signaling pathways. To determine whether regulation of
p38 was involved in acute insulin activation of ERK1/2, serum deprived
cells were pretreated with the p38 inhibitor SB202190 (SB) at
concentrations of 2 or 10 µM over a period of 5-270 min
and compared with control (vehicle-treated) cells. Western blots
revealed that, by itself, 2 µM SB, and to a greater
extent 10 µM SB, induced ERK1/2 activation in a
time-dependent manner (P-ERK1/2; Fig. 1, B and
C). The levels of total ERK1/2 immunoreactive protein were
not significantly or consistently altered by any of the treatments
(Fig. 1, A-C, lower panels), indicating that changes in P-ERK1/2 bands reflected changes in the fraction of activated kinase. The results of three or more experiments with each
concentration and at each time of SB treatment were compiled and
expressed as the average fold increases of band intensity compared with
untreated control cell lysate within each experiment (Fig.
1D). Treatment with 2 µM SB resulted in a
maximum of 15.6-fold induction of P-ERK1/2 over control by 30 min
compared with 33-fold above control by 60 min in cells treated with 10 µM SB. For both concentrations of SB, P-ERK1/2 remained
elevated above control levels even at 150 and 270 min, the longest time
points tested.
Studies were performed to ensure that our measurements of P-ERK1/2 by
Western blot were linear within the range of proteins used and the
amount of P-ERK1/2 generated. Using a highly sensitive digital video
camera, chemiluminescent Western blots were imaged directly. Over a
1-30-fold range, P-ERK1/2 band intensity in Western blots correlated
with whole cell lysate concentration and, thus, P-ERK1/2 concentration
in a linear fashion (Fig. 1E).
In addition, to ensure that insulin-induced phosphorylation of ERK1/2
correlated well with ERK activity, we performed ERK1/2 kinase assays
(Fig. 1F). We found an identical pattern of ERK1/2 activity
compared with ERK1/2 dual phosphorylation (P-ERK1/2) as described
previously (50, 51). Because there was such consistency between
P-ERK1/2 and ERK1/2 activity, the simpler (P-ERK1/2) assay was used
throughout the remainder of the studies.
To examine whether the p38 inhibitor alters the ability of insulin to
activate ERK1/2, H4IIE cells were pretreated with either 2 or 10 µM SB for 30 min. The cells were then either harvested immediately or treated with insulin for 5-240 min. Pretreatment with
SB alone caused the expected, concentration-dependent large increase in P-ERK1/2. In addition, the induction of P-ERK1/2 was greatly augmented by insulin treatment (Fig.
2, A and B).
Insulin treatment alone caused a maximum increase in ERK1/2
phosphorylation of ~28-fold by 5 min followed by a decrease to a
plateau of around 2-3-fold above control levels between 120 and 240 min (Fig. 2C, left panel).
Pretreatment with the lower concentration of SB (2 µM)
caused an approximate doubling of the maximum level of insulin-induced P-ERK1/2 (28 versus 59-fold), and the higher
concentration of SB (10 µM) caused an ~4-fold increase
in maximum ERK1/2 phosphorylation compared with insulin alone (110-fold
above serum free control). These increases in response to insulin are
in addition to the elevated P-ERK1/2 at the beginning of insulin
treatment that results from the 30-min incubation with SB. For
comparison, in the right panel of Fig. 2C is the
effect of SB alone (replicated from Fig. 1C, but on the
expanded scale necessary for these experiments) or the combination of
SB and insulin as presented in the left panel. Regulation of
ERK activity in in vitro kinase assays parallels the
induction of P-ERK1/2 in H4IIE cells treated with SB alone or prior to
insulin addition (Fig. 2D).
This indicates a greater than additive effect of 2 and 10 µM SB in combination with insulin to induce P-ERK1/2 as
compared with the effects of the agents added separately (Fig.
2C). For example, if the effects are compared (treatment
with SB 2 µM plus insulin versus the addition
of the effects of SB 2 µM alone plus the effects of
insulin alone), at 5, 15, and 120 min, there were 42, 122, and 266%
increases above additivity, respectively, for induction of P-ERK1/2,
suggestive of a synergistic effect of the combination of low dose SB
and insulin. The effect of pretreatment with the higher dose of SB (10 µM), followed by insulin addition resulted in 81, 161, and 150% increases above additivity for induction of P-ERK1/2 at 5, 15, and 120 min, respectively, above the simple addition of the effects
of 10 µM SB alone and insulin alone. Throughout all of
these experiments, there were no significant changes in the amount of
total ERK1/2 protein, indicating that changes in the phosphorylation of
ERK1/2 reflected changes in the fraction of activated kinase (Fig. 2,
A and B, lower panels).
To ensure that the effects of p38 inhibition by SB are not a unique
feature of this compound, a separate but chemically similar inhibitor
was also tested. The distinct inhibitor, SB203580, was also found to
induce ERK1/2 phosphorylation when added alone (Fig. 3A, lane 1).
SB203580 also acted synergistically with insulin to increase ERK1/2
activation (Fig. 3A, lanes 2-4). As a further control, a structurally related but inactive compound, SB202474, which
does not inhibit p38 kinase activity, was also tested on H4IIE cells
and was unable to increase ERK1/2 phosphorylation on its own or in
combination with insulin treatment (Fig. 3B).
Insulin, MEK/ERK, and p38 Regulation of Gene Expression--
To
examine the effects of activation of the ERK1/2 pathway and the
possible cross-talk of the ERK and p38 pathways in insulin regulation
of gene expression, nuclear run-on assays were performed. H4IIE cells
were either not treated or treated with 10 nM insulin, and
the transcription rate of c-Fos and Pip92 was measured. In addition,
other cells were pretreated either with vehicle (control) or with the
MEK1 inhibitor (PD) to inhibit signaling through the MEK1/ERK1/2
pathway. As we have previously reported (27, 33), following 30 min of
insulin treatment, there were significant increases in transcription of
both genes (Fig. 4). There was no significant effect on the transcription of either gene when cells were
treated with PD alone (50 µM). In cells pretreated with
PD, the effects of insulin on c-Fos and Pip92 were reduced ~70-80%, to levels not significantly different from basal (control) levels of
transcription. These results indicate that MEK1, and most likely ERK1/2
activation, is necessary for the full effect of insulin-mediated induction of both c-Fos and Pip92 genes. A control gene,
We then determined whether regulation of p38 could effect
insulin-mediated regulation of c-Fos and Pip92 transcription.
Serum-deprived cells were pretreated with the p38 inhibitor (SB) at
concentrations of 2 or 10 µM and compared with vehicle
control. Following a pretreatment period, the cells were stimulated
acutely with 10 nM insulin for 30 min or left untreated.
Low dose (2 µM) SB alone increased c-Fos transcription
5.5-fold while having a slight but nonsignificant effect on Pip92 (Fig.
5A; insulin data from Fig.
4B added to this graph for comparison). Pretreatment with 2 µM SB followed by stimulation with insulin for 30 min
resulted in a 26.5-fold increase in c-Fos and a 12.2-fold induction of
Pip92 transcription (Fig. 5A). For the two induced genes,
the effect of 2 µM SB alone was less than that of insulin
(48% for c-Fos and 21% for Pip92), whereas the combination of insulin
and SB on transcription is greater than additive. If the effects are
compared (treatment with SB 2 µM plus insulin
versus the addition of the effects of SB 2 µM
alone plus the effects of insulin alone), there was a 55% increase
above additivity for c-Fos and a 61% increase above additivity for
Pip92, suggestive of a synergistic effect (Fig. 5A), similar
to that found in P-ERK1/2 induction. There was no significant effect of any of these treatments on transcription of the control gene,
The higher dose of SB (10 µM) by itself caused a dramatic
36-fold increase in c-Fos transcription, much larger than the 11.5-fold effect of insulin alone (the insulin data is again shown in Fig. 5B for comparison). Unlike the lower dose of SB, which was
ineffective, the 10 µM concentration of SB induced Pip92
transcription 5.5-fold, similar to the 6.3-fold effect of insulin
alone. When H4IIE cells were pretreated with the higher dose of SB and
were then exposed to insulin for 30 min, there was no further increase
in c-Fos transcription compared with 10 µM SB alone.
However, this regimen resulted in an induction of Pip92 transcription
that is 83% larger (21.5-fold) than the sum of the effects of 10 µM SB alone (5.5-fold) and insulin alone (6.3-fold),
again suggesting a synergistic interaction of insulin and SB. The
synergistic effects of SB and insulin on gene transcription coincided
well with the synergistic effects of the p38 inhibitor and insulin on
the induction of P-ERK1/2.
Previous studies from our laboratory (27) have shown that other potent
inducers or combinations of inducers of immediate-early gene expression
cause a maximum induction of 35-45-fold in c-Fos transcription in
H4IIE cells and could never induce transcription beyond the 35-45-fold
that is also observed in the present experiments. This suggests that
the increase in transcription induced by 10 µM SB (alone)
may achieve the maximum possible induction of c-Fos transcription in
H4IIE cells. Thus, the combination of SB and insulin could not cause a
further increase.
It is clear that the addition of SB can increase phosphorylation of
ERK1/2 and result in the increased transcription of insulin-regulated, ERK-dependent genes. Additionally, there is a synergistic
induction of P-ERK1/2 and the ERK-dependent genes, c-Fos
and Pip92, when SB and insulin are combined. However, whether a common
pathway was used by both insulin and p38 inhibitors to activate ERK1/2 and subsequently induce c-Fos and Pip92 expression was tested by again
using the MEK1 inhibitor, PD98059. After 30 min of pretreatment with
PD, the cells were treated or not for a further 30 min with SB. Greatly
overexposed Western blots (Fig. 5C) indicated that the MEK1
inhibitor completely blocked SB-induced increases in ERK1/2
phosphorylation, as well as slightly reducing basal phosphorylation, suggesting that MEK1 activity is required for the activation of ERK1/2
by the p38 inhibitor.
When the effects of MEK1 inhibition were studied on the
stimulation of c-Fos and Pip92 expression, there was an inhibition of
the SB (10 µM) induction of both genes by PD pretreatment
(Fig. 5B). In addition, when cells were pretreated with PD
prior to the combination of SB and insulin, the large induction of both c-Fos and Pip92 transcription were completely inhibited (Fig. 5B). This suggests that SB induces transcription of these
ERK1/2-dependent genes, acting at the level of MEK1 or at a
point in the insulin signaling pathway upstream of MEK1.
The similarities in the induction of ERK1/2 activation/phosphorylation
and transcriptional regulation of both c-Fos and Pip92 was striking. To
further examine the relationship between these two variables, the
correlation was quantitated (52). Linear regression lines were obtained
for induction of each gene (represented as the y variable)
and the corresponding level of P-ERK induction (represented as the
x variable) for each treatment described. The slopes of the
lines obtained for c-Fos and Pip92 were 0.32 and 0.21, respectively
(Fig. 6). The strengths of the
correlation described by these regression lines
(rs = 0.9084 and 0.8772, respectively) and their
significance (p < 0.0001) (53) further support the idea that insulin alone, p38 inhibitor alone, or the combination of
treatments all regulate c-Fos and Pip92 via ERK1/2.
We then measured whether insulin had a direct effect on p38 activity in
H4IIE cells. Consistent with our cross-talk hypothesis, basal activity
of p38 was observed in quiescent cells. A modest, but not statistically
significant increase in p38 activity was sustained over a time course
of insulin treatment at all times greater than 5 min (Fig.
7). Small changes in p38 activity in response to insulin are consistent with recent reports of studies in a
cultured skeletal muscle cell line (10).
To study the regulation of insulin signaling, the activation
(phosphorylation) state of ERK1/2 was examined in rat H4IIE hepatoma cells. Insulin treatment alone resulted in a rapid increase in ERK1/2
phosphorylation and activity, whereas pretreatment with the MEK1
inhibitor, PD98059, abolished the effect of insulin on P-ERK1/2 as well
as insulin induction of the c-Fos and Pip92 genes, implying that
insulin control of these genes proceeded primarily through the
MEK1-ERK1/2 pathway.
Our findings agree with work in rat hippocampal neurons indicating that
Raf- or fibroblast growth factor-mediated activation of ERK1/2 via MEK1
is required for the rapid activation of Pip92 transcription (34). In
3T3-L1 adipocytes, insulin induction of a c-Fos reporter construct was
prevented by MEK1 inhibition (54). Thus, MEK/ERK activation is
important for the activation of these genes and, as presented here, is
of specific importance for insulin induction of c-Fos and Pip92 in rat
H4IIE hepatoma cells.
When H4IIE cells were treated with SB alone, there was a
dose-dependent induction of P-ERK1/2 and of c-Fos and Pip92
transcription. Further, when cells pretreated with SB were given a
secondary insulin stimulus, there was a greater-than-additive
activation of ERK1/2 and of c-Fos and Pip92 transcription. It is not
entirely clear why the lower concentration of SB alone is more potent
with respect to transcription of c-Fos than Pip92, but presumably there are other factors involved in c-Fos and Pip92 transcription because they are not uniformly induced in these studies. One possibility is
that Pip92 has a higher "threshold" of ERK1/2 activity necessary for induction than does c-Fos.
The activation of ERK1/2 observed by treatment of cells with the p38
inhibitor SB202190 was also observed when cells were treated with a
different p38 inhibitor, SB203580. The chemically related compound
SB202474, which does not inhibit p38, has no effect on ERK1/2
phosphorylation either alone or in combination with insulin. This
implies that it is the inhibition of p38 itself and not a nonspecific
action of one of the p38 inhibitors that results in induction of
P-ERK1/2.
One could hypothesize that p38 activity inhibits transcription through
a mechanism separate from the ERK1/2 pathway used by insulin in the
regulation of these two genes and that the synergistic actions of the
p38 inhibitors and insulin were due to SB having effects downstream of
ERK1/2 activity. Thus, when p38 was inhibited by SB, this separate
inhibitory pathway would have been neutralized, and the c-Fos and Pip92
genes would be induced to a greater degree. Clearly, this hypothesis
was incorrect because the p38 inhibitors resulted in activation of
ERK1/2 and acted to synergistically induce P-ERK1/2 in combination with insulin.
Another hypothesis initially considered was that basal p38 activity was
responsible for a separate and distinct "off" signal for
transcription. In this model, transcription would be turned on by
insulin, but the off signal would be deficient in the presence of p38
inhibition, and transcription would continue unchecked. This also was
not supported by the evidence, because the p38 inhibitors alone could
induce P-ERK1/2 and transcription of the c-Fos and Pip92 genes, even in
the absence of insulin.
Alternatively, the p38 pathway may have a tonic, inhibitory effect on
MEK/ERK signaling, and inhibition of p38 by SB increased ERK1/2 pathway
activity resulting in induction of ERK1/2-dependent genes.
When a stimulator of the MEK/ERK pathway was added in combination with
the p38 inhibitors, a greater fold induction of P-ERK1/2 and the c-Fos
and Pip92 genes was obtained. This is a potential explanation of the
data obtained in the present work, because the addition of an active,
but not the inactive p38 inhibitor by itself resulted in activation of
ERK1/2 signaling and subsequent transcription of c-Fos and Pip92. Our
data also suggested that the activation occurs at or above the level of
MEK1, because inhibition of MEK1 activity blocked the induction of
transcription and P-ERK1/2 by insulin alone, SB alone, and the
combination of SB and insulin. Conversely we found the basal level of
p38 activity is modestly induced by insulin. The fact that this occurs
with a slightly delayed time course, corresponding to the downward
slope of the peak of insulin-induced ERK1/2 activity, fits well with
the idea that p38 may play a negative regulatory role.
Data regarding the activation of other, parallel, MAPK pathways by
insulin has been somewhat contradictory. However, recent studies have
indicated a major role of the p38 signaling pathway in
insulin-inducible glucose transport. In these studies, insulin induced
p38 activity in L6 myotubes. The p38 inhibitors, SB202190 and SB203580,
reduced insulin-stimulated glucose transport by 40-60% without
altering insulin-induced Akt phosphorylation (8-11). In the present
studies, the same inhibitors activated ERK and worked synergistically
with insulin to stimulate ERK-dependent genes. This
suggests a potential complex role of the p38 signaling pathway in the
multiple actions of insulin. This may also imply a role for p38 that
varies in the different insulin target tissues.
Several recent reports suggest possible cross-talk between the MEK/ERK
and p38 pathways, in ways supportive of our findings. For instance, in
PANC-1, HL-60, L1210, and ECV304 cells, ERK1/2 activity or
phosphorylation is increased by SB203580 or SB202190 (55-58). In human
skin fibroblasts, phorbol ester- and Raf-mediated induction of MEK1 and
ERK1/2 are inhibited when p38 is activated by constitutively active
MAPK kinase 3b, the kinase upstream of p38 (59), and in the human
hepatoma cell line, HepG2, low density lipoprotein receptor mRNA
and P-ERK1/2 are increased by SB treatment and reduced by
constitutively active MAPK kinase 6b, another kinase upstream of p38
(60). These observations suggest a role for the p38 pathway in
MEK-ERK-dependent processes.
However, several other reports obtained results different from those
presented here; in baboon smooth muscle cells and Swiss 3T3 fibroblasts
use of the p38 inhibitor activates Raf, the upstream kinase for MEK1,
but somehow fails to activate MEK1 and ERK1/2 (61, 62). Treatment with
SB203580 did not alter epidermal growth factor-induced ERK activation
in Swiss 3T3 cells or IGF-1-induced ERK activation in L6 myotubes (62),
and in mIMCD3 cells, inhibition of p38 blocks activation of P-ERK1/2 by
hypertonic NaCl treatment (63). Thus, there is extensive heterogeneity
in the nature of the interactions between the MEK-ERK and p38 pathways.
There appear to be cell type differences, differences in interactions
with various stimulators of intracellular signaling, and differences in
the effects of p38 activation or inhibition on the downstream effects
being measured following activation of the MEK-ERK pathway.
The present studies have identified potential cross-talk between the
MEK-ERK and p38 pathways and a role of this interaction in insulin
signaling and insulin action. The modest and slightly delayed effect of
insulin on p38 activity may indicate that it functions to limit ERK1/2
activation and subsequent gene transcription rather than actively
decreasing ERK1/2 activity and transcription. Future studies are
required to identify the target at or upstream of MEK1 in the MEK-ERK
signaling pathway affected by addition of p38 inhibitors, and why these
effects may be tissue-specific. Future experiments will also be needed
to determine whether insulin regulation of other insulin-sensitive
genes is modulated by changes in p38 activity and subsequent changes in
MEK1/ERK signaling.
We thank Drs. S. Frank, S. Ji, and W. Bennett
for helpful and insightful discussions and suggestions.
*
This work was supported by a grant from the American
Diabetes Association and by National Institutes of Health Grant DK40456 (to J. L. M.).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.
Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M207837200
The abbreviations used are:
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
ERK, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
PD, PD098059;
SB, SB202190;
GST, glutathione S-transferase;
P-, phosphorylated.
Insulin Signal Transduction Pathways and Insulin-induced Gene
Expression*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 0.1 mM
Na3VaO4, and 2 mM dithiothreitol). The immune complexes were then incubated at 30 °C for 30 min with 200 µM ATP and 2 µg of the purified recombinant
substrate, GST-ATF-2 or GST-Elk, respectively (47-49). The kinase
reaction was terminated by the addition of 1% SDS loading buffer and
boiling for 5 min. Phosphorylated substrate was detected by Western
blotting with antisera specific for the phosphorylated form of ATF-2 or Elk1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
MEK1-dependent induction of
ERK1/2 phosphorylation and kinase activity by insulin and p38
inhibitors. H4IIE cells were serum-deprived and treated for the
indicated times, and protein was prepared and separated as described
under "Experimental Procedures." A, a representative autoradiograph of a Western blot of whole cell
lysates with the treatments as indicated above the
lanes. Ins, insulin. B and
C, serum-deprived H4IIE cells were treated for the indicated
times with 2 or 10 µM SB. The upper panels
were probed with anti-P-ERK1/2, and the lower panels were
probed with the total ERK1/2 antiserum. D, averages of three
to five separate experiments at each time point with error
bars (S.E.) indicated. The band densitometric intensity for the
controls was arbitrarily set to 1 in each experiment, and other
treatment values were expressed as fold increases versus
control. #, greater than control (p < 0.05); *,
greater than 2 µM (p < 0.05).
E, serial dilutions of identically treated whole cell
lysates were probed with the anti-P-ERK1/2 antiserum (left
panel) and visualized using direct digital imaging. In the
right panel, the band intensity corresponding to each lysate
concentration is plotted on the y axis, and the resultant
curve fit is represented as a solid line connecting each
point. The extrapolated correlation is shown as a dashed
line. F, in vitro ERK kinase assays were
performed on cells treated as indicated. Following incubation with
insulin, the immunoprecipitated ERK1/2 was incubated with GST-Elk, and
P-GST-Elk was detected by Western blot with P-Elk antiserum followed by
direct digital image analysis of the chemiluminescent blot.
View larger version (40K):
[in a new window]
Fig. 2.
Effect of p38 inhibition on induction of
ERK1/2 phosphorylation and activity by insulin. H4IIE cells were
serum-deprived and then left untreated or pretreated for 30 min with 2 µM SB (A) or 10 µM SB
(B) prior to treatment for the indicated times with insulin
(Ins). A and B, representative
autoradiographs of Western blots of whole cell lysates as described
under "Experimental Procedures." The upper panels were
probed with anti-P-ERK1/2, and the lower panels were probed
with the total ERK1/2 antiserum. C, averages of three to
five separate experiments at each time point with error bars
(S.E.). In the left panel, the fold increase in ERK1/2
phosphorylation by insulin treatment alone (from Fig. 1) is compared
with the time course of the combination of SB plus insulin treatments.
In the right panel, ERK1/2 phosphorylation by SB alone (from
Fig. 1) is compared with the time course of the combination of SB plus
insulin treatments. The portion of the graph to the left of
the zero time point indicates the activation of ERK1/2 that occurs
during the pretreatment with SB; insulin treatments begin at time 0. #,
greater than SB alone (p < 0.02); *, greater than
insulin alone (p < 0.03). D, after
incubation with SB alone or followed by insulin, kinase activity of
immunopurified ERK was determined by Western blot with P-Elk antiserum
followed by direct digital image analysis of the chemiluminescent
blot.
View larger version (26K):
[in a new window]
Fig. 3.
Effect of different SB compounds on ERK1/2
phosphorylation alone and in combination with insulin. H4IIE cells
were serum-deprived and then left untreated or pretreated for 30 min
prior to treatment for the indicated times with insulin
(Ins). Shown are representative autoradiographs of Western
blots of whole cell lysates probed with anti-P-ERK1/2 as described
under "Experimental Procedures." A, cells were
pretreated with 2 µM SB203580 prior to treatment with 10 nM insulin for the indicated times. B, cells
were pretreated with SB202474 or SB202190 at the indicated
concentrations alone ( 
) or prior to treatment (+) with 10 nM insulin for 5 min.
-tubulin, was not significantly altered by insulin or PD alone or by the combination of the two.
View larger version (21K):
[in a new window]
Fig. 4.
Effect of MEK1 inhibition on induction of
c-Fos and Pip92 transcription by insulin. H4IIE cells were serum
deprived and then treated for 30 min with 10 nM insulin
(Ins 30) alone or after pretreatment with 50 µM PD98059. Transcriptionally active nuclei were then
isolated and used in run-on assays. A, representative
autoradiograph of a run-on experiment as described under
"Experimental Procedures," with cells treated as indicated. The
results graphed in B are the averages of four to eight
separate experiments with error bars (S.E.). The dot
densitometric intensity for the control was arbitrarily set to 1 in
each experiment, and other treatment values were expressed as fold
increase versus control. #, greater than control
(p < 0.001); §, decreased from insulin alone
(p < 0.001).
-tubulin.
View larger version (17K):
[in a new window]
Fig. 5.
MEK1-dependent effect
of p38 inhibition on induction of c-Fos and Pip92 transcription and
ERK1/2 phosphorylation. H4IIE cells were serum-deprived, and
nuclear run-on experiments performed. A, cells were treated
with 2 µM SB for 30 min prior to treatment with or
without 10 nM insulin for 30 min (Ins 30).
B, cells were treated with 10 µM SB for 30 min
prior to treatment with or without 10 nM insulin for 30 min
or pretreated with 50 µM PD98059 prior to further
additions. The results are the average fold increases of three to five
separate experiments with error bars (S.E.). Fold increases
induced by insulin alone in previous experiments are included for
reference in both A and B. #, greater than
control (p < 0.05); *, different from insulin alone
(p < 0.05). C, cells were serum-deprived
and then left untreated or pretreated for 30 min with 50 µM PD98059 before the addition of 2 µM SB.
A representative Western blot of three separate experiments is
shown.
View larger version (14K):
[in a new window]
Fig. 6.
Correlation between ERK1/2 activation and
induction of c-Fos and Pip92 transcription. Mean fold induction
values of transcription for each treatment (Figs. 4 and 5) were plotted
on the y axis, and the corresponding mean fold increases in
ERK1/2 phosphorylation were plotted along the x axis. The
strengths of the correlation for c-Fos and Pip92 with ERK1/2
phosphorylation (Spearman correlation coefficients) were
rs = 0.9084 and 0.8772, respectively, and both
of these correlations were highly significant (p 
0.0001).
View larger version (30K):
[in a new window]
Fig. 7.
Effect of insulin treatment on p38 kinase
activity. In vitro p38 kinase assays were performed on
cells treated as indicated. Following incubation with insulin
(Ins), the immunoprecipitated p38 was incubated with
GST-ATF-2 and P-GST-ATF-2 was detected by Western blot with P-ATF-2
antiserum followed by direct digital image analysis of the
chemiluminescent blot. A, a representative blot is shown.
B, the results graphed are the averages of six separate
experiments at each time point with error bars (S.E.)
indicated. The band densitometric intensity for the controls was
arbitrarily set to 1 in each experiment, and other treatment values
were expressed as fold change.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pathology,
Div. of Molecular and Cellular Pathology, Volker Hall, G019, 1670 University Blvd., University of Alabama at Birmingham, Birmingham, AL
35294-0019. Tel.: 205-934-4921; Fax: 205-934-1775; E-mail: messina@path.uab.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Messina, J. L.
(1999)
in
Volume V: Hormonal Control of Growth
(Kostyo, J. L., ed)
, pp. 783-811, Oxford University Press, New York
2.
Virkamaki, A.,
Ueki, K.,
and Kahn, C. R.
(1999)
J. Clin. Invest.
103,
931-943[Medline]
[Order article via Infotrieve]
3.
Whitmarsh, A. J.,
and Davis, R. J.
(1998)
Trends Biochem. Sci.
23,
481-485[CrossRef][Medline]
[Order article via Infotrieve]
4.
Robinson, M. J.,
and Cobb, M. H.
(1997)
Curr. Opin. Cell Biol.
9,
180-186[CrossRef][Medline]
[Order article via Infotrieve]
5.
Heidenreich, K. A.,
and Kummer, J. L.
(1996)
J. Biol. Chem.
271,
9891-9894 6.
Conejo, R.,
and Lorenzo, M.
(2001)
J. Cell. Physiol.
187,
96-108[CrossRef][Medline]
[Order article via Infotrieve]
7.
Conejo, R.,
Valverde, A. M.,
Benito, M.,
and Lorenzo, M.
(2001)
J. Cell. Physiol.
186,
82-94[CrossRef][Medline]
[Order article via Infotrieve]
8.
Sweeney, G.,
Somwar, R.,
Ramlal, T.,
Volchuk, A.,
Ueyama, A.,
and Klip, A.
(1999)
J. Biol. Chem.
274,
10071-10078 9.
Sweeney, G.,
Keen, J.,
Somwar, R.,
Konrad, D.,
Garg, R.,
and Klip, A.
(2001)
Endocrinology
142,
4806-4812 10.
Somwar, R.,
Kim, D. Y.,
Sweeney, G.,
Huang, C.,
Niu, W.,
Lador, C.,
Ramlal, T.,
and Klip, A.
(2001)
Biochem. J.
359,
639-649[CrossRef][Medline]
[Order article via Infotrieve]
11.
Somwar, R.,
Niu, W.,
Kim, D. Y.,
Sweeney, G.,
Randhawa, V. K.,
Huang, C.,
Ramlal, T.,
and Klip, A.
(2001)
J. Biol. Chem.
276,
46079-46087 12.
Butler, P. C.,
Kryshak, E. J.,
Schwenk, W. F.,
Haymond, M. W.,
and Rizza, R. A.
(1990)
Diabetes
39,
217-225[Abstract]
13.
Michael, M. D.,
Kulkarni, R. N.,
Postic, C.,
Previs, S. F.,
Shulman, G. I.,
Magnuson, M. A.,
and Kahn, C. R.
(2000)
Mol. Cell
6,
87-97[CrossRef][Medline]
[Order article via Infotrieve]
14.
Mukhopadhyay, N. K.,
Price, D. J.,
Kyriakis, J. M.,
Pelech, S.,
Sanghera, J.,
and Avruch, J.
(1992)
J. Biol. Chem.
267,
3325-3335 15.
Kim, S. J.,
and Kahn, C. R.
(1997)
Biochem. J.
323,
621-627[Medline]
[Order article via Infotrieve]
16.
Hofmann, C.,
Marsh, J. W.,
Miller, B.,
and Steiner, D. F.
(1980)
Diabetes
29,
865-874[Abstract]
17.
Granner, D.,
Andreone, T.,
Sasaki, K.,
and Beale, E.
(1983)
Nature
305,
549-551[CrossRef][Medline]
[Order article via Infotrieve]
18.
Rosella, G.,
Zajac, J. D.,
Kaczmarczyk, S. J.,
Andrikopoulos, S.,
and Proietto, J.
(1993)
Mol. Endocrinol.
7,
1456-1462 19.
Andreone, T. L.,
Beale, E. G.,
Bar, R. S.,
and Granner, D. K.
(1982)
J. Biol. Chem.
257,
35-38 20.
Streeper, R. S.,
Svitek, C. A.,
Chapman, S.,
Greenbaum, L. E.,
Taub, R.,
and O'Brien, R. M.
(1997)
J. Biol. Chem.
272,
11698-11701 21.
Beale, E. G.,
Koch, S. R.,
Brotherton, A. F.,
Sheorain, V. S.,
and Granner, D. K.
(1986)
Diabetes
35,
546-549[Abstract]
22.
O'Brien, R. M.,
and Granner, D. K.
(1990)
Diabetes Care
13,
327-339[Abstract]
23.
Sutherland, C.,
O'Brien, R. M.,
and Granner, D. K.
(1995)
J. Biol. Chem.
270,
15501-15506 24.
Potter, V. R.,
Evanson, T. R.,
Gayda, D. P.,
and Gurr, J. A.
(1984)
In Vitro
20,
723-731[Medline]
[Order article via Infotrieve]
25.
Messina, J. L.
(1990)
in
Handbook of Experimental Pharmacology: Insulin
(Cuatrecasas, P.
, and Jacobs, S. J., eds)
, pp. 399-419, Springer-Verlag, New York
26.
O'Brien, R. M.,
and Granner, D. K.
(1996)
Physiol. Rev.
76,
1109-1161 27.
Messina, J. L.
(1990)
J. Biol. Chem.
265,
11700-11705 28.
Messina, J. L.,
Standaert, M. L.,
Ishizuka, T.,
Weinstock, R. S.,
and Farese, R. V.
(1992)
J. Biol. Chem.
267,
9223-9228 29.
Lau, L. F.,
and Nathans, D.
(1985)
EMBO J.
4,
3145-3151[Medline]
[Order article via Infotrieve]
30.
Charles, C. H.,
Simske, J. S.,
O'Brien, T. P.,
and Lau, L. F.
(1990)
Mol. Cell. Biol.
10,
6769-6774 31.
Coleclough, C.,
Kuhn, L.,
and Lefkovits, I.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
1753-1757 32.
Shimizu, N.,
Ohta, M.,
Fujiwara, C.,
Sagara, J.,
Mochizuki, N.,
Oda, T.,
and Utiyama, H.
(1991)
J. Biol. Chem.
266,
12157-12161 33.
Bortoff, K. D.,
Zhu, C. C.,
Hrywna, Y.,
and Messina, J. L.
(1997)
Endocrine
7,
199-207[Medline]
[Order article via Infotrieve]
34.
Chung, K. C.,
Gomes, I.,
Wang, D.,
Lau, L. F.,
and Rosner, M. R.
(1998)
Mol. Cell. Biol.
18,
2272-2281 35.
Yamauchi, K.,
Holt, K.,
and Pessin, J. E.
(1993)
J. Biol. Chem.
268,
14597-14600 36.
Messina, J. L.
(1989)
Endocrinology
124,
754-761 37.
Ji, S.,
Guan, R.,
Frank, S. J.,
and Messina, J. L.
(1999)
J. Biol. Chem.
274,
13434-13442 38.
Laemmli, U. K.
(1970)
Nature
227,
680-685[CrossRef][Medline]
[Order article via Infotrieve]
39.
Burnette, W. H.
(1981)
Anal. Biochem.
112,
195-203[CrossRef][Medline]
[Order article via Infotrieve]
40.
Towbin, H.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354 41.
Whitehead, T. P.,
Kricka, L. J.,
Carter, T. J.,
and Thorpe, G. H.
(1979)
Clin. Chem.
25,
1531-1546 42.
Nelson, J. W.
(ed)
(2002)
Alpha Innotech Hands On Application Note 26
, Alpha Innotech Corp., San Leandro, CA
43.
Nelson, J. W.
(ed)
(2002)
Alpha Innotech Hands On Application Note 9
, Alpha Innotech Corp., San Leandro, CA
44.
Kendrick, N. C.,
Johansen, J. J.,
Lee, P. R.,
and Santek, D. A.
(1994)
Anal. Biochem.
219,
297-304[CrossRef][Medline]
[Order article via Infotrieve]
45.
Tarlton, J. F.,
and Knight, P. J.
(1996)
Anal. Biochem.
237,
123-128[CrossRef][Medline]
[Order article via Infotrieve]
46.
Malek, A. M.,
Izumo, S.,
and Alper, S. L.
(1997)
BioTechniques
22,
1150-1153[Medline]
[Order article via Infotrieve]
47.
Kumar, S.,
McDonnell, P. C.,
Gum, R. J.,
Hand, A. T.,
Lee, J. C.,
and Young, P. R.
(1997)
Biochem. Biophys. Res. Commun.
235,
533-538[CrossRef][Medline]
[Order article via Infotrieve]
48.
Livingstone, C.,
Patel, G.,
and Jones, N.
(1995)
EMBO J.
14,
1785-1797[Medline]
[Order article via Infotrieve]
49.
Marais, R.,
Wynne, J.,
and Treisman, R.
(1993)
Cell
73,
381-393[CrossRef][Medline]
[Order article via Infotrieve]
50.
Anderson, N. G.,
Maller, J. L.,
Tonks, N. K.,
and Sturgill, T. W.
(1990)
Nature
343,
651-653[CrossRef][Medline]
[Order article via Infotrieve]
51.
Payne, D. M.,
Rossomando, A. J.,
Martino, P.,
Erickson, A. K.,
Her, J. H.,
Shabanowitz, J.,
Hunt, D. F.,
Weber, M. J.,
and Sturgill, T. W.
(1991)
EMBO J.
10,
885-892[Medline]
[Order article via Infotrieve]
52.
Motulsky, H. J.,
and Searle, P.
(1998)
InStat Guide to Choosing and Interpreting Statistical Tests
, Graphpad Software, Inc., San Diego, CA
53.
Rosner, B. A.
(1990)
Fundamentals of Biostatistics
, 3rd Ed.
, PWS-KENT Publishing Company, Boston, MA
54.
Lazar, D. F.,
Wiese, R. J.,
Brady, M. J.,
Mastick, C. C.,
Waters, S. B.,
Yamauchi, K.,
Pessin, J. E.,
Cuatrecasas, P.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
20801-20807 55.
Ding, X. Z.,
and Adrian, T. E.
(2001)
Biochem. Biophys. Res. Commun.
282,
447-453[CrossRef][Medline]
[Order article via Infotrieve]
56.
Flamigni, F.,
Facchini, A.,
Giordano, E.,
Tantini, B.,
and Stefanelli, C.
(2001)
Biochem. Pharmacol.
61,
25-32[CrossRef][Medline]
[Order article via Infotrieve]
57.
Flamigni, F.,
Facchini, A.,
Stanic, I.,
Tantini, B.,
Bonavita, F.,
and Stefanelli, C.
(2001)
Biochem. Pharmacol.
62,
319-328[CrossRef][Medline]
[Order article via Infotrieve]
58.
Ishii, Y.,
Sakai, S.,
and Honma, Y.
(2001)
Leukocyte Res.
25,
813-820
59.
Westermarck, J., Li, S. P.,
Kallunki, T.,
Han, J.,
and Kahari, V. M.
(2001)
Mol. Cell. Biol.
21,
2373-2383 60.
Singh, R. P.,
Dhawan, P.,
Golden, C.,
Kapoor, G. S.,
and Mehta, K. D.
(1999)
J. Biol. Chem.
274,
19593-19600 61.
Kalmes, A.,
Deou, J.,
Clowes, A. W.,
and Daum, G.
(1999)
FEBS Lett.
444,
71-74[CrossRef][Medline]
[Order article via Infotrieve]
62.
Hall-Jackson, C. A.,
Goedert, M.,
Hedge, P.,
and Cohen, P.
(1999)
Oncogene
18,
2047-2054[CrossRef][Medline]
[Order article via Infotrieve]
63.
Yang, X. Y.,
Zhang, Z.,
and Cohen, D. M.
(1999)
Am. J. Physiol.
277,
F176-F185[Medline]
[Order article via Infotrieve]
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