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(Received for publication, April 21, 1995; and in revised form, June 8, 1995) From the
Insulin stimulates the activity of mitogen-activated protein
kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual
specificity kinase that phosphorylates MAPK on threonine and tyrosine.
The potential role of MAPK activation in insulin action was
investigated with the specific MEK inhibitor PD98059. Insulin
stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6
myotubes (1.4-fold) was completely abolished by pretreatment of cells
with the MEK inhibitor, as was the phosphorylation of MAPK and
pp90
Insulin is the most potent physiological anabolic agent known.
It promotes the synthesis and storage of carbohydrates, lipids, and
proteins and inhibits their degradation and release into the
circulation. While the precise intracellular events that mediate
insulin action are not well understood, regulation of protein
phosphorylation is believed to play a critical role (Saltiel, 1994).
The insulin receptor, a heterotetrameric protein complex, undergoes
autophosphorylation on tyrosine residues upon binding of hormone,
thereby increasing its tyrosine kinase activity and the tyrosine
phosphorylation of specific intracellular proteins (Kasuga et
al., 1982; Rees-Jones and Taylor, 1985). Distal to receptor
activation, insulin regulates serine and threonine phosphorylation,
paradoxically stimulating the phosphorylation of some proteins while
causing the dephosphorylation of others (Czech et al., 1988;
Rosen, 1987; Saltiel, 1990). Many of the serine/threonine
phosphorylations induced by insulin are shared by other growth factors.
In contrast, the dephosphorylation of proteins observed with insulin is
unique. Indeed, many of the rate-limiting enzymes involved in glucose
and lipid metabolism, such as glycogen synthase, hormone-sensitive
lipase, and pyruvate dehydrogenase are regulated through
dephosphorylation mechanisms. Thus, these dephosphorylations are likely
to be critical to many of the metabolic effects of insulin, including
stimulation of glycogen and lipid synthesis, and inhibition of
lipolysis. The best characterized pathway leading to
insulin-dependent serine phosphorylation is the MAPK ( This
model has provided an attractive link between the activation of MAPK by
insulin and the stimulation of glycogen synthesis, resolving the
apparent paradox of simultaneous stimulation of protein phosphorylation
and dephosphorylation by insulin. However, there are inconsistencies
with this model. We have further evaluated the role of the MAPK pathway
in insulin action in the highly responsive 3T3-L1 adipocytes and L6
myotubes. Using a specific inhibitor of MEK, we demonstrate that MAPK
activation is not required for insulin stimulation of PP1 activity and
glucose metabolism, including glycogen synthesis, glucose uptake, and
lipogenesis.
Figure 1:
Structure of
PD98059.
Figure 2:
PD98059 blocks the activation of MAPK by
insulin. Serum-deprived 3T3-L1 adipocytes (A) and L6 myotubes (B) were treated with (shaded bars) and without (hatched bars) 10 µM PD98059 for 30 min prior to
the addition of insulin (A, 100 nM; B, 300
nM) for 5 min. Cells were lysed, and MAPK activity was assayed
as described under ``Experimental Procedures.'' Shown are the
means + S.E. of three separate experiments, each performed in
duplicate. Basal activities were 76 and 127 Cerenkov/µg of protein
for 3T3-L1 adipocytes and L6 myotubes,
respectively.
Figure 3:
Concentration-dependent blockade of MAPK
phosphorylation and activation by PD98059. 3T3-L1 adipocytes were
treated for 30 min with increasing concentrations of PD98059 followed
by 100 nM insulin for 5 min. A, MAPK activity in cell
lysates was determined. Shown are the means + S.E. from three
separate experiments, each performed in duplicate. B,
anti-MAPK immunoprecipitates were resolved on SDS, 8% PAGE and
subjected to Western blotting with anti-phosphotyrosine antibody. C, following pretreatment with 100 µM PD98059 for
60 min, cells were treated with insulin. Cell lysates (75 µg of
protein) were resolved by SDS-PAGE, then immunoblotted with anti-ERK1/2
antisera.
Activation of MAPK is known to involve increased
tyrosine and threonine phosphorylation of the kinase. To verify the
inhibitory effect of PD98059 on MAPK activation by insulin, we examined
the tyrosine phosphorylation of the enzyme. 3T3-L1 adipocytes were
treated with 100 nM insulin in the presence or absence of
PD98059. MAPK was immunoprecipitated, and tyrosine phosphorylation was
evaluated by SDS-PAGE followed by immunoblotting with
anti-phosphotyrosine antibody (Fig. 3B). Insulin
treatment produced a significant increase in the tyrosine
phosphorylation of pp44 Treatment of cells with
insulin has also been shown to increase the activities of other
kinases, including pp90
Figure 4:
PD98059 blocks insulin-stimulated
phosphorylation of both pp90
Previous studies have
demonstrated that the c-fos serum response element (SRE)
mediates the insulin-stimulated transcription of the c-fos gene (Stumpo et al., 1988). This is generally believed to
occur via MAPK-dependent phosphorylation of the TCF/Elk-1 and SRF
transcription factors (Gille et al., 1992). We therefore
examined the effect of the MEK inhibitor on c-fos transcription using the SRE-luciferase (Luc) reporter gene
construct (Yamauchi et al., 1993). 3T3-L1 adipocytes
transfected with this construct demonstrate 1.6-fold and 1.8-fold
increases in luciferase activity following insulin treatments of 1 and
2 h, respectively (Fig. 4C). Pretreatment of cells with
PD98059 completely blocked the stimulation of luciferase activity at
these time points, in agreement with insulin stimulation of c-fos transcription by a MAPK-dependent pathway.
Figure 5:
PD98059 differentially blocks
insulin-stimulated tyrosine phosphorylation. Serum-deprived 3T3-L1
adipocytes and L6 myotubes were treated with or without 10 µM PD98059, followed by insulin for 5 min, as indicated. Cell lysates
(100 µg of protein) were resolved by SDS, 8% PAGE and immunoblotted
with anti-phosphotyrosine antibody. Shown are the predicted positions
of the insulin receptor and the 42- and 44-kDa isoforms of MAPK
protein.
Upon activation, the insulin receptor
catalyzes the tyrosine phosphorylation of its major substrate, insulin
receptor substrate 1, resulting in its selective association with
proteins containing SH2 domains (Sun et al., 1991, 1993). One
such protein, PI 3`-kinase, undergoes activation upon occupancy of the
SH2 domains of its 85-kDa regulatory subunit (Myers et al.,
1992). Pretreatment of 3T3-L1 adipocytes with 10 µM
PD98059 did not reduce activation of PI 3`-kinase by insulin, as
detected in anti-phosphotyrosine immunoprecipitates (Fig. 6).
Moreover, the MEK inhibitor had no effect on PI 3`-kinase activity when
added directly to the in vitro assay (data not shown).
Figure 6:
PD98059 does not block activation of PI
3`-kinase by insulin. 3T3-L1 adipocytes were treated with and without
PD98059, followed by insulin treatment for 5 min. PI 3`-kinase activity
associated with anti-phosphotyrosine immunoprecipitates was determined
as described under ``Experimental Procedures.'' Shown is a
representative result obtained in two separate determinations. PI3P, phosphatidylinositol
3`-phosphate.
Figure 7:
Insulin-stimulated 2-deoxyglucose uptake
and lipid synthesis are insensitive to PD98059. Insulin (100
nM) stimulation of
2-[U-
Figure 8:
PD98059 does not affect insulin
stimulation of glycogen synthesis. 3T3-L1 adipocytes and L6 myotubes
were treated with (solid bars) and without (hatched
bars) 10 µM PD98059 for 30 min, followed by insulin
treatment. [U-
The
hormonal regulation of glycogen synthesis is primarily mediated by
modulation of the activity of glycogen synthase. This enzyme is
stimulated by its allosteric activator, glucose 6-phosphate, and by
dephosphorylation. Glycogen synthase activity was assayed in lysates
from 3T3-L1 adipocytes treated with 100 nM insulin for 20 min
in the absence of extracellular glucose to eliminate the allosteric
activation by glucose 6-phosphate that is produced upon
insulin-stimulated glucose uptake. Insulin treatment produced a 3-fold
increase in the glycogen synthase activity ratio, regardless of whether
or not cells were pretreated with 10 µM PD98059 (Fig. 8B). Insulin (300 nM) produced a
1.7-fold increase in the glycogen synthase activity ratio in the
myotubes, which also was unaffected by pretreatment with 10 µM PD98059 (Fig. 8B). Incubation with inhibitor alone
had no effect on basal glycogen synthase activity, and total activity
was not significantly altered by insulin and/or PD98059 treatment (data
not shown). These results clearly demonstrate that MAPK activation is
not required for insulin stimulation of glycogen synthase activity and
the accumulation of glycogen in 3T3-L1 adipocytes and L6 myotubes.
Figure 9:
PD98059 does not affect insulin
stimulation of PP1 activity. Serum-deprived 3T3-L1 adipocytes and L6
myotubes were treated with (solid bars) and without (hatched bars) 10 µM PD98059 for 30 min prior to
the addition of insulin (A, 100 nM; B, 10
nM) for 10 min. Cell extracts were prepared, and PP1 activity
was assayed as described under ``Experimental Procedures.''
Shown are the means + S.E. of four separate experiments, each
performed in triplicate.
The regulation of protein phosphorylation appears to be a
central component in the pleiotropic actions of insulin (Saltiel,
1994). The insulin-dependent autophosphorylation of the receptor and
activation of its tyrosine kinase activity leads to the subsequent
tyrosine phosphorylation of several intracellular proteins, including
insulin receptor substrate 1 (Sun et al., 1991) and Shc (Pronk et al., 1993). It is likely that the phosphorylation of these
and other receptor substrates induces a series of protein-protein
interactions, leading ultimately to changes in serine/threonine
phosphorylation levels, paradoxically increasing the activities of both
kinases and phosphatases that target numerous intracellular proteins
(Czech et al., 1988; Rosen, 1987; Saltiel, 1990). Studies with
mutant insulin receptors (McClain, 1990; Moller et al., 1991;
Pang et al., 1993b; Pang et al., 1994; Rolband et
al., 1993; Takata et al., 1991), wild-type receptors in
particular cell lines (Ohmichi et al., 1993), or anti-receptor
antibodies (Sung, 1991; Wilden et al., 1992) indicate that the
activation of protein serine kinases and phosphatases may diverge at or
near the receptor. One pathway leading to serine kinase activation
which has been fairly well defined is activation of MAPK. The activity
of this enzyme, first detected in insulin-treated 3T3-L1 cells (Ray and
Sturgill, 1987), and later found to be activated by a number of other
growth factors and mitogens, results from a well characterized cascade
of events. While many of the molecular components involved in the
activation of downstream serine/threonine kinases such as MAPK have
been elucidated, less progress has been made in understanding the
events that are more relevant to the metabolic effects of insulin, the
activation of serine/threonine phosphatase activity. An attractive
model has emerged (Dent et al., 1990) linking MAPK with
stimulation of the type 1 protein phosphatase (PP1) responsible for
activation of glycogen synthase and inactivation of phosphorylase
kinase and glycogen phosphorylase. The MAPK-activated pp90 In order to determine whether
MAPK activation is required for insulin stimulation of glucose
metabolism in more classical insulin-responsive cell lines, we have
studied insulin action and the involvement of MAPK activation in 3T3-L1
adipocytes and L6 myotubes. 3T3-L1 adipocytes are well-suited for the
study of insulin-stimulated glucose metabolism. In addition to glycogen
and lipid synthesis, glucose transport is insulin-sensitive in these
cells due to expression of the insulin-responsive glucose transporter,
Glut4 (Garcia de Herreros and Birnbaum, 1989). L6 myotubes are also a
useful model system for studies of insulin action. Although these cells
do not express Glut4, the regulation of glycogen synthesis by insulin
via dephosphorylation of glycogen synthase resembles that observed in
intact muscle. Using the specific MEK inhibitor PD98059, which blocks
the phosphorylation and activation of MAPK in both cell-based and
cell-free assays, we have found that complete blockade of MAPK
activation and subsequent pp90 The molecular
mechanisms by which metabolic enzymes such as glycogen synthase are
regulated by insulin remains one of the crucial, unresolved issues in
insulin action. While there is considerable evidence that these enzymes
are modulated via dephosphorylation mechanisms likely to be catalyzed
by protein phosphatase 1 activity, the precise pathway linking the
insulin receptor to this activity requires further study.
Volume 270,
Number 35,
Issue of September 01, pp. 20801-20807, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, and the transcriptional activation of
c-fos. Insulin receptor autophosphorylation on tyrosine
residues and activation of phosphatidylinositol 3`-kinase were
unaffected. Pretreatment of cells with PD98059 had no effect on basal
and insulin-stimulated glucose uptake, lipogenesis, and glycogen
synthesis. Glycogen synthase activity in extracts from 3T3-L1
adipocytes and L6 myotubes was increased 3-fold and 1.7-fold,
respectively, by insulin. Pretreatment with 10 µM PD98059
was without effect. Similarly, the 2-fold activation of protein
phosphatase 1 by insulin was insensitive to PD98059. These results
indicate that stimulation of the MAPK pathway by insulin is not
required for many of the metabolic activities of the hormone in
cultured fat and muscle cells.
)cascade. This pathway is initiated by tyrosine
phosphorylation of insulin receptor substrate 1 or Shc proteins by the
receptor kinase, inducing their association with the SH2 domain of the
adapter protein Grb2 (Sasaoka et al., 1994b; Skolnik et
al., 1993b). This association with phosphorylated Shc induces Grb2
to target the nucleotide exchange factor SOS, which in turn associates
with and activates the GTP-binding protein p21
(Rozakis-Adcock et al., 1992; Sasaoka et al., 1994a;
Skolnik et al., 1993a). p21
activation leads to
the stimulation of Raf and other kinases (Thomas et al., 1992;
Wood et al., 1992). These kinases can phosphorylate MAPK
kinase, or MEK (Dent et al., 1992; Kyriakis et al.,
1992; Zheng et al., 1994), a dual specificity kinase that
catalyzes the phosphorylation of MAPK on threonine and tyrosine
residues, causing its activation (Crews et al., 1992; Kosako et al., 1992). MAPK has a number of substrates, including
transcription factors (Gille et al., 1992; Pulverer et
al., 1991), phospholipase A
(Lin et al.,
1993; Nemenoff et al., 1993), and other kinases, such as
ribosomal S6 kinase II, or pp90 (Sturgill et
al., 1988). Dent et al.(1990) have suggested that the
phosphorylation and activation of pp90
by activated MAPK
increases its activity toward site 1 on the regulatory glycogen-binding
subunit (PP1G) of type 1 protein phosphatase (PP1), based on a series
of reconstitution experiments. Increased phosphorylation of this site
has also been detected in PP1G isolated from rabbit skeletal muscle
following insulin treatment. The phosphorylation of this regulatory
subunit produces a 2-fold increase in the PP1-catalyzed
dephosphorylation of glycogen synthase and phosphorylase kinase,
thereby increasing the overall rate of glycogen accumulation.
Materials
3T3-L1 and L6 cells were purchased
from ATCC (Rockville, MD). DMEM and calf serum were purchased from Life
Technologies, Inc., while fetal bovine serum (FBS) was obtained from
Hyclone. Porcine insulin was a generous gift from Eli Lilly.
[-
P]ATP (3000 Ci/mmol),
[U-
C]glucose (298 mCi/mmol), and
2-[U-C]deoxyglucose (323 mCi/mmol) were
from DuPont NEN, while UDP-[U-C]glucose
(254 mCi/mmol) was from ICN. Mouse anti-phosphotyrosine monoclonal
antibody was purchased from Upstate Biotechnology Inc. (Lake Placid,
NY). Anti-MAPK antiserum used for immunoprecipitations was prepared
from rabbits immunized with a C-terminal peptide (amino acids
425-445) of pp44 expressed as a GST fusion
protein. Anti-ERK1/2 for immunoblotting was obtained from Zymed (San
Francisco, CA). SOS polyclonal antibody was from Upstate Biotechnology
Inc. For ECL detection, horseradish peroxidase-conjugated goat
anti-mouse and goat anti-rabbit IgG were purchased from Life
Technologies, Inc. Phosphatidylinositol was obtained from Avanti Polar
Lipids. Anti-pp90
and anti-pp70
rabbit
antisera were generous gifts of Dr. John Blenis (Harvard Medical
School). Glycogen phosphorylase and phosphorylase kinase were purchased
from Sigma, and okadaic acid was obtained from Calbiochem. Other
reagents were from Sigma and were of the highest quality available.
Tissue Culture
3T3-L1 fibroblasts were maintained
in Dulbecco's modified Eagle's medium (DMEM) with 10% calf
serum prior to initiation of the differentiation protocol.
Differentiation to adipocytes was induced by incubating confluent
monolayers for 2 days in DMEM containing 10% FBS, 0.5 mM
3-isobutyl-1-methylxanthine, and 0.4 µg/ml dexamethasone.
Subsequently, cells were incubated for 2 days with 1 µM insulin in DMEM containing 10% FBS. One day after transfer to the
same medium without insulin, greater than 85% of the cells expressed
the adipocyte phenotype. L6 myoblasts were maintained in DMEM
containing 10% FBS until approximately 50% confluent, at which point
differentiation was initiated by conversion to DMEM with 2% FBS. Cell
fusion was apparent at day 5, and greater than 85% of the culture
expressed the myotube phenotype by day 12. Prior to assay, both
adipocyte and myotube cultures were serum-starved for 3 h in
Krebs-Ringer buffer with 30 mM Hepes (KRBH; pH 7.4) containing
0.5% bovine serum albumin and 2.5 mM glucose.Immunoblots
After insulin treatment, cells (100-mm
dishes) were washed twice with ice-cold phosphate-buffered saline
(PBS), then lysed in HNTG buffer (50 mM Hepes, pH 7.5, 150
mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 1 mM EDTA, 10 mM sodium
pyrophosphate, 1 mM Na
VO
, 30 mMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10
µg/ml leupeptin, 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride (Margolis et al., 1990).
Cell lysates were centrifuged (10,000 g; 10 min) to
preclear insoluble material, then diluted directly into Laemmli sample
buffer. Lysates were then resolved by sodium dodecyl sulfate (SDS), 8%
polyacrylamide gel electrophoresis (PAGE), transferred to
nitrocellulose paper, and immunoblotted with anti-phosphotyrosine,
anti-pp90
, anti-ERK1/2, and anti-SOS antibodies, followed
by horseradish peroxidase-goat anti-mouse or horseradish
peroxidase-goat anti-rabbit IgG, respectively. pp44
was
immunoprecipitated from denatured cell lysates, as described previously
(Mastick et al., 1994), prior to anti-phosphotyrosine
immunoblotting.
Assay of MAPK Activity
Following insulin treatment
of cultures in 12-well dishes, cells were washed twice in ice-cold PBS,
then lysed in 50 µl of buffer containing 50 mM
-glycerol phosphate, 10 mM Hepes, pH 8.0, 70 mM NaCl, 1 mM NaVO, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride. Determination of MAPK activity in lysates was performed as
described previously (Pang et al., 1993a). After
centrifugation (10,000 g; 10 min) to preclear
insoluble material, 10-µl aliquots of cell lysate (5-10
µg of protein) were incubated with approximately 5 µg of
microtubule-associated protein 2 (MAP2) for 15 min at 25 °C in a
final volume of 25 µl containing 50 mM Tris-HCl, pH 7.4, 2
mM EGTA, 10 mM MgCl
, and 40 µM [-
P]ATP (2 µCi). After termination
of activity by the addition of 4
Laemmli sample buffer,
phosphorylation of MAP2 was determined by resolution on SDS, 6% PAGE,
Coomassie Blue staining, excision of MAP2 protein from the gel, and
Cerenkov counting of incorporated radioactivity.
Transcriptional Activation of c-fos
3T3-L1
adipocytes were transfected using the calcium phosphate
co-precipitation method with CsCl double-banded DNA as described
previously (Yamauchi et al., 1993). Briefly, 10-day fully
differentiated 3T3-L1 adipocytes were transfected with 15 µg of the
serum response element-luciferase (SRE-Luc) and 5 µg of the Rous
sarcoma virus--galactosidase reporter plasmid DNAs. Twelve hours
after transfection, the cells were placed into serum-free Ham's
F12 medium for 12 h and incubated with or without PD98059 for 1 h prior
to the addition of 100 nM insulin. Whole cell extracts were
prepared at various times for the determination of luciferase (Luc) and
-galactosidase activities. To correct for differences in
transcription efficiencies between plates within an experiment, the
luciferase activity in each extract was normalized to
-galactosidase activity.Assay of Phosphatidylinositol (PI) 3`-Kinase
Activity
This was determined as described previously (Ohmichi et al., 1992). Cells were lysed in 0.5 ml of buffer containing
20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5
mM EDTA, 1 mM NaVO, 10
µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Cell lysates were precleared of
insoluble material by centrifugation (10,000 g, 10
min, 4 °C), then preincubated with Pansorbin cells and rabbit
IgG-agarose before being subjected to immunoprecipitation with
anti-phosphotyrosine antibody and Protein A-agarose. The resulting
pellets were thoroughly washed, and associated PI 3`-kinase activity
was determined by the incubation of immunoprecipitates with
[
P]ATP and phosphatidylinositol.
Phosphatidylinositol 3-phosphate produced was resolved by thin layer
chromatography and visualized by autoradiography.
Determination of 2-Deoxyglucose Uptake
Cells in
12-well dishes were serum-deprived and insulin-treated as above. Assay
of glucose uptake was initiated by the addition of
2-[U-C]deoxyglucose (1 µCi/well)
and 5 mM glucose. After a 15-min incubation at 37 °C,
cells were washed three times with ice-cold PBS containing 10 mM glucose, then solubilized in 0.5 M NaOH. Samples were
then assessed for radioactivity by scintillation counting in Ready Gel
(Beckman).Glycogen Synthesis and Lipogenesis Assays
The
accumulation of glycogen in intact cells was determined by an
adaptation of the method of Lawrence et al.(1977), as
described previously (Hess et al., 1991). After serum
deprivation and pretreatment with or without PD98059, cells in 6-well
dishes were incubated in the presence or absence of insulin for an
additional 15 min. Subsequently, cells were incubated with 5
mMD-[U-C]glucose (2 µCi
per well) for 60 min at 37 °C. Cells were then washed three times
with ice-cold PBS, solubilized in 30% KOH, and radiolabeled glucose
incorporation into glycogen was determined. Alternatively, in 3T3-L1
adipocytes (6-well dishes), radiolabeled glucose incorporation into
lipid was assessed by scraping cells into 1 ml of PBS and shaking
vigorously with 5 ml of Betafluor scintillant (National Diagnostics,
Manville, NJ). After samples settled overnight, radioactivity which
partitioned into the organic phase was determined by scintillation
counting.Assay of Glycogen Synthase Activity
Insulin
stimulation of glycogen synthase activity was determined as described
previously (Robinson et al., 1993; Thomas et al.,
1968) with some modifications. Cells (100-mm dishes) were
serum-deprived in KRBH with 0.5% bovine serum albumin in the absence of
glucose for 3 h, pretreated with or without PD98059 for an additional
30 min, then incubated with insulin for 20 min at 37 °C. After
three washes with ice-cold PBS, cells were scraped into 500 µl of
glycogen synthase assay buffer (50 mM Tris-HCl, pH 7.8, 10
mM EDTA, and 100 mM KF) and homogenized with a
glass-glass dounce homogenizer prior to centrifugation (10,000 g, 20 min). To measure glycogen synthase activity, 50 µl
of the supernatant (50-200 µg of protein) was added to an
equal volume of original buffer containing 10 mM UDP-[
C]glucose (0.05-0.15
µCi/µmol) and 15 mg/ml glycogen, in the presence or absence of
10 mM glucose 6-phosphate. After a 15-min incubation at 37
°C, assay tubes were chilled for 15 min in an ice bath. Tube
contents were then spotted on prelabeled Whatman filter papers (GF/A;
2.4 cm) which were immediately immersed in 500 ml of 70% ethanol (4
°C), mixed 40 min, then washed two more times in 250 ml of 70%
ethanol (15 min and 60 min, respectively) to remove unincorporated
substrate from precipitated glycogen. Filters were air-dried, and
radioactivity was counted with 5 ml of Ready Gel scintillant. For
optimal stimulation of glycogen synthase in 3T3-L1 adipocytes, cells
were used 8-12 days post-differentiation.Assay of PP1 Activity
Following insulin treatment,
cell extracts were prepared in homogenization buffer (25 mM Hepes, pH 7.2, 2 mM EDTA, 0.2% -mercaptoethanol, 2
mg/ml glycogen, 40 µM leupeptin, 0.1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin) as described
previously (Srinivasan and Begum, 1994). PP1 activity in cell extracts
(1-3 µg of protein) was determined against 20 µg of
substrate ([P]phosphorylase a) for 7
min at 30 °C in 60 µl of homogenization buffer containing 3
nM okadaic acid to block type 2A protein phosphatase activity.
After resolution by SDS, 8% PAGE, substrate was excised from the gel,
and residual radioactivity was determined by scintillation counting.
P-Labeled phosphorylase a was prepared as
described (Cohen et al., 1988). Free
[
-
P]ATP was removed from radiolabeled
substrate by the use of Bio-Gel P-6 spin columns (Bio-Rad). As per
glycogen synthase activation, optimal activation of PP1 by insulin was
observed in 3T3-L1 adipocytes that were 8-12 days
post-differentiation.
PD98059 Inhibits the Activation of MAPK by
Insulin
The structure of PD98059 is shown in Fig. 1. This
compound is a selective inhibitor of MAPK kinase, or MEK, and is
noncompetitive with respect to ATP binding to MEK. ()3T3-L1
adipocytes and L6 myotubes were incubated for 30 min in the presence or
absence of PD98059 prior to treatment with insulin. MAPK activity was
assessed in cell extracts by examining in vitro phosphorylation of the specific substrate MAP2. Insulin treatment
of 3T3-L1 adipocytes resulted in a 2.7-fold increase in MAP2
phosphorylation (Fig. 2A) which was maximal at 5 min
and gradually decreased thereafter (Wiese et al., 1995).
Pretreatment of adipocytes with PD98059 completely abolished the
stimulation of MAPK by insulin, with an IC of
approximately 1 µM and a maximal inhibitory effect
obtained with 10 µM (Fig. 3A). 10
µM PD98059 prevented stimulation of MAPK activity by
insulin up to 60 min after hormone treatment (data not shown). Insulin
produces a more modest (35%) stimulation of MAPK activity in L6
myotubes. This increase was also completely blocked upon pretreatment
of cells with 10 µM inhibitor (Fig. 2B).
Basal activities were not significantly affected by incubation with
inhibitor alone in either cell line. Ion-exchange chromatography of
lysates from 3T3-L1 adipocytes revealed ERK-1 and ERK-2 as the only
MAPK family members activated by insulin. PD98059 completely blocked
activation by insulin of both forms of the enzyme. (
)
. This increased phosphorylation
was inhibited in a concentration-dependent manner by pretreatment of
cells with PD98059, with negligible tyrosine phosphorylation of MAPK
remaining after incubation with 10 µM concentration of the
compound. The activation of MAPK also results in a characteristic
change in its SDS-PAGE mobility due to threonine and tyrosine
phosphorylation (de Vries-Smits et al., 1992). Pretreatment of
3T3-L1 adipocytes with PD98059 completely blocked the insulin-induced
gel shift of MAPK (Fig. 3C).
(Erikson, 1991) and pp70
(Thomas, 1992). Activation of these kinases requires
serine/threonine phosphorylation, also reflected by reduced mobility on
SDS-PAGE (Blenis et al., 1991). pp90
is thought
to be directly activated by a MAPK-catalyzed phosphorylation (Sturgill et al., 1988). To evaluate the role of MAPK in pp90
phosphorylation, 3T3-L1 adipocytes were treated with insulin, and
pp90
and pp70
were detected by Western
blotting. Insulin caused a shift in electrophoretic mobilities of both
pp90
and pp70
. Prior incubation of cells
with 10 µM PD98059 completely prevented the
insulin-stimulated shift in pp90
mobility (Fig. 4A), consistent with the successful blockade of
MAPK activation in vivo. The mobility shift of pp70
was unaffected by inhibitor pretreatment (data not shown),
confirming a lack of involvement of MAPK in this particular response to
insulin (Ballou et al., 1990; Blenis et al., 1991).
In addition to pp90
, the guanine nucleotide exchange
factor SOS is also believed to be a direct substrate of MAPK (Waters et al., 1995; Cherniack et al., 1994). Pretreatment
of 3T3-L1 adipocytes with the MEK inhibitor completely blocked the
insulin-stimulated SOS gel shift characteristic of serine/threonine
phosphorylation (Fig. 4B).
and SOS and insulin
stimulation of c-fos transcription. A, serum-deprived
3T3-L1 adipocytes were treated with or without 10 µM PD98059 for 30 min, followed by 100 nM insulin for 5 min.
Cell lysates were resolved by SDS, 8% PAGE then immunoblotted with
anti-pp90
. B, serum-deprived 3T3-L1 adipocytes
were treated with 100 µM PD98059 for 60 min, followed by
100 nM insulin for 15 min. Cell lysates were resolved by SDS,
5-10% PAGE and immunoblotted with anti-SOS. C, 3T3-L1
adipocytes were transfected with SRE-Luc and RSV-
-galactosidase as
described under ``Experimental Procedures,'' serum-deprived
for 12 h, then treated with (solid bars) or without (hatched bars) 100 µM PD98059 for 60 min.
Following the treatment of cells with 100 nM insulin for the
indicated times, luciferase and -galactosidase activities were
determined in cell extracts. Shown are the means + S.E. of two
independent determinations, each performed in
triplicate.
Specificity of the Inhibitory Effects of
PD98059
PD98059 inhibits MEK activity in a manner which is not
competitive with either substrate (MAPK) or ATP binding and has been
shown to be without effect on MAPK activity itself, as well as the
activity of other serine kinases. Furthermore, nerve growth
factor-, epidermal growth factor-, and platelet-derived growth
factor-receptor tyrosine autophosphorylation is completely insensitive
to PD98059 pretreatment of cells. To evaluate the
specificity of this compound in blocking MAPK activation in both 3T3-L1
adipocytes and L6 myotubes, insulin receptor autophosphorylation was
evaluated. PD98059 treatment was without effect on insulin-dependent
phosphorylation of the receptor, as determined by anti-phosphotyrosine
immunoblotting (Fig. 5). In the same immunoblot of cell lysates,
insulin-stimulated tyrosine phosphorylation of both the 42- and 44-kDa
isoforms of MAPK was effectively blocked by pretreatment with 10
µM PD98059.
Stimulation of Glucose Uptake and Lipid Synthesis by
Insulin Does Not Require MAPK Activation
Differentiated 3T3-L1
cells respond to insulin treatment with marked increases in glucose
uptake and metabolism. Incubation of cells with 100 nM insulin
for 15 min produced a 9-fold increase in the uptake of the
nonmetabolizable glucose analog, 2-deoxy-D-glucose in the
presence of 5 mM unlabeled glucose (Fig. 7A).
This stimulation of glucose transport was unaffected by prior treatment
of cells with 10 µM PD98059. Similar results were obtained
when 2-deoxy-D-glucose uptake was determined in the presence
of 100 µM unlabeled glucose (data not shown). These data
demonstrate that MAPK activation is not required for insulin-stimulated
glucose uptake in 3T3-L1 adipocytes. Insulin also increases the rate of
lipid synthesis in these cells. Exposure of 3T3-L1 adipocytes to
insulin produced a 7-fold increase in the conversion of radiolabeled
glucose into lipid (Fig. 7B). This stimulation of
lipogenesis was unaffected by pretreatment of cells with 10 µM PD98059. Moreover, the dose-response for insulin stimulation of
lipid synthesis (EC approximately 3 nM) was not
affected by the MEK inhibitor (data not shown). Basal activities were
unaltered by the presence of inhibitor in both glucose uptake and
lipogenesis assays.
C]deoxyglucose uptake (A)
and [U-C]glucose incorporation into
lipid (B) were determined in 3T3-L1 adipocytes following
pretreatment with (solid bars) and without (hatched
bars) 10 µM PD98059. Results are the means +
S.E. from individual experiments performed in triplicate and are
representative of three separate
experiments.
The Stimulation of Glycogen Synthesis by Insulin Does Not
Require MAPK Activation
The potential role of MAPK activation in
the stimulation of glycogen synthesis by insulin was examined in both
3T3-L1 adipocytes and L6 myotubes by determining the incorporation of C-labeled glucose into glycogen in the presence and
absence of 10 µM PD98059 (Fig. 8A).
Insulin stimulated the rate of glycogen synthesis in a dose-dependent
manner in both cell lines, with an EC of approximately 5
nM in 3T3-L1 adipocytes and 30 nM in L6 myotubes.
Concentrations of PD98059 sufficient to completely block insulin
stimulation of MAPK activity had no effect on either the sensitivity or
maximal stimulation of glycogen synthesis in these cells.
C]Glucose incorporation
into glycogen in intact cells (A) and glycogen synthase
activity (±10 mM glucose 6-phosphate) in broken cell
extracts (B) were determined as described under
``Experimental Procedures.'' Results are the means +
S.E. of three separate experiments, each performed in
triplicate.
Stimulation of Type 1 Protein Phosphatase Activity by
Insulin Does Not Require MAPK Activation
Stimulation of PP1
activity is believed to be critical for many of the metabolic effects
of insulin, including the stimulation of glycogen synthesis (Hess et al., 1991; Tanti et al., 1991). This enzyme was
assayed by following the in vitro dephosphorylation of P-labeled glycogen phosphorylase in cell lysates (Fig. 9). In order to specifically assay PP1 activity, release
of
P was monitored in the presence of 3 nM okadaic acid, which completely inhibits type 2A protein
phosphatase. Insulin treatment of 3T3-L1 adipocytes produced a 1.8-fold
activation of PP1. This enzyme activity was similarly stimulated by
insulin in L6 myotubes. In both cell lines, insulin stimulation of PP1
activity was unaffected by prior treatment of cells with 10 µM PD98059, and little or no effect was observed on basal activity.
kinase can phosphorylate site 1 on the regulatory G subunit of
PP1 in vitro, increasing the activity of the phosphatase
toward glycogen synthase and phosphorylase kinase. However, evidence
from several studies contradicts a central role for MAPK activation in
this particular response. Agents such as phorbol esters or okadaic acid
can activate MAPK, yet they antagonize the metabolic effects of insulin
(Corvera et al., 1991; Hess et al., 1991). Moreover,
platelet-derived growth factor and epidermal growth factor potently
activate the MAPK pathway in 3T3-L1 adipocytes, but are ineffective in
stimulating glycogen synthesis, suggesting that MAPK activation is not
sufficient to produce this response (Robinson et al., 1993;
Wiese et al., 1995). Furthermore, experiments in a number of
cell lines expressing wild-type or mutant insulin receptors (Moller et al., 1991; Ohmichi et al., 1993; Pang et
al., 1993b; Pang et al., 1994) or downstream effectors
(Sakaue et al., 1995) have dissociated MAPK activation from
metabolic responses, indicating that activation of this enzyme is not
even required for insulin stimulation of glycogen synthesis. However,
these latter studies were performed in cell lines not considered
representative of the primary target tissues of insulin, especially
with regard to glucose metabolism.
phosphorylation was
without effect on insulin stimulation of glucose utilization, although
both SOS phosphorylation and transcriptional activation of c-fos were completely inhibited. The stimulation of glucose uptake,
lipogenesis, and glycogen synthesis were unaltered by blockade of MAPK
activation. Moreover, stimulation of glycogen synthase and PP1
activities by insulin were also unaffected by MEK inhibition. The
possibility remains that significant activation of PP1 via
MAPK-activated pp90
does occur, but is not required due
to the potential existence of an alternative pathway for the
stimulation of PP1. In the event of such redundant signaling, one might
expect the MEK inhibitor to produce decreased insulin sensitivity or
maximal response for insulin stimulation of lipid or glycogen
accumulation by insulin. However, the dose-response for insulin
stimulation of glycogen synthesis was completely unaffected by the
abolishment of MAPK activation in both 3T3-L1 adipocytes and L6
myotubes. These results, obtained in highly responsive fat and muscle
cell lines, clearly demonstrate that activation of MAPK is not required
for insulin stimulation of glycogen synthesis.
)))
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P. Elsner, B. Quistorff, T. S. Hermann, J. Dich, and N. Grunnet Regulation of glycogen accumulation in L6 myotubes cultured under optimized differentiation conditions Am J Physiol Endocrinol Metab, December 1, 1998; 275(6): E925 - E933. [Abstract] [Full Text] [PDF]
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 J. R. Crawford and B. S. Jacobson Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2 Mol. Biol. Cell, December 1, 1998; 9(12): 3429 - 3443. [Abstract] [Full Text]
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 S. M. Rodems and D. H. Spector Extracellular Signal-Regulated Kinase Activity Is Sustained Early during Human Cytomegalovirus Infection J. Virol., November 1, 1998; 72(11): 9173 - 9180. [Abstract] [Full Text] [PDF]
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 R. A. Hegele, S. B. Harris, B. Zinman, J. Wang, H. Cao, A. J. G. Hanley, L.-C. Tsui, and S. W. Scherer Variation in the AU(AT)-Rich Element within the 3'-Untranslated Region of PPP1R3 Is Associated with Variation in Plasma Glucose in Aboriginal Canadians J. Clin. Endocrinol. Metab., November 1, 1998; 83(11): 3980 - 3983. [Abstract] [Full Text]
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D. Wang and H. S. Sul Insulin Stimulation of the Fatty Acid Synthase Promoter Is Mediated by the Phosphatidylinositol 3-Kinase Pathway. INVOLVEMENT OF PROTEIN KINASE B/Akt J. Biol. Chem., September 25, 1998; 273(39): 25420 - 25426. [Abstract] [Full Text] [PDF]
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C. E. Pierreux, B. Ursø, P. De Meyts, G. G. Rousseau, and F. P. Lemaigre Inhibition by Insulin of Glucocorticoid-Induced Gene Transcription: Involvement of the Ligand-Binding Domain of the Glucocorticoid Receptor and Independence from the Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase Pathways Mol. Endocrinol., September 1, 1998; 12(9): 1343 - 1354. [Abstract] [Full Text]
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M. F. Favata, K. Y. Horiuchi, E. J. Manos, A. J. Daulerio, D. A. Stradley, W. S. Feeser, D. E. Van Dyk, W. J. Pitts, R. A. Earl, F. Hobbs, et al. Identification of a Novel Inhibitor of Mitogen-activated Protein Kinase Kinase J. Biol. Chem., July 17, 1998; 273(29): 18623 - 18632. [Abstract] [Full Text] [PDF]
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A. Wagle, S. Jivraj, G. L. Garlock, and S. R. Stapleton Insulin Regulation of Glucose-6-phosphate Dehydrogenase Gene Expression Is Rapamycin-sensitive and Requires Phosphatidylinositol 3-Kinase J. Biol. Chem., June 12, 1998; 273(24): 14968 - 14974. [Abstract] [Full Text] [PDF]
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M. J. Brady, F. J. Bourbonais, and A. R. Saltiel The Activation of Glycogen Synthase by Insulin Switches from Kinase Inhibition to Phosphatase Activation during Adipogenesis in 3T3-L1 Cells J. Biol. Chem., June 5, 1998; 273(23): 14063 - 14066. [Abstract] [Full Text] [PDF]
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R. H. Weiss, E. A. Maga, and A. Ramirez MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells Am J Physiol Cell Physiol, June 1, 1998; 274(6): C1521 - C1529. [Abstract] [Full Text] [PDF]
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A. G. Kayali, J. Eichhorn, T. Haruta, A. J. Morris, J. G. Nelson, P. Vollenweider, J. M. Olefsky, and N. J. G. Webster Association of the Insulin Receptor with Phospholipase C-gamma (PLCgamma ) in 3T3-L1 Adipocytes Suggests a Role for PLCgamma in Metabolic Signaling by Insulin J. Biol. Chem., May 29, 1998; 273(22): 13808 - 13818. [Abstract] [Full Text] [PDF]
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Y Shimoni, H S Ewart, and D Severson Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin J. Physiol., March 1, 1998; 507(2): 485 - 496. [Abstract] [Full Text] [PDF]
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T.-Z. Su, M. Wang, L.-J. Syu, A. R. Saltiel, and D. L. Oxender Regulation of System A Amino Acid Transport in 3T3-L1 Adipocytes by Insulin J. Biol. Chem., February 6, 1998; 273(6): 3173 - 3179. [Abstract] [Full Text] [PDF]
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V. Ribon, J. A. Printen, N. G. Hoffman, B. K. Kay, and A. R. Saltiel A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes Mol. Cell. Biol., February 1, 1998; 18(2): 872 - 879. [Abstract] [Full Text]
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K. N. Dalby, N. Morrice, F. B. Caudwell, J. Avruch, and P. Cohen Identification of Regulatory Phosphorylation Sites in Mitogen-activated Protein Kinase (MAPK)-activated Protein Kinase-1a/p90rsk That Are Inducible by MAPK J. Biol. Chem., January 16, 1998; 273(3): 1496 - 1505. [Abstract] [Full Text] [PDF]
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J. Wijkander, T. R. Landstrom, V. Manganiello, P. Belfrage, and E. Degerman Insulin-Induced Phosphorylation and Activation of Phosphodiesterase 3B in Rat Adipocytes: Possible Role for Protein Kinase B But Not Mitogen-Activated Protein Kinase or p70 S6 Kinase Endocrinology, January 1, 1998; 139(1): 219 - 227. [Abstract] [Full Text] [PDF]
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F. Bost, R. McKay, N. Dean, and D. Mercola The JUN Kinase/Stress-activated Protein Kinase Pathway Is Required for Epidermal Growth Factor Stimulation of Growth of Human A549 Lung Carcinoma Cells J. Biol. Chem., December 26, 1997; 272(52): 33422 - 33429. [Abstract] [Full Text] [PDF]
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 J. Mills, D. Laurent Charest, F. Lam, K. Beyreuther, N. Ida, S. L. Pelech, and P. B. Reiner Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway J. Neurosci., December 15, 1997; 17(24): 9415 - 9422. [Abstract] [Full Text] [PDF]
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 Y. Takeuchi, J. Yamada, T. Yamada, and A. Todisco Functional role of extracellular signal-regulated protein kinases in gastric acid secretion Am J Physiol Gastrointest Liver Physiol, December 1, 1997; 273(6): G1263 - G1272. [Abstract] [Full Text] [PDF]
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M. J. Brady, A. C. Nairn, and A. R. Saltiel The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes. EVIDENCE FOR A POTENTIAL ROLE FOR DARPP-32 IN INSULIN ACTION J. Biol. Chem., November 21, 1997; 272(47): 29698 - 29703. [Abstract] [Full Text] [PDF]
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 K.-C. Leung, M. J. Waters, I. Markus, W. R. Baumbach, and K. K. Y. Ho Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: A novel mechanism of growth hormone receptor regulation PNAS, October 14, 1997; 94(21): 11381 - 11386. [Abstract] [Full Text] [PDF]
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N. Hemati, S. E. Ross, R. L. Erickson, G. E. Groblewski, and O. A. MacDougald Signaling Pathways through Which Insulin Regulates CCAAT/Enhancer Binding Protein alpha (C/EBPalpha ) Phosphorylation and Gene Expression in 3T3-L1 Adipocytes. CORRELATION WITH GLUT4 GENE EXPRESSION J. Biol. Chem., October 10, 1997; 272(41): 25913 - 25919. [Abstract] [Full Text] [PDF]
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T. Bellido, V. Z. C. Borba, P. Roberson, and S. C. Manolagas Activation of the Janus Kinase/STAT (Signal Transducer and Activator of Transcription) Signal Transduction Pathway by Interleukin-6-Type Cytokines Promotes Osteoblast Differentiation Endocrinology, September 1, 1997; 138(9): 3666 - 3676. [Abstract] [Full Text] [PDF]
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M. J. Brady, J. A. Printen, C. C. Mastick, and A. R. Saltiel Role of Protein Targeting to Glycogen (PTG) in the Regulation of Protein Phosphatase-1 Activity J. Biol. Chem., August 8, 1997; 272(32): 20198 - 20204. [Abstract] [Full Text] [PDF]
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M. Ohmichi, K. Koike, A. Kimura, K. Masuhara, H. Ikegami, Y. Ikebuchi, T. Kanzaki, K. Touhara, M. Sakaue, Y. Kobayashi, et al. Role of Mitogen-Activated Protein Kinase Pathway in Prostaglandin F2{alpha}-Induced Rat Puerperal Uterine Contraction Endocrinology, August 1, 1997; 138(8): 3103 - 3111. [Abstract] [Full Text] [PDF]
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L. Ragolia and N. Begum The Effect of Modulating the Glycogen-Associated Regulatory Subunit of Protein Phosphatase-1 on Insulin Action in Rat Skeletal Muscle Cells Endocrinology, June 1, 1997; 138(6): 2398 - 2404. [Abstract] [Full Text] [PDF]
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A. W. Kao, S. B. Waters, S. Okada, and J. E. Pessin Insulin Stimulates the Phosphorylation of the 66- and 52-Kilodalton Shc Isoforms by Distinct Pathways Endocrinology, June 1, 1997; 138(6): 2474 - 2480. [Abstract] [Full Text] [PDF]
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Y. Takahashi, Y. Okimura, I. Mizuno, K. Iida, T. Takahashi, H. Kaji, H. Abe, and K. Chihara Leptin Induces Mitogen-activated Protein Kinase- dependent Proliferation of C3H10T1/2 Cells J. Biol. Chem., May 16, 1997; 272(20): 12897 - 12900. [Abstract] [Full Text] [PDF]
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C. Ravanat, M. Morales, D. O. Azorsa, S. Moog, S. Schuhler, P. Grunert, D. Loew, A. Van Dorsselaer, J.-P. Cazenave, and F. Lanza Gene Cloning of Rat and Mouse Platelet Glycoprotein V: Identification of Megakaryocyte-Specific Promoters and Demonstration of Functional Thrombin Cleavage Blood, May 1, 1997; 89(9): 3253 - 3262. [Abstract] [Full Text] [PDF]
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H. S. Camp and S. R. Tafuri Regulation of Peroxisome Proliferator-activated Receptor gamma Activity by Mitogen-activated Protein Kinase J. Biol. Chem., April 18, 1997; 272(16): 10811 - 10816. [Abstract] [Full Text] [PDF]
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C. Knall, G. S. Worthen, and G. L. Johnson Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases PNAS, April 1, 1997; 94(7): 3052 - 3057. [Abstract] [Full Text] [PDF]
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 R. W. Brownsey, A. N. Boone, and M. F. Allard Actions of insulin on the mammalian heart: metabolism, pathology and biochemical mechanisms Cardiovasc Res, April 1, 1997; 34(1): 3 - 24. [Full Text] [PDF]
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S. A. Coolican, D. S. Samuel, D. Z. Ewton, F. J. McWade, and J. R. Florini The Mitogenic and Myogenic Actions of Insulin-like Growth Factors Utilize Distinct Signaling Pathways J. Biol. Chem., March 7, 1997; 272(10): 6653 - 6662. [Abstract] [Full Text] [PDF]
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V. I. Shifrin, R. J. Davis, and B. G. Neel Phosphorylation of Protein-tyrosine Phosphatase PTP-1B on Identical Sites Suggests Activation of a Common Signaling Pathway during Mitosis and Stress Response in Mammalian Cells J. Biol. Chem., January 31, 1997; 272(5): 2957 - 2962. [Abstract] [Full Text] [PDF]
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L. Gnudi, E. U. Frevert, K. L. Houseknecht, P. Erhardt, and B. B. Kahn Adenovirus-Mediated Gene Transfer of Dominant Negative Rasasn17 in 3T3L1 Adipocytes Does Not Alter Insulin-Stimulated PI3-Kinase Activity or Glucose Transport Mol. Endocrinol., January 1, 1997; 11(1): 67 - 76. [Abstract] [Full Text]
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K. Graf, X.-P. Xi, D. Yang, E. Fleck, W. A. Hsueh, and R. E. Law Mitogen-Activated Protein Kinase Activation Is Involved in Platelet-Derived Growth Factor-Directed Migration by Vascular Smooth Muscle Cells Hypertension, January 1, 1997; 29(1): 334 - 339. [Abstract] [Full Text] [PDF]
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A. D. Kohn, S. A. Summers, M. J. Birnbaum, and R. A. Roth Expression of a Constitutively Active Akt Ser/Thr Kinase in 3T3-L1 Adipocytes Stimulates Glucose Uptake and Glucose Transporter 4Translocation J. Biol. Chem., December 6, 1996; 271(49): 31372 - 31378. [Abstract] [Full Text] [PDF]
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C. M. Moxham, A. Tabrizchi, R. J. Davis, and CraigC. Malbon jun N-terminal Kinase Mediates Activation of Skeletal Muscle Glycogen Synthase by Insulin in Vivo J. Biol. Chem., November 29, 1996; 271(48): 30765 - 30773. [Abstract] [Full Text] [PDF]
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S. Harada, R. M. Smith, J. A. Smith, M. F. White, and L. Jarett Insulin-induced egr-1 and c-fos Expression in 32D Cells Requires Insulin Receptor, Shc, and Mitogen-activated Protein Kinase, but Not Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase Activation J. Biol. Chem., November 22, 1996; 271(47): 30222 - 30226. [Abstract] [Full Text] [PDF]
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J.-F. Tanti, T. Gremeaux, S. Grillo, V. Calleja, A. Klippel, L. T. Williams, E. Van Obberghen, and Y. Le Marchand-Brustel Overexpression of a Constitutively Active Form of Phosphatidylinositol 3-Kinase Is Sufficient to Promote Glut 4Translocation in Adipocytes J. Biol. Chem., October 11, 1996; 271(41): 25227 - 25232. [Abstract] [Full Text] [PDF]
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V. Lefebvre, M.-C. Mechin, M. P. Louckx, M. H. Rider, and L. Hue Signaling Pathway Involved in the Activation of Heart 6-Phosphofructo-2-kinase by Insulin J. Biol. Chem., September 13, 1996; 271(37): 22289 - 22292. [Abstract] [Full Text] [PDF]
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G. J. Bhat, S. T. Abraham, and K. M. Baker Angiotensin II Interferes with Interleukin 6-induced Stat3 Signaling by a Pathway Involving Mitogen-activated Protein Kinase Kinase 1 J. Biol. Chem., September 13, 1996; 271(37): 22447 - 22452. [Abstract] [Full Text] [PDF]
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J. Si, Z. Luo, and L. Mei Induction of Acetylcholine Receptor Gene Expression by ARIA Requires Activation of Mitogen-activated Protein Kinase J. Biol. Chem., August 16, 1996; 271(33): 19752 - 19759. [Abstract] [Full Text] [PDF]
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O. Aharonovitz and Y. Granot Stimulation of Mitogen-activated Protein Kinase and Na+/H+ Exchanger in Human Platelets. DIFFERENTIAL EFFECT OF PHORBOL ESTER AND VASOPRESSIN J. Biol. Chem., July 12, 1996; 271(28): 16494 - 16499. [Abstract] [Full Text] [PDF]
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J. K. Klarlund, A. D. Cherniack, M. McMahon, and M. P. Czech Role of the Raf/Mitogen-activated Protein Kinase Pathway in p21ras Desensitization J. Biol. Chem., July 12, 1996; 271(28): 16674 - 16677. [Abstract] [Full Text] [PDF]
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