(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, 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.
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 ()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.
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. 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).
Treatment of cells with
insulin has also been shown to increase the activities of other
kinases, including pp90 (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).
Figure 4:
PD98059 blocks insulin-stimulated
phosphorylation of both pp90 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.
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-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.
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-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.
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 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.
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 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.
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.