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Department of Biochemistry, Faculty of Medicine, Universidad Autónoma de Madrid, and Department of Experimental Endocrinology, Puerta de Hierro Hospital, 28029 Madrid, Spain
Address all correspondence and requests for reprints to: Juan E. Felíu, M.D., Ph.D., Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain. E-mail: juane.feliu{at}uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and other cytosolic proteins
(4). There is also evidence that stimulation of PI 3-K is
involved in the activation of p70 S6-kinase (p70 S6-K) by insulin
(8). On the other hand, tyrosine-phosphorylated IRSs and
Shc also interact with SH-2 domains of growth factor receptor-binding
protein 2 (1, 2). This protein binds proline-rich
motifs on SOS through its SH-3 domains, which results in the activation
of Ras. This is followed by translocation of Raf-1 to the plasma
membrane and activation of the mitogen-activated protein kinase (MAPK)
cascade (9, 10). The involvement of the above-mentioned signaling cascades in the long- and short-term metabolic effects elicited by insulin, such as stimulation of glucose transport (11), acceleration of both glycogen (12, 13) and protein synthesis (14), inhibition of lipolysis (15), and repression of both phosphoenolpyruvate carboxykinase and glucose 6-phosphatase gene transcription (16, 17) has been recently established. However, the signaling pathways involved in the short-term control of key regulatory steps of hepatic gluconeogenesis by insulin have not been elucidated. In the liver, the glycolytic/gluconeogenic pathway is tightly regulated by hormones such as insulin, glucagon, and epinephrine, acting at both the fructose 6-P/fructose 1,6-P2 and the phosphoenolpyruvate/pyruvate substrate cycles (18, 19). In the latter substrate cycle, the short-term hormonal control is exerted by modulating PK-L activity. This enzyme is regulated by allosteric effectors and phosphorylation/dephosphorylation mechanisms. In the liver, glucagon can elicit the phosphorylation and inactivation of PK-L through the stimulation of a cAMP-dependent protein kinase (18, 19). Dephosphorylation and reactivation of PK-L appear to be mediated by protein phosphatases 2A and/or 2C (20, 21, 22). It is known that insulin may antagonize the effects of glucagon on PK-L by decreasing the hepatic concentration of cAMP (18, 19). Furthermore, insulin may induce the activation of PK-L without significant changes in the hepatic content of cAMP (19, 23, 24) by a process in which the dephosphorylation of the enzyme has been implicated (23, 24, 25). In connection with this, it has been demonstrated that the activation of PK-L by insulin in isolated rat hepatocytes is accompanied by a transient increase in pyruvate kinase phosphatase activity (22, 25).
In this work we have investigated the potential involvement of PI 3-K/PKB and p44/p42 MAPK [extracellular signal-regulated kinase 1/2 (ERK-1/ERK-2)] signaling pathways as well as p70 S6-K in the insulin-induced activation of PK-L in primary cultures of rat hepatocytes. Our results show that the presence of PI 3-K inhibitors, such as wortmannin or LY294002, in the incubation medium significantly reduced the activation of PK-L by insulin. Furthermore, PD98059 at a concentration that completely blocked insulin signaling through the MAPK cascade caused a 50% reduction in insulin-induced activation of PK-L, an effect that was further reinforced by the additional presence of a suboptimal concentration of wortmannin. It was also observed that the blockade of p70 S6-K by rapamycin did not significantly affect the activation of PK-L by insulin. These results indicate that both p44/p42 MAPK and PI 3-K signaling pathways, but not p70 S6-K, are implicated in the short-term regulation of PK-L by insulin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-phosphatidylinositol were
obtained from Sigma (St. Louis, MO). Rapamycin, PD98059,
and LY294002 were purchased from Calbiochem (San Diego,
CA). Insulin was from Novo Industri (Copenhagen, Denmark). Earle’s
medium 199 was obtained from ICN Iberica, S.A. (Barcelona, Spain).
Collagenase A, substrates, auxiliary enzymes, and coenzymes were
purchased from Roche Molecular Biochemicals (Mannheim,
Germany). [-32P]ATP (3 Ci/µmol) and
enhanced chemiluminescence reagents were provided by Amersham Pharmacia Biotech (Barcelona, Spain). The remaining reagents,
all of analytical grade, were obtained from Roche,
Sigma, or Merck & Co., Inc. (Darmstadt,
Germany).
Animals
Fed male Wistar rats from our inbred colony were used. The
animals were maintained on standard chow (A.04 Panlab SL, Barcelona,
Spain) and water ad libitum and were housed in animal
quarters at constant temperature (23 C) with a fixed light cycle (12
h). The animal experimentation described has been conducted in
accordance with the highest standards of humane animal care. All
animals were anesthetized with sodium pentobarbital (5 mg/100 g BW)
immediately before the experiments.
Culture conditions and cell incubations
Hepatocytes were isolated by perfusion of the liver with
collagenase (26) and suspended in medium 199 supplemented
with 10% FBS, 0.1% BSA, 1 µM T3, 1
µM dexamethasone, and glucose up to 10 mM.
Hepatocytes were plated to attain a cell density of 1.2 x
105 cells/cm2 and cultured
in a cell incubator at 37 C in an atmosphere of 5%
CO2. Four hours after plating, the medium was
replaced by fresh supplemented medium 199. After 19 h of culture,
hepatocyte monolayers were washed three times with PBS and incubated
for an additional 2 h in medium 199 supplemented with 20
mM HEPES (pH 7.4), 0.1% BSA, and 10 mM
glucose. Then, cells were treated with insulin for 5 or 10 min, as
indicated. Wortmannin, LY294002, and rapamycin were added to the
incubation medium 10 min before the addition of insulin, whereas
PD98059 was incorporated into the medium 30 min before the addition of
hormone. Appropriate vehicle controls were included in every
experiment.
Analytical procedures
For the measurement of pyruvate kinase activity, hepatocyte
monolayers were washed once with ice-cold 0.9% NaCl and immediately
frozen in liquid N2 at the indicated times. Then,
cell monolayers were thawed in a medium containing 50 mM
glycylglycine, 15 mM EGTA, and 100 mM potassium
fluoride, pH 7.4, and homogenized with a Polytron PT 1200C
(Kinematica AG, Lucerne, Switzerland). After centrifugation (10,000 x
g for 5 min), pyruvate kinase activity was determined in the
supernatants using a spectrophotometric assay as described by
Felíu et al. (27). The incubation
mixture contained 50 mM glycylglycine (pH 7.4),
0.1 M KCl, 10 mM
MgCl2, 1.25 mM Mg-ADP, 0.15
mM NADH, and 1 U lactate dehydrogenase in a total
volume of 1 ml. Aliquots of the supernatant were added to the mixture
and allowed to stand for 10 min at 30 C; the reaction was started by
the addition of either 0.15 mM
(v0.15) or 5 mM
phosphoenolpyruvate (Vmax).
For immunoprecipitations, hepatocytes were washed once with ice-cold 0.9% NaCl and immediately homogenized in a lysis medium containing 50 mM HEPES (pH 7.4), 10 mM Na4P2O7, 100 mM NaF, 10 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM benzamidine, and 2 mM phenylmethylsuflonylfluoride. Cell lysates were centrifuged (10,000 x g for 30 min), and the supernatants were incubated for 3 h with the corresponding antibody. Then, the immunocomplexes were captured with protein A-agarose (Sigma), as indicated by the manufacturer. All of these procedures were carried out at 4 C. PI 3-K activity was assayed in either anti-IRS1 or anti-IRS2 immunoprecipitates by 32P incorporation into phosphatidylinositol (28).
For immunoblotting, cell lysates or immunoprecipitates were subjected to SDS-PAGE. Proteins were transferred to PVDF membranes and blocked with 5% nonfat dried milk dissolved in a solution (TTBS) containing 20 mM Tris (pH 7.4), 0.05% Tween-20, and 150 mM NaCl. Then, PVDF membranes were probed with the corresponding antibody and washed four times (5 min each) with TTBS. Afterward, membranes were incubated with horseradish peroxidase conjugated to antirabbit or antimouse IgG (1:2000). Finally, membranes were washed four times with TTBS and developed by enhanced chemiluminescence. All immunoprecipitations, immunoblots, and PI 3-K activity assays were performed in at least two separate experiments.
Statistical analysis
Where applicable, data are expressed as the mean ±
SEM, with n equal to the number of different experiments.
Paired Student’s t test was performed when two groups were
compared. One-way ANOVA with repeated measures was used, followed by
Tukey-Kramer’s test, when multiple groups were compared (Instat,
GraphPad Software, Inc., San Diego, CA). Differences were
considered statistically significant when P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This was
accompanied by a dose-dependent association of p85 with phosphorylated
IRSs and an activation of PI 3-K. It is of note that clear increases in
tyrosine phosphorylation of both IRS-1 and IRS-2, in p85 association,
and in PI 3-K activity were observed at the lowest insulin
concentration tested (0.1 nM).
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). It is well established that the PK-L
activity ratio is inversely related to the degree of phosphorylation of
the enzyme (18, 29). Moreover, in good agreement with
other reports (23, 25), the activation of PK-L by insulin
was still evident after ammonium sulfate precipitation of the enzyme
(data not shown), suggesting that a covalent change in the PK-L
molecule (possibly dephosphorylation) occurs in response to insulin
(22, 23, 24, 25).
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).
The differences in insulin-induced PK-L activation observed in the
presence of 100 and 500 nM wortmannin could be related to
distinct degrees of PI 3-K inhibition. To check this possibility, PI
3-K activity was measured in anti-IRS-1 and anti-IRS-2
immunoprecipitates obtained from hepatocytes incubated with increasing
concentrations of insulin in the absence and presence of the inhibitor.
As shown in Fig. 3, both 100 and 500
nM wortmannin strongly inhibited insulin-dependent
activation of PI 3-K associated with IRS-1 and IRS-2, at all of the
assayed insulin concentrations. However, slight differences appeared to
exist between residual PI 3-K activities found in hepatocytes incubated
in the presence of 100 and 500 nM wortmannin. To assess the
consequence of these slight differences in PI 3-K activity in steps of
the insulin signaling cascade downstream of this enzyme, we examined
the degree of phosphorylation of PKB on both
Thr308 and Ser473 residues,
which is known to be dependent on PI 3-K activation
(5, 6, 7). As shown in Fig. 4A, cultured hepatocytes incubated with different concentrations of insulin
for 5 min showed a dose-dependent increase in the phosphorylation
status of both Thr308 and
Ser473 residues. The presence of 100
nM wortmannin completely blocked the phosphorylation of PKB
elicited by 0.1 nM insulin. However, some residual PKB
phosphorylation was detected in hepatocytes treated with 1 and 10
nM insulin. When 500 nM wortmannin was used, a
complete suppression of both Thr308 and
Ser473 phosphorylation was observed at all the
assayed insulin concentrations (Fig. 4B). A good direct correlation
appeared to exist between insulin-mediated PK-L activation and the
extent of PKB phosphorylation in the presence of 100 and 500
nM wortmannin.
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, insulin increased the extent of phosphorylation of both ERK-1
and ERK-2 in a dose-dependent manner. The effect on ERK-1 was far lower
than that observed on ERK-2; this is in agreement with the data
reported by Peak et al. (13), who found a
barely detectable phosphorylation and activation of ERK-1 in response
to either insulin or epidermal growth factor in rat hepatocytes. When
cultured hepatocytes were preincubated with 100
nM wortmannin, insulin-induced phosphorylation of
ERK-1/ERK-2 was partially inhibited (Fig. 4A), whereas it was
completely abolished, at all the assayed insulin concentrations, in
hepatocytes treated with 500 nM wortmannin (Fig. 4B). All of these findings indicate that in cultured rat hepatocytes
wortmannin is a powerful inhibitor of both PI 3-K- and
MAPKdependent pathways.
Effect of LY294002 on insulin-induced PK-L activation
To further support the involvement of PI 3-K in the activation of
PK-L by insulin, LY294002, a PI 3-K inhibitor not structurally related
to wortmannin (3), was used. As shown in Fig. 4C, treatment of cultured hepatocytes with LY294002 (50 µM)
completely blocked the insulin-dependent activation of the PI 3-K/PKB
pathway at all of the assayed hormone concentrations. When the
influence of LY294002 on the MAPK signaling pathway was tested (Fig. 4C), it was observed that, in contrast to what occurred with
wortmannin, insulin-induced phosphorylation of ERK-1/ERK-2 was not
reduced, but even appeared to be increased. These results were in good
agreement with those reported by Zimmerman and Moelling
(33), who showed that the blockade of PKB activation by
LY294002 increased insulin-like growth factor I-mediated activation of
the MAPK cascade in HEK-293 and MCF-7 cells.
When the influence of LY294002 on the insulin-dependent activation of
PK-L was studied, it was observed that 50 µM LY294002
significantly reduced the PK-L activity ratio in hepatocytes incubated
under basal conditions (0.168 ± 0.012 vs. 0.105
± 0.014, for control and LY294002-treated hepatocytes, respectively;
by paired Student’s t test, P < 0.001;
n = 4 experiments). This effect was not due to any interference of
LY294002 with the assay of PK-L activity (data not shown). Furthermore,
LY294002 clearly blocked the activation of PK-L by subsaturating
concentrations of insulin (0.1 nM). At higher
concentrations of hormone (1 and 10 nM), the PK-L
activity ratio increased, but to levels far below those reached in
hepatocytes incubated in the absence of LY294002 (Fig. 5).
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).
When hepatocytes were incubated with 50 µM PD98095 in the
absence of insulin, a small, but statistically significant, increase
was observed in the basal PK-L activity ratio (0.154 ± 0.08
vs. 0.173 ± 0.08 for control and PD98059-treated
hepatocytes, respectively; by paired Student’s t test,
P < 0.05; n = 6 experiments; Fig. 6). We have no clear explanation for this
effect, but the presence of this inhibitor in the reaction mixture did
not interfere with the assay of PK-L activity (data not shown).
Nevertheless, as also shown in Fig. 6, PD98059, at a concentration that
completely blocked the MAPK cascade (Fig. 4D), significantly reduced
the activation of PK-L elicited by saturating concentrations of insulin
(1 and 10 nM). Moreover, when insulin-induced
PK-L activation was quantified as a percentage of the respective basal
value, a partial, but statistically significant, reduction of the
insulin effect was observed at all of the assayed hormone
concentrations in hepatocytes treated with PD98059 (50%, 47%, and
45% reduction vs. the corresponding control incubations for
0.1, 1, and 10 nM insulin, respectively;
P < 0.001). The additional presence of 100
nM wortmannin in the incubation medium enhanced
the inhibitory effect elicited by PD98059 on the insulin-mediated
activation of PK-L. This enhancement was statistically significant at
0.1 and 1 nM insulin. All of these findings
suggest that both PI 3-K and MAPK pathways are involved in the
short-term activation of PK-L by insulin.
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, maximal PKB phosphorylation in
response to 10 nM insulin was achieved as early as 2 min
after hormone treatment, and this kinase remained phosphorylated for at
least 20 min. Nevertheless, phosphorylation of p70 S6-K on
Thr389 residue, which is essential to bring about
full kinase activation (37), was barely detected 4 min
after insulin stimulation and increased gradually until min 20 (Fig. 7A). As shown in Fig. 7B, the phosphorylation of p70 S6-K by insulin
was also dose dependent and was completely suppressed at all of the
assayed hormone concentrations when cultured hepatocytes were treated
with 100 nM rapamycin. Under these conditions, the
phosphorylation of PKB at Ser473 was not
inhibited. As shown in Fig. 8, this same
concentration of rapamycin did not significantly affect the activation
of PK-L by insulin. These findings indicate that the activation of p70
S6-K is not required for the insulin-dependent activation of PK-L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PI 3-K/PKB signaling pathway has been shown to play a central role in the modulation of glucose metabolism by insulin. Thus, activation of this pathway has been shown to mediate insulin effects on GLUT-4 translocation to the plasma membrane in L6 myoblasts (11) as well as on the activation of glycogen synthase and the stimulation of glycogen synthesis in different cell types, including cultured hepatocytes (12, 13). To investigate the possible involvement of the PI 3-K-dependent signaling pathway in the insulin-mediated activation of PK-L, wortmannin, a fungal metabolite that is a known inhibitor of PI 3-K activity (3, 30), was used. Thus, 100 nM wortmannin totally suppressed the activation of PK-L elicited by a subsaturating concentration of insulin (0.1 nM), whereas the inhibitory effect of this agent was only partial at 1 and 10 nM insulin. These findings were paralleled by an inhibition of insulin-stimulated PI 3-K activity and PKB phosphorylation, elicited by this concentration of wortmannin in cultured rat hepatocytes. When a higher concentration of wortmannin (500 nM) was used, the activation of PK-L was completely blocked at all of the assayed insulin concentrations. Under these conditions, insulin signaling through the PI 3-K/PKB pathway was totally suppressed in cultured hepatocytes, as indicated by the complete blockade of PKB phosphorylation. It is of note that PKB phosphorylation on both Thr308 and Ser473 is a well known PI 3-K-dependent process (5, 6, 7). All of these findings suggest that the PI 3-K pathway is involved in the short-term activation of PK-L by insulin.
However, it must be mentioned that wortmannin has been reported to reduce the activation of the MAPK cascade in response to insulin in L6 cells (31), but not in human skeletal muscle (38). Considering these controversial data, we also investigated the possible influence of wortmannin on the activation of the MAPK signaling pathway by insulin in cultured rat hepatocytes. We observed that at a concentration of 100 nM, this inhibitor partially antagonized insulin effects on the phosphorylation of ERK-1/ERK-2, whereas 500 nM wortmannin completely suppressed these insulin effects. Taking into account the fact that wortmannin was able to inhibit both PI 3-K and MAPK pathways in cultured rat hepatocytes, the wortmannin effect on insulin-mediated PK-L activation could be due to the blockade of either of these two pathways, or both. The mechanism by which wortmannin inhibits the MAPK pathway is not well understood; it has been suggested that this agent may block the activation of this pathway between Ras and Raf in L6 cells (31), whereas a decrease in GTP loading of Ras has been observed in 3T3L1 adipocytes (39).
To further investigate the involvement of PI 3-K in insulin-induced PK-L activation, LY294002, a selective PI 3-K inhibitor, was used. In our cell system, this inhibitor blocked insulin signaling through the PI 3-K/PKB pathway, and in contrast to the results obtained with wortmannin, LY294002 did not reduce but, rather, increased the insulin effect on the MAPK cascade. In this respect it has been demonstrated in different types of cells that the activation of PKB results in Raf phosphorylation on Ser259, which reduces its kinase activity and consequently the activation of the p44/p42 MAPK pathway (33, 40). Furthermore, inhibition of PKB activation by LY294002 has been shown to increase Raf and ERK activities in HEK 293 and MCF-7 cells (33). Our results suggest that this cross-regulation between PI 3-K/PKB and MAPK pathways may also occur in cultured hepatocytes.
When the influence of LY294002 on insulin-induced PK-L activation was investigated, it was observed that this inhibitor, in contrast to what occurred with wortmannin, caused a significant reduction in the PK-L activity ratio in hepatocytes incubated in the absence of insulin, although we have no clear explanation for this phenomenon. In hepatocytes treated with a subsaturating concentration of insulin (0.1 nM), LY294002 almost completely blocked the activation of PK-L, whereas at higher concentrations of insulin (1 and 10 nM), the effect of LY294002 was markedly reduced. However, in these conditions the values for the PK-L activity ratio attained in response to insulin were far lower than those reached in control hepatocytes. From these data and from the studies carried out with wortmannin, it can be concluded that the PI 3-K signaling pathway is implicated in the short-term activation of PK-L by insulin. In addition, it must be pointed out that the difference between the degrees of inhibition of insulin-mediated PK-L activation elicited by 500 nM wortmannin and 50 µM LY294002 may have been due to the fact that wortmannin at this concentration blocked the activation of the MAPK cascade, whereas LY294002 did not.
p70 S6-K is an enzyme that lies downstream of PI 3-K in the insulin signaling pathway (9). In previous studies (12, 13), rapamycin, a potent inhibitor of this kinase, has been used to study the possible involvement of p70 S6-K in the regulation of glycogen synthase activity by insulin. In our work we observed that 100 nM rapamycin, a concentration that completely blocked the phosphorylation of p70 S6-K in Thr389, did not significantly affect insulin-mediated activation of PK-L in cultured rat hepatocytes. This finding allows us to rule out the implication of this kinase in the signaling cascade that leads to the short-term activation of PK-L by insulin.
To analyze the possible involvement of the p44/p42 MAPK cascade in the activation of PK-L by insulin, PD98059 was used. Thus, we observed that 50 µM PD98059, which completely blocked ERK-1/ERK-2 phosphorylation without affecting PI 3-K/PKB activation, led to a 50% inhibition of the insulin-induced activation of PK-L. This finding strongly suggests that the activation of the p44/p42 MAPK pathway plays a relevant role in the short-term activation of PK-L by insulin. The additional presence of 100 nM wortmannin in the incubation medium significantly reinforced the inhibitory effect of PD98059 on insulin-induced activation of PK-L. As the signal transduction through the MAPK pathway was completely suppressed by the presence of PD98059, this wortmannin action must be attributed to the partial inhibition of PI 3-K elicited by this agent. In good agreement with this, when both the PI 3-K and MAPK pathways were completely blocked by the presence of 500 nM wortmannin, activation of PK-L by insulin was totally suppressed.
The activation of the PI 3-K/PKB pathway has been shown to be essential for most of the metabolic actions of insulin (3), whereas the MAPK cascade has been considered to be implicated mainly in mitogenic signals (10). Nevertheless, recent reports have affirmed that both pathways are involved in different actions of insulin and insulin-like growth factor I, such as protection of cells from apoptosis (41), stimulation of protein synthesis (42), and regulation of insulin receptor trafficking (43). Moreover, cross-talk between the PI 3-K and MAPK pathways has been reported at different levels in several cell systems (33, 39, 40, 44), suggesting that integration of signals through different pathways is crucial for the coordinated responses of cells to hormonal stimuli.
In summary, our findings provide evidence that both p44/p42 MAPK and PI 3-K pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin.
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Acknowledgments |
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Footnotes |
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Received July 27, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and 500 nM (
)
wortmannin or vehicle (0.005% dimethylsulfoxide; •). Afterward,
insulin was added at the indicated concentrations; 10 min later, cell
monolayers were frozen and kept in liquid nitrogen until PK-L activity
was assayed. Results are the mean ± SEM (n = 4
experiments). **, P < 0.01; ***,
P < 0.001 (vs. the corresponding
vehicle incubations).
) or
vehicle (0.3% dimethylsulfoxide; •). Afterward, insulin was added at
the indicated concentrations; 10 min later, cell monolayers were frozen
and kept in liquid nitrogen until PK-L activity was assayed. Results
are the mean ± SEM (n = 4 experiments). ***,
P < 0.001 vs. the corresponding
basal incubations; ***, P < 0.001
vs. respective control incubations.
