(Received for publication, October 31, 1994; and in revised form, January 23, 1995)
From the
The signaling pathways whereby glucose and hormonal
secretagogues regulate insulin-secretory function, gene transcription,
and proliferation of pancreatic -cells are not well defined. We
show that in the glucose-responsive
-cell line INS-1, major
secretagogue-stimulated signaling pathways converge to activate 44-kDa
mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin
secretion was found to be associated with a small stimulatory effect on
44-kDa MAP kinase, which was synergistically enhanced by increased
levels of intracellular cAMP and by the hormonal secretagogues
glucagon-like peptide-1 and pituitary adenylate cyclase-activating
polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent
on Ca
influx and may in part be mediated by MEK-1, a
MAP kinase kinase. Stimulation of Ca
influx by KCl
was in itself sufficient to activate 44-kDa MAP kinase and MEK-1.
Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP
kinase by both Ca
-dependent and -independent
pathways. Nerve growth factor, independently of changes in cytosolic
Ca
, efficiently stimulated 44-kDa MAP kinase without
causing insulin release, indicating that activation of this kinase is
not sufficient for secretion. In the presence of glucose, however,
nerve growth factor potentiated insulin secretion. In INS-1 cells,
activation of 44-kDa MAP kinase was partially correlated with the
induction of early response genes junB, nur77, and zif268 but not with stimulation of DNA synthesis. Our findings
suggest a role of 44-kDa MAP kinase in mediating some of the
pleiotropic actions of secretagogues on the pancreatic
-cell.
Insulin secretion from the pancreatic -cells is the result
of an interplay of multiple nutrient and hormonal stimuli (reviewed in (1) and (2) ). Glucose, a major physiological stimulus
for insulin release, signals through the production of metabolic
coupling factors of mainly mitochondrial origin, which leads to
membrane depolarization and the opening of voltage-gated Ca
channels (reviewed in (3) and (4) ). The
subsequent rise in cytosolic Ca
is thought to trigger
secretion(5) . Glucose-induced insulin secretion is potentiated
by hormones and neurotransmitters that activate adenylyl cyclase or
phospholipase C, thereby generating signals that enhance the action of
glucose at various points in the secretory pathway (reviewed in Refs.
1, 2, and 6). Secretagogue action is not confined to control of the
exocytotic process; it also includes the regulation of gene
transcription, proliferation, and other processes of the
-cell (7, 8, 9) . However, since many signaling
pathways remain ill-defined, a major task is the further delineation of
signaling events, especially of processes participating in signal
integration.
Mitogen-activated protein (MAP) ()kinase
(also called extracellular signal-regulated kinase) comprises a family
of serine/threonine kinases activated by growth factors, hormones, and
neurotransmitters in a cell type-specific manner ( (10) and (11) and reviewed in 12-14). MAP kinase has been
implicated in the regulation of proliferation, differentiation, and
cellular metabolism. MAP kinase is activated through phosphorylation on
tyrosine and threonine by the dual specificity kinase MEK(15) .
MEK, in turn, is phosphorylated and activated by Raf-1 (16, 17, 18) and
B-Raf(19, 20) . The Raf kinases are regulated by Ras
in its GTP-bound, active state(21, 22) , which, at
least in the case of Raf-1, appears to occur via recruitment to the
plasma membrane, where it becomes activated by an unknown mechanism (23, 24) . In addition to Raf, another family of MEK
kinases has been discovered that may be regulated by Ras(22) .
Although Ras is a major entry point on which diverse extracellular
signals converge to activate MAP kinase, some pathways may activate
Raf-1 or MEK in a Ras-independent
manner(24, 25, 26, 27) . Signaling
via the Ras-MAP kinase pathway is thought to diverge primarily at the
level of MAP kinase, which appears to phosphorylate a multitude of
substrates, including protein kinases, protein phosphatases, structural
proteins, and transcription factors, thereby linking cell surface
signals with gene
transcription(12, 13, 14, 28) . This
signal diversification may in part explain the ability of the Ras-MAP
kinase pathway to elicit a complex cellular
response(29, 30) .
In the present study, we have
investigated the regulation of MAP kinase in INS-1 insulinoma cells,
which exhibit a secretory and enzymatic profile characteristic of
normal -cells(31, 32) . We show that the MAP
kinase cascade can be added to the list of signaling pathways
stimulated by nutrient and hormonal secretagogues, a finding that may
help explain the pleiotropic actions of secretagogues on the
-cell.
Figure 1: Effect of glucose and CPT-cAMP on 44-kDa MAP kinase activity (A) and insulin secretion (B) by INS-1 cells. Monolayers of INS-1 cells were incubated with glucose (15 mM) and CPT-cAMP (1 mM) in combination as indicated and for the periods of time shown. Thereafter, the medium was collected, and the cells were solubilized. A, 44-kDa MAP kinase was immunopurified from the cell extracts, and its kinase activity was measured in vitro using myelin basic protein as a substrate as described under ``Experimental Procedures.''. MAP kinase activity is expressed as -fold stimulation compared with its activity in untreated cells. B, the amount of insulin in the incubation medium was determined by radioimmunoassay as described under ``Experimental Procedures'' and expressed as ng/ml. Data are means ± S.D. of triplicate determinations. The experiment was performed twice with similar results.
Figure 2: Dose-response relationship of glucose-induced stimulation of 44-kDa MAP kinase and insulin secretion by INS-1 cells. Monolayers of INS-1 cells were incubated with glucose at the concentration indicated in the absence (A) or presence of 1 mM CPT-cAMP (B). After 30 min of incubation, the medium was collected, and the cells were solubilized. The kinase activity of 44-kDa MAP kinase immunopurified from the cell extracts and the insulin content of the incubation medium were measured and expressed in percent of the value obtained with 15 mM glucose. Data are means ± S.D. of three experiments performed in triplicates.
We next investigated MAP kinase
activation by two hormonal/neurotransmitter secretagogues,
glucagon-like peptide-1 (GLP-1) (38) and pituitary adenylate
cyclase-activating polypeptide 38 (PACAP38)(39) , both of which
stimulate cAMP synthesis in -cells. GLP-1 and PACAP38 increased by
2-5-fold, depending on the experiment, the effect of glucose on
MAP kinase activity, while having little effect when added alone (Fig. 3). PACAP27, an alternatively processed PACAP form, also
stimulated MAP kinase in the presence of glucose (data not shown).
Figure 3: Effect of glucose, GLP-1, and PACAP38 on 44-kDa MAP kinase activity in INS-1 cells. INS-1 cells were incubated for 15 min with glucose (15 mM), GLP-1 (50 nM), or PACAP38 (10 nM) as indicated and thereafter solubilized. The kinase activity of immunopurified 44-kDa MAP kinase was measured and expressed as -fold stimulation compared with its activity in untreated cells. Data are means ± S.D. of triplicate determinations. The experiment was performed twice with similar results.
Activation of the insulin receptor leads to stimulation of MAP kinase in the physiological target tissues of insulin. Addition of insulin at concentrations ranging from 10 nM to 5 µM, however, did not significantly stimulate MAP kinase in INS-1 cells. An experiment with 5 µM insulin is shown in Fig. 4A. When experiments were performed at 20 °C, glucose plus CPT-cAMP failed to induce any measurable insulin secretion but still activated MAP kinase (data not shown). These experiments suggest that secreted insulin is not involved in secretagogue-induced activation of MAP kinase.
Figure 4:
Effect of phorbol ester, insulin, and KCl
on 44-kDa MAP kinase activity in INS-1 cells. INS-1 cells were exposed
to (A) 1 µM PMA () or 5 µM insulin (
) or (B) 24 mM KCl in the absence
(
) or presence (
) of 5 mM EGTA added 2 min before
the KCl. After incubation for the periods of time shown, the cells were
solubilized, and the activity of immunopurified 44-kDa MAP kinase was
measured and expressed as -fold stimulation compared with its activity
in untreated cells. Data are means ± S.D. of three experiments
performed in triplicates.
Phorbol ester (PMA), an activator of protein kinase C (PKC) and a secretagogue in INS-1 cells (see Fig. 6A), was found to activate MAP kinase (Fig. 4A). Maximal stimulation was reached within 15 min and was sustained thereafter.
Figure 6:
Effect of NGF, PMA, and glucose on insulin
secretion by INS-1 cells. Monolayers of INS-1 cells were incubated for
30 min with glucose (A) at the concentration indicated, added
either alone () or together with 100 ng/ml 2.5 S NGF (
) or 1
µM PMA (
), or with 2.5 S NGF (B), at the
concentration indicated, together with glucose (15 mM).
Thereafter, the medium was collected, and its insulin content was
measured and expressed as percent of insulin secretion in response to
15 mM glucose. Data are means ± S.E. of four to eight
observations.
Finally, MAP kinase was activated
after exposure of INS-1 cells to KCl, which depolarizes the membrane
potential, leading to influx of extracellular Ca through voltage-gated Ca
channels (Fig. 4B). A swift peak of MAP kinase activity was
observed at 2.5 min of exposure to KCl, followed by a rapid decline to
a low level sustained for up to 30 min. Chelation of extracellular
Ca
with EGTA abolished the activation of MAP kinase
by KCl, suggesting that the effect of KCl was mediated by
Ca
influx.
Figure 5: Effect of prolactin, NGF, and fetal calf serum (FCS) on 44-kDa MAP kinase activity in INS-1 cells. INS-1 cells were incubated with prolactin (1 nM), 2.5S NGF (100 ng/ml), or fetal calf serum (10%) for the periods of time shown. Thereafter, the cells were solubilized, and the activity of immunopurified 44-kDa MAP kinase was measured and expressed as -fold stimulation compared with its activity in untreated cells. Data are means ± range of two experiments performed in triplicates.
Figure 7:
Effect of glucose, CPT-cAMP, and NGF on
cytosolic Ca, measured in single fura 2-loaded INS-1
cells. The basal superfusion medium contained 2.8 mM glucose
in A, B, and D but contained no glucose in C. As indicated by bars, glucose (at the
concentration shown), CPT-cAMP (1 mM), 2.5 S NGF (100 ng/ml),
or EGTA (2.5 mM) were added in a square-wave manner from a
pipette placed close to the cell. Cytosolic Ca
([Ca
]
) is
expressed in nM. Each trace was reproduced from 5 to 18 times
with similar results as described in the
text.
In the
absence of glucose, NGF failed to raise
[Ca]
(Fig. 7C,
5 out of 5 cells examined). Furthermore, activation of MAP kinase by
NGF was not inhibited by EGTA (Table 1). Thus, NGF utilizes a
Ca
-independent pathway to activate the MAP kinase
cascade in INS-1 cells. In the presence of 15 mM glucose,
however, NGF was found to induce [Ca
]
transients (Fig. 7, B and C, 15 out of
17 cells examined). The NGF-induced rise in
[Ca
]
was due to influx of
extracellular Ca
, since it was abolished by EGTA (Fig. 7D, 18 out of 18 cells examined) and 20
µM verapamil (10 out of 10 cells examined, not shown).
Note that reexposure to Ca
after withdrawal of EGTA
caused a transient rise in [Ca
]
in the INS-1 cells (Fig. 7D).
Figure 8: Effect of glucose, CPT-cAMP, and KCl on MEK-1 activity in INS-1 cells. Monolayers of INS-1 cells were incubated for 15 min with CPT-cAMP (1 mM), glucose (15 mM), or KCl (24 mM) as indicated. When present, EGTA (5 mM) was added 2 min before the other agents. After the incubation period, the cells were solubilized, and MEK-1 was immunoprecipitated from the cell extracts. The activity of immunopurified MEK-1 was measured in a reconstitution assay by its ability to activate recombinant 44-kDa MAP kinase, the activity of which was measured using myelin basic protein as a substrate. The activity of MEK-1 is expressed as -fold stimulation compared with its activity in untreated INS-1 cells. Data are means ± S.D. of triplicate determinations. The experiment was performed three times with similar results.
Figure 9:
Effect of secretagogues, prolactin (PRL), and NGF on DNA synthesis by INS-1 cells. Serum-starved
INS-1 cells were incubated for 72 h in supplemented, serum-free culture
medium in the presence of test agent as indicated. DNA synthesis in the
cultures was assessed by a pulse of [H]thymidine
during the final 24 h of incubation and expressed in percent of basal
[
H]thymidine incorporation (firstbar, set to 100%). Data are means ± S.E. of three
to six experiments performed in triplicates. FCS, fetal calf
serum.
The present findings in INS-1 cells suggest that three major
signaling pathways employed by nutrient and hormonal secretagogues, i.e. the Ca-, cAMP-, and PKC-stimulated
pathways, converge on the MAP kinase cascade in the
-cell. Because
of a high degree of cross-talk among signaling pathways in this cell
type, it is difficult to dissect the mechanism by which any given
secretagogue activates MAP kinase. For instance, our results indicate
that Ca
influx is a prerequisite for glucose-induced
activation of MAP kinase. Subsequent to Ca
influx,
however, divergent pathways, e.g. PKC or
Ca
/calmodulin-dependent protein kinase
II(42) , may propagate the signal(s) leading to activation of
MAP kinase. In the case of phorbol ester,
Ca
-dependent as well as -independent pathways
contributed to the activation of MAP kinase. The
Ca
-independent mechanism may involve direct
phosphorylation and activation of Raf-1 by PKC(26) . Increased
levels of cAMP per se had little effect on MAP kinase in INS-1
cells, contrasting to a large potentiating effect on MAP kinase
activation by glucose as well as by PMA or NGF. (
)The
potentiation by cAMP of glucose-induced stimulation of MAP kinase may
in part be due to cAMP enhancement of Ca
influx.
However, cAMP was able to potentiate MAP kinase activation by NGF in
the presence of EGTA,
suggesting that cAMP acts also at a
point distal to Ca
influx to enhance MAP kinase
activation. Our data suggest that glucose and the cAMP- and
Ca
-stimulated pathways act at least at the level of
MEK to activate MAP kinase in INS-1 cells. Upstream of MEK, multiple
divergent pathways for activation of MAP kinase may be operating. It
will be of particular interest to determine whether cytosolic
Ca
activates the MAP kinase cascade through Ras in
INS-1 cells, as shown recently in rat pheochromocytoma PC12
cells(43) .
In smooth muscle cells(44) , adipocytes(45) , Chinese hamster ovary cells(45) , and fibroblasts(46, 47, 48) , cAMP inhibits the activation of MAP kinase by external stimuli, whereas only in PC12 cells cAMP has so far been shown to stimulate MAP kinase, acting, at least partly, at the level of MEK(49) . cAMP inhibits activation of the MAP kinase cascade by interfering with Ras activation of several MAP kinase kinase kinases, including Raf-1, B-Raf, and 98-kDa MEK kinase, and this inhibitory mechanism(s) seems to operate even in PC12 cells(19, 20, 22, 47) , leaving the question, how cAMP activates MEK and MAP kinase, unresolved. The finding that cAMP stimulates MEK and MAP kinase also in INS-1 cells suggests that cAMP activation of the MAP kinase cascade is a more widely occurring response than previously believed.
INS-1
cells and other insulin-secreting cell lines express two types of NGF
receptors, the trkA receptor tyrosine kinase and the p75 NGF receptor
(p75)(50) . In PC12 cells, trkA-mediated
activation of Ras constitutes a major pathway by which NGF activates
MAP kinase. The role of p75
in NGF signaling is
controversial, but this receptor may stimulate cAMP synthesis in PC12
cells(51) , thereby generating additional signaling pathways
for activation of MAP kinase in this cell type (49) and
possibly also in INS-1 cells. Our data show that in INS-1 cells,
Ca
influx is dispensable for MAP kinase activation by
NGF. In contrast, the inability of NGF to stimulate insulin secretion
by these cells in the absence of glucose probably relates to its
failure to stimulate Ca
influx under these
conditions. The mechanism by which NGF potentiates glucose-induced
insulin secretion remains to be established. Enhancement of
Ca
influx, activation of MAP kinase, or generation of
cAMP are possible mechanisms. The physiological importance of NGF as an
insulin secretagogue is unclear, as it was not detected in islets of
adult mouse(40) . Furthermore, long-term (3 days) exposure of
INS-1 cells to NGF did not alter responsiveness to glucose or insulin
production. (
)
The finding that MAP kinase is activated by
major secretagogue signaling pathways, some of them acting
synergistically, and the close correlation between MAP kinase
activation and insulin secretion in response to some secretagogues
suggest that MAP kinase may regulate the secretory function of
-cells. MAP kinase activation, however, was clearly not sufficient
for secretion, since NGF activated MAP kinase without stimulating
secretion in the absence of glucose. cAMP-dependent protein kinase
activation, however, is also not sufficient for secretion but
nevertheless believed to play an important role by potentiating
glucose-induced insulin release(52, 53) . Similarly,
MAP kinase could have a modulatory function in secretion. Alternatively
to a role in the stimulus-secretion coupling mechanism, MAP kinase may
regulate secretion-related processes, such as glucose metabolism or
insulin synthesis, possibly at the transcriptional level. Although
secretagogues induce the transcription of genes implicated in
-cell function, many regulatory pathways remain
unknown(7, 8) . The induction of early response genes junB, nur77, and zif268 was correlated with
activation of MAP kinase by glucose and CPT-cAMP, suggesting that MAP
kinase may mediate secretagogue regulation of these genes.
Based on
the poor correlation with DNA synthesis, our data do not support a role
of the MAP kinase pathway in INS-1 cell proliferation. However, since
INS-1 cells are tumor cells with aberrant growth regulation, the data
must be interpreted with reservation. It is possible that MAP kinase
mediates glucose- and secretagogue-stimulated proliferation of normal
-cells(9) . Alternatively, MAP kinase might regulate
-cell differentiation. In this respect, NGF has been proposed to
be implicated in the development of the endocrine pancreas (50) , where evidence for its presence has been obtained in
fetal and neonatal mouse(40) . Moreover, we have found that
basic fibroblast growth factor, which promotes islet development in
vitro(54) , efficiently stimulates (10-20-fold) MAP
kinase in INS-1 cells.
A role of MAP kinase in
-cell
differentiation could imply that secretagogues may also regulate this
process.
Finally, the observation that glucose stimulates MEK and MAP kinase in INS-1 cells adds a nutrient to the list of extracellular signals that activate the MAP kinase cascade, illustrating further the versatility of this signal transduction pathway in multicellular organisms.