©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucose, Other Secretagogues, and Nerve Growth Factor Stimulate Mitogen-activated Protein Kinase in the Insulin-secreting -Cell Line, INS-1 (*)

(Received for publication, October 31, 1994; and in revised form, January 23, 1995)

Morten Frödin (1)(§) Nobuo Sekine (2)(¶) Enrique Roche (2) Chantal Filloux (1) Mark Prentki (2) Claes B. Wollheim (2) Emmanuel Van Obberghen (1)(**)

From the  (1)From INSERM, Unité 145, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cédex 2, France and the (2)Division de Biochimie Clinique, Département de Médecine, University of Geneva, CH-1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-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 beta-cell.


INTRODUCTION

Insulin secretion from the pancreatic beta-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 beta-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) (^1)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 beta-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 beta-cell.


EXPERIMENTAL PROCEDURES

Materials

Rabbit polyclonal antisera to the 12-amino acid carboxyl-terminal of 44-kDa MAP kinase and the 17-amino acid amino-terminal of MEK-1, synthesized by Neosystem (Strasbourg, France), were generated as described(33) . Mouse 2.5 S NGF, 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP), forskolin, phorbol 12-myristate 13-acetate, prolactin, human growth hormone, verapamil, sodium orthovanadate, bovine serum albumin, phenylmethylsulfonyl fluoride, bovine brain myelin basic protein, bovine serum albumin, cell culture reagents, leupeptin, protein A-Sepharose, and Triton X-100 were from Sigma. Pituitary adenylate cyclase-activating polypeptide 38 of ovine origin was from Peninsula Laboratories, Inc. Synthetic human glucagon-like peptide-1 was a kind gift from Dr. G. K. Hendrick (Boston, MA). Fura 2 acetoxymethyl ester was from Molecular Probes. [-P]ATP was from ICN.

Cell Culture and Experimental Conditions

The INS-1 cell line, established from an x-ray-induced rat transplantable insulinoma, was cultured in RPMI 1640 supplemented with 10% fetal calf serum and other additions as described(31) . The following procedure was used to prepare the cells for experimentation, with all incubations being at 37 °C in 92.5% atmospheric air, 7.5% CO(2). Cell monolayers were washed once with modified glucose-free Krebs-Ringer-bicarbonate-HEPES buffer (KRBH), pH 7.4, composed of 134 mM NaCl, 3.5 mM KCl, 1.2 mM KH(2)PO(4), 0.5 mM MgSO(4), 1.5 mM CaCl(2), 5 mM NaHCO(3), 10 mM HEPES, and 0.1% (w/v) bovine serum albumin. Cells were preincubated for 1 h in KRBH with 1 mM glucose and then shifted to glucose-free KRBH. Within the following 1.5 h, experiments were performed by adding test agents to this medium.

Immunopurification of 44-kDa MAP Kinase and MEK-1 from INS-1 Cell Extracts

After incubation with test agent of confluent cell monolayers (4.5 cm^2 and 10 cm^2/condition for MAP kinase and MEK assay, respectively), experiments were stopped by quickly aspirating the medium (which was saved for measurement of secreted insulin) and solubilizing cells for 15 min on ice with solubilization buffer containing 1% (v/v) Triton X-100, 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM Na(4)P(2)O(7), 2 mM Na(3)VO(4), 100 mM NaF, 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride. Cell extracts were clarified by centrifugation for 15 min at 18,000 times g and then incubated for 2 h with antibody to 44-kDa MAP kinase or MEK-1 preadsorbed to protein A-Sepharose beads. Following the immunoprecipitation period, the beads were washed three times with solubilization buffer. All manipulations were performed at 4 °C.

MAP Kinase Assay

Protein A-Sepharose beads with immunoprecipitated 44-kDa MAP kinase were washed two times with HNTG buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100) with 0.2 mM Na(3)VO(4), dehydrated with a syringe, and resuspended in 50 µl of HNTG buffer supplemented with 0.2 mM Na(3)VO(4), 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride. The kinase reaction was started by the addition of the following, given at final concentrations: 150 µg/ml myelin basic protein (as a MAP kinase substrate), 10 mM magnesium acetate, 1 mM dithiothreitol, and [-P]ATP (5 µM, 33 Ci/mmol). The phosphorylation reaction was allowed to proceed for 15 min at room temperature (linear assay condition) and was stopped by spotting Whatman P-81 filter papers with an aliquot of the reaction mixture and dropping them into 0.1% (v/v) orthophosphoric acid. The papers were washed overnight in this solution with several changes, washed once in ethanol, and dried. Radioactivity associated with the papers was determined by Cerenkov counting. The reaction blank (a sample in which cell lysate had been omitted during immunoprecipitation but otherwise treated like the rest of the samples) was subtracted from all values.

MEK Assay

The kinase activity of MEK-1 was measured in a reconstitution assay by the ability of immunopurified MEK-1 to activate bacterially expressed, recombinant rat 44-kDa MAP kinase, the activity of which was measured using myelin basic protein as a substrate. Protein A-Sepharose beads with immunoprecipitated MEK-1 were washed two times with 50 mM HEPES, pH 7.4, dehydrated with a syringe, and resuspended in 50 µl of HEPES buffer, pH 7.4, containing recombinant 44-kDa MAP kinase, 0.2 mM Na(3)VO(4), 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl fluoride. The in vitro phosphorylation cascade was started by the addition of (final concentrations) 50 µM [-P]ATP (33 Ci/mmol), 150 µg/ml myelin basic protein, 15 mM MgCl(2), and 1 mM EGTA. The phosphorylation reaction was allowed to proceed for 10 min at room temperature and was stopped by spotting Whatman P-81 filter papers with an aliquot of the reaction mixture and dropping them into 0.1% (v/v) orthophosphoric acid. The papers were washed overnight in this solution with several changes, washed once in ethanol, and dried. Radioactivity associated with the papers was determined by Cerenkov counting. The reaction blank (a sample in which cell lysate had been omitted during immunoprecipitation but otherwise treated like the rest of the samples) was subtracted from all values. Control experiments showed that omission of myelin basic protein or recombinant MAP kinase from the phosphorylation reaction mixture reduced radioactivity in the filter papers by 90%.

Measurement of Insulin Secretion

After incubation of monolayers of INS-1 cells with test agents, the medium was collected and immediately centrifuged at 4 °C for 3 min at 3000 times g to remove any detached cells. Thereafter, the medium was frozen until the insulin assay was performed. In experiments where MAP kinase activation was not concomitantly measured, insulin secretion was assayed in 96-well microtiter plates (approx100,000 cells/well). In these experiments, cell monolayers were washed twice with glucose-free KRBH, preincubated in the same buffer at 37 °C for 30 min, and thereafter incubated for 30 min with fresh KRBH buffer supplemented with test agents. After the incubation period, the medium was collected and centrifuged at 4 °C for 2 min at 3000 times g. Insulin content of the INS-1 cell media was determined by radioimmunoassay using rat insulin as a standard(34) .

[^3H]Thymidine Incorporation Assay

INS-1 cells in microtiter plates (20,000 cells/well) were cultured for 3 days in normal culture medium where serum had been replaced by a supplement modified from Clark and Chick (35) composed of 0.1% (w/v) human serum albumin (fraction V), 10 µg/ml transferrin, 0.1 nM triiodothyronine, 0.65 nM insulin-like growth factor-I, 50 µM ethanolamine, and 50 µM phosphoethanolamine. After two washes, the cells were incubated for another 3 days in the same medium containing agents to be tested. During the final 24 h, the cells were pulsed with [methyl-^3H]thymidine (0.5 µCi/well). Thereafter, the cells were collected on filter papers in a multichannel cell harvester (PHD, Cambridge Technology, Cambridge, MA). Radioactivity associated with the filters, containing the cell nuclei, was quantitated by liquid scintillation counting.

Measurement of Cytosolic Ca

INS-1 cells were plated at 50,000 cells/glass coverslip (21 times 26 mm) precoated with poly-L-ornithine. After 3 days, cells were loaded with 1 µM fura 2 acetoxymethyl ester added to the culture medium for 30 min preceding the experiment. The coverslips were then mounted in a recording chamber held at 37 °C. Cells were superfused with KRBH containing 2.8 mM glucose. Stimuli were delivered as square pulses by two large orifice glass pipettes connected to microsyringe pumps(36) . The microfluorimeter system consisted of a Nikon Diafut microscope and a rotating filter-wheel fluorimeter (constructed in the workshop of the Geneva University Medical Center). The cells were excited at 340 and 380 nm and emitted light recorded at 500 nm. Cytosolic Ca ([Ca](i)) was calculated from the ratio 340/380 using equations and constants as previously defined(36) .

Northern Blot Analysis

After incubation with test agents, 80% confluent INS-1 cells were lysed with guanidinium thiocyanate, and total RNA was extracted(37) . junB, nur77, and zif268 mRNAs were detected by Northern blot hybridization with antisense P-labeled riboprobes. The linearized plasmids used were pGEM-2-junB containing a 1180-base pair BamHi-EcoRI fragment (positions 423-1576), pGEM-2-nur77 containing a nur77 cDNA probe kindly provided by Dr L. Lau (University of Illinois, Chicago), and pBS-KS-zif268 containing a 690-base pair NdeI-BglII fragment (positions 1273-1963). The DNA probe of the ribosomal RNA 18 S, kindly provided by Dr I. Oberbaümer (Max-Planck-Institut, Martinsried, Germany), was used as unvariant control.


RESULTS

Secretagogues Activate MAP Kinase in INS-1 Cells

We first investigated the effect of glucose and various secretagogues on the activity of 44-kDa MAP kinase in INS-1 cells maintained in monolayer. Following exposure to secretagogues, INS-1 cells were solubilized, 44-kDa MAP kinase was immunopurified from the cell extract, and its activity was measured in vitro using myelin basic protein as a substrate. The exposure of INS-1 cells to glucose led to a small 2.5-fold increase in the activity of MAP kinase (Fig. 1A). Activation of MAP kinase by glucose was increased 3-4-fold by CPT-cAMP, a membrane-permeant cAMP analogue that activates cAMP-dependent protein kinase. In the absence of glucose, CPT-cAMP had little stimulatory effect. Activation of MAP kinase by glucose, in the absence or presence of CPT-cAMP, was sustained from 12 to 30 min (and remained at the same level for up to 60 min; not shown). Forskolin, which increases intracellular cAMP by activating adenylyl cyclase, reproduced the effect of CPT-cAMP (data not shown). To compare activation of MAP kinase with insulin secretion, we measured the amount of insulin that accumulated in the incubation medium during the experiment. We found that the 2.5-fold stimulation of MAP kinase by glucose was associated with a 2.5-fold increase in secreted insulin as measured after 30 min of incubation (Fig. 1B). Likewise, the potentiation of glucose-induced activation of MAP kinase by CPT-cAMP was associated with a large increase in insulin secretion. CPT-cAMP alone induced no detectable insulin release. To further investigate the correlation between stimulation of MAP kinase and secretion of insulin, we determined the concentration dependence of the two processes with respect to glucose, in the absence as well as in the presence of CPT-cAMP. The dose-response curves for stimulation of MAP kinase and for secretion of insulin were found to be superimposable (Fig. 2, A and B). Maximal stimulation was obtained at 10 mM glucose.


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 beta-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 (bullet) or 5 µM insulin (circle) or (B) 24 mM KCl in the absence (bullet) or presence (circle) 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 (circle) or together with 100 ng/ml 2.5 S NGF () or 1 µM PMA (bullet), 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.

Effect of Mitogens and Other Factors on MAP Kinase in INS-1 Cells

Prolactin and growth hormone (the latter not shown), which promote beta-cell proliferation(9) , did not stimulate MAP kinase in INS-1 cells, whereas fetal calf serum had a modest and transient stimulatory effect (Fig. 5). In contrast, nerve growth factor (NGF), a neurotrophic factor that is present in fetal and neonatal mouse islets(40) , was found to be a strong activator of MAP kinase in INS-1 cells (Fig. 5). NGF stimulated MAP kinase in a transient manner, showing a peak at 5 min followed by a rapid decline to a lower level sustained for 15-30 min.


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.



NGF Stimulates Insulin Secretion by INS-1 Cells in the Presence of Glucose

The strong activation of MAP kinase by NGF led us to investigate whether NGF promotes insulin secretion by INS-1 cells. In the absence of glucose (the experimental condition of Fig. 5), NGF failed to stimulate insulin secretion (Fig. 6A). In the presence of glucose, however, NGF enhanced by 2-fold the secretion of insulin (Fig. 6A). A dose-response analysis of NGF-stimulated insulin secretion showed that the factor was near optimal at 100-500 ng/ml (Fig. 6B). In contrast to NGF, PMA was found to induce insulin secretion when added alone, in addition to enhancing glucose-induced secretion by INS-1 cells (Fig. 6A).

The Role of Cytosolic Cain the Activation of MAP Kinase by Various Stimuli

Exposure to glucose leads to an increase in cytosolic Ca ([Ca](i)) in INS-1 cells due to stimulation of Ca influx through voltage-gated Ca channels as described (31, 41) and illustrated for single INS-1 cells in Fig. 7A. CPT-cAMP was found to transiently enhance glucose-induced Ca influx (Fig. 7A, 14 out of 18 cells examined). Moreover, chelation of extracellular Ca with EGTA inhibited by 70-90% the activation of MAP kinase by glucose added alone or together with CPT-cAMP (Table 1). Similarly, MAP kinase activation by these agents was attenuated by 30-60% by verapamil, a blocker of L-type, voltage-gated Ca channels (Table 1). EGTA inhibited the activation of MAP kinase by PMA by some 40% (Table 1). These findings suggest that Ca influx is required for MAP kinase activation by glucose, whereas in the case of PMA, it contributes but is not essential.


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](i) (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](i) transients (Fig. 7, B and C, 15 out of 17 cells examined). The NGF-induced rise in [Ca](i) 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](i) in the INS-1 cells (Fig. 7D).

Glucose, CPT-cAMP, and KCl Activate MEK-1 in INS-1 Cells

We next investigated the effect of glucose, CPT-cAMP, and KCl on MEK-1 in INS-1 cells. Exposure to glucose was found to activate MEK-1 in these cells (Fig. 8). Glucose-induced stimulation of MEK-1 was increased by CPT-cAMP, which had a small effect by itself. MEK-1 activation by glucose plus CPT-cAMP was inhibited by chelation of extracellular Ca with EGTA. Finally, KCl activated MEK-1 in a Ca-dependent manner. In conclusion, the similar activation pattern of MEK and MAP kinase suggests that glucose-, cAMP-, and Ca-stimulated pathways activate MAP kinase by activating MEK-1.


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.



Stimulation of DNA Synthesis in INS-1 Cells

MAP kinase is implicated in the regulation of proliferation of various cell types. We therefore measured DNA synthesis by INS-1 cells in response to secretagogues and other agents to investigate whether this process was correlated with MAP kinase activity. Raising the concentration of glucose from 3 to 15 mM stimulated DNA synthesis as measured after a 72-h incubation with a [^3H]thymidine pulse during the final 24 h (Fig. 9). CPT-cAMP had no effect on DNA synthesis in low (3 mM) glucose medium but inhibited high (15 mM) glucose-induced INS-1 cell DNA synthesis. This inhibition, however, was not seen with forskolin, raising the possibility that the effect of CPT-cAMP was not cAMP specific. In any case, the effect of glucose and of increased cAMP on DNA synthesis was not well correlated with the effect of these agents on MAP kinase activity. Prolactin and growth hormone (the latter not shown) were efficient mitogens for INS-1 cells, both under low and high glucose conditions (Fig. 9), although these factors did not activate MAP kinase (confer Fig. 5). Conversely, NGF did not stimulate DNA synthesis by INS-1 cells, neither under low nor high glucose conditions (Fig. 9), despite being a strong activator of MAP kinase in these cells (confer Fig. 5). Finally, for comparison is shown DNA synthesis in response to 10% fetal calf serum and 11 mM glucose, which are the normal culture conditions for INS-1 cells.


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 [^3H]thymidine during the final 24 h of incubation and expressed in percent of basal [^3H]thymidine incorporation (firstbar, set to 100%). Data are means ± S.E. of three to six experiments performed in triplicates. FCS, fetal calf serum.



Induction of Early Response Genes in INS-1 Cells

Finally, we investigated the induction of junB, nur77, and zif268, which are early response genes that code for transcription factors. Glucose caused no or very weak induction of either of the genes (Table 2). CPT-cAMP had a small effect on junB and nur77. Glucose and CPT-cAMP added together, however, caused synergistic induction of all three genes, which in the case of junB was markedly attenuated by EGTA, thus showing a correlation with MAP kinase activation by these agents. KCl, on the other hand, had only little effect on the expression of the early response genes, possibly due to the swift and transient action of KCl on INS-1 cells (confer Fig. 4), whereas gene induction was measured after 1 h of stimulation. NGF caused a small induction of the genes, most markedly of zif268. For comparison is shown early gene induction by a mixture of PMA, forskolin, and ionomycin, which in the case of junB was shown to be inhibited by EGTA.




DISCUSSION

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 beta-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. (^2)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,^2 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. (^3)

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 beta-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 beta-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 beta-cells(9) . Alternatively, MAP kinase might regulate beta-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.^2 A role of MAP kinase in beta-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.


FOOTNOTES

*
This work was supported by funds from INSERM, Université de Nice-Sophia-Antipolis, Association pour la Recherche contre le Cancer Grant 6760, Ligue Nationale Française contre le Cancer, Fédération des Comités Départementaux, Comité Départemental du Var, Groupe LIPHA (Lyon, France; Contract 93123), the Danish Cancer Society Grant 90-070, the Swiss National Science Foundation Grant 32-32376.91, 32-32343.91, and 32-32505.91, and a grant from the U. S. Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The recipient of an EMBO fellowship. Present address: Dept. of Clinical Chemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark.

On leave from the 4th Dept. of Internal Medicine, University of Tokyo, Japan.

**
To whom all correspondence and reprint requests should be addressed. Tel.: 33-93-81-54-47; Fax: 33-93-81-54-32.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; CPT-cAMP, 8-(4-chlorophenylthio)cyclic AMP; GLP-1, glucagon-like peptide-1; MEK, MAP kinase/ERK kinase; NGF, nerve growth factor; PACAP38, pituitary adenylate cyclase-activating polypeptide 38-amino acid form; PKC, protein kinase C; PMA, 12-myristate 13-acetate.

(^2)
M. Frödin and E. Van Obberghen, unpublished observation.

(^3)
N. Sekine and C. B. Wollheim, unpublished observation.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Joseph Avruch (Boston, MA) for providing bacteria expressing rat GST-44-kDa MAP kinase fusion protein. We thank Dr. Guodong Li (Geneva, Switzerland) for help with the initial measurements of cytosolic Ca.


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