Protein Kinase C
Activation Mediates Glucagon-Like Peptide-1Induced Pancreatic ß-Cell Proliferation
Jean Buteau1,
Sylvain Foisy1,
Christopher J. Rhodes2,
Lee Carpenter3,
Trevor J. Biden3, and
Marc Prentki1
1 Molecular Nutrition Unit, Department of Nutrition, University of Montreal, the Centre de Recherche du CHUM and Institut du Cancer, Montreal, Quebec, Canada
2 Pacific Northwest Research Institute & Department of Pharmacology, University of Washington, Seattle, Washington
3 Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia
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ABSTRACT
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Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic ß-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on ß-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic ß(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC)
isoform in INS as well as in dissociated normal rat ß-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1induced ß-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (
, ß, and
) or
aPKC isoforms. The PKC
pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKC
acting as a dominant negative protein suppressed GLP-1induced proliferation. In addition, ectopic expression of a constitutively active PKC
mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1induced activation of PKC
is implicated in the ß-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1induced PI-3K activation results in PKC
translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.
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INTRODUCTION
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Glucagon-like peptide-1 (GLP-1)-(7-36) amide, a potent glucoincretin hormone (1,2), is secreted by the intestinal L-cells in response to fat meals and carbohydrates (3,4). It is a potentially important drug in the treatment of diabetes in view of its ability to improve insulin secretion in both patients with impaired glucose tolerance and type 2 diabetes (5,6). GLP-1 is also an insulinotropic agent through its ability to stimulate insulin gene expression and proinsulin biosynthesis (7) and acts as a potent ß-cell growth factor (8). GLP-1 increases the expression level of the ß-cell specific transcription factor pancreatic and duodenal homeobox gene-1 (PDX-1) (8,9). In addition, the glucoincretin increases ß-cell proliferation nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)dependent manner in ß(INS-1) cells (8) as well as islet mass in mouse pancreas (9). However, the precise signal transduction pathway that mediates the proliferative action of GLP-1 is not completely elucidated.
PI-3K is a family of proteins known to be activated in response to various growth factors in different cell types (10). Many downstream effectors of PI-3K mediate proliferative signals. Extracellular signal-related kinases (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) are in some instances downstream targets of PI-3K (11), mediating the proliferative response of a variety of external signals (12). ERK 1/2 and p38 MAPK also promote cell growth by being involved in antiapoptotic processes (13). Glucose activates p38 MAPK in pancreatic ß-cell, an action that may be causally implicated in insulin gene induction by the sugar via phosphorylation of the transcription factor PDX-1 (14). However, the involvement of p38 in PDX-1 activation was challenged recently (15). Among other downstream effectors of PI-3K are phosphoinositide-dependent kinases (PDK), which in turn activate protein kinase B (PKB) (also named Akt) (16). PKB participates in proliferative signals in response to many stimuli in different cell types possibly via the activation of the mammalian target of rapamycin and p70s6 kinase (17). Other targets of PDK that could play a role in cell growth regulation include the atypical isoform
of protein kinase C (PKC) (18,19). PKC is a multigene family divided into three classes depending on their cofactor requirements: classical PKCs (cPKCs), which are sensitive to calcium/diacylglycerol and tumor-promoting phorbol esters; novel PKCs, which are sensitive to diacylglycerol and tumor-promoting phorbol esters only; and atypical PKCs (aPKCs), which are insensitive to all three regulators (20).
We report here that GLP-1 increases the PI-3K downstream targets ERK 1/2, p38 MAPK, and PKB activities nonadditively with glucose in INS(832/13) cells. GLP-1 also causes PKC
nuclear translocation. However, only PKC
and p38 MAPK are likely to be involved in GLP-1induced proliferation as revealed by tritiated thymidine incorporation measurements in the presence of specific inhibitors. The implication of the aPKC isoform
in the proliferative action of GLP-1 is demonstrated using recombinant adenoviruses, which allow expression of various PKC
constructs.
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RESEARCH DESIGN AND METHODS
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Reagents.
Pharmacological inhibitors (SB203580, PD98059, LY294002, KN-93, myristoylated PKC
, and cPKC [20,21,22,23,24,25,26,27,28] peptide inhibitors) were purchased from Biomol (Plymouth Meeting, PA). Human GLP-1 fragment 736 amide was obtained from Sigma (St. Louis, MO). The anti-PKC
antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-insulin antibody was from Sigma. RPMI-1640 and the supplements, including fetal calf serum, were purchased from Gibco BRL (Burlington, Ontario, Canada). Methyl [3H]-thymidine was from ICN (Costa Mesa, CA).
Cell culture and incubation.
INS(832/13) (21) cells (passages 3670) were grown in monolayer cultures as described previously (22) in regular RPMI-1640 medium supplemented with 10 mmol/l HEPES, 10% heat-inactivated fetal calf serum, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 50 µmol/l ß-mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified (5% CO2, 95% air) atmosphere. This clone (832/13) of INS-1 cell was used because it shows better differentiation characteristics in term of glucose-stimulated insulin secretion than the original INS-1 cells (21). When cells reached 80% confluence after
7 days, they were washed with phosphate-buffered saline (PBS) and preincubated at 37°C for 90 min in a Krebs-Ringer bicarbonate HEPES medium containing 1 mmol/l CaCl2, 5 mmol/l NaHCO3, 25 mmol/l HEPES (pH 7.4) supplemented with 3 mmol/l glucose and 0.1% defatted bovine serum albumin (BSA) (Fraction V; Sigma). Cells were then washed with PBS and incubated for the indicated times in the same supplemented Krebs-Ringer bicarbonate HEPES medium containing the substances to be tested.
In vitro kinase assays.
In vitro kinase activities were evaluated using MAPK (ERK 1/2), p38 MAPK, and PKB/Akt kinase assay kits from New England Biolab (Beverly, MA) according to the manufacturers protocol. In brief, cells that were cultured as described above were homogenized in lysis buffer (1% SDS, 60 mmol/l Tris-HCl [pH 6.8], 10% glycerol). The different kinases (ERK 1/2, p38 MAPK, and PKB/Akt) were then immunoprecipitated from 200 µg of cell lysate and resuspended in 40 µl of kinase buffer (25 mmol/l Tris [pH 7.5], 5 mmol/l ß-glycerophosphate, 2 mmol/l dithiothreitol [DTT], 0.1 mmol/l sodium-orthovanadate, and 10 mmol/l MgCl2) supplemented with 200 µmol/l ATP. Specific substrates (respectively Elk-1, ATF-2, and GSK3a for ERK 1/2, p38 MAPK, and PKB) were added, and the reactions were stopped after 30 min by adding SDS sample buffer containing 62.5 mmol/l Tris (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/l DTT, and 0.1% bromophenol blue. Twenty microliters of samples were loaded on SDS-PAGE gels for Western immunoblottings. Substrate phosphorylation was detected by incubation of the membranes with phospho-specific antibodies.
Immunofluorescence and confocal microscopy.
INS cells were cultured overnight on polyornithine-coated coverslips and stimulated as described in the legend to Fig. 2. After washing with PBS, the cells were fixed with 3.7% paraformaldehyde/PBS for 15 min at room temperature before incubation for 5 min with 0.1 mol/l glycine/PBS and permeabilization with 0.2% Triton X-100 in PBS for 2 min. For immunofluorescence, cells were blocked with 1% BSA/PBS for 10 min, incubated with PKC
primary antibodies at 10 µg/ml for 1 h, washed three times with PBS, stained with a goat anti-rabbit fluorescein secondary antibody (Pierce, Rockford, IL) for 1 h, and washed three times with PBS. Image acquisition was performed using a LSM-410 confocal microscope (Carl Zeiss).
Rat islets were isolated from 200-g Wistar rats as described previously (23) and trypsinized to obtain dissociated islet cells (24). Dissociated islet cells (corresponding to 100 islets per condition) were seeded on polyornithine-coated coverslips in six-well plates and cultured in regular RPMI for 24 h. Cells were then washed with PBS, incubated in the absence and presence of GLP-1, and subsequently fixed as described above for INS cells. For immunofluorescence, cells were blocked with 1% BSA/PBS for 10 min, incubated for 1 h with both a polyclonal PKC
(10 µg/ml) and a monoclonal mouse anti-insulin (10 µg/ml) primary antibody, washed three times with PBS, incubated for 1 h with both a goat anti-rabbit fluorescein secondary antibody (Pierce) and a rhodamine-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA), and finally washed three times with PBS. Image acquisition was performed using an LSM-410 confocal microscope (Carl Zeiss).
Preparation of nuclear extracts and immunoblot analysis of PKC
.
Nuclear extracts were isolated using a published procedure (25). Briefly, cells (40 x 106 per condition) previously grown in 225 cm2 Petri dishes were harvested with a rubber policeman in cold PBS, sedimented at 3,500 g for 4 min, and lysed in 1 ml of ice-cold buffer A (15 mmol/l KCl, 2 mmol/l MgCl2, 10 mmol/l HEPES [pH 7.4], 0.1% phenylmethylsulfonyl fluoride [PMSF], and 0.5% Nonidet P-40). After a 10-min incubation on ice, nuclei were collected by centrifugation (1,000g for 5 min) and washed with buffer A without Nonidet P-40. Nuclei were lysed in a buffer containing 2 mmol/l KCl, 25 mmol/l HEPES (pH 7.4), 0.1% EDTA, and 1 mmol/l DTT. After a 15-min incubation period on ice, a dialysis buffer (25 mmol/l HEPES [pH 7.4], 1 mmol/l DTT, 0.1% PMSF, 2 µg/ml aprotinin, 0.1 mmol/l EDTA, and 11% glycerol) was added to the nuclei preparations. Samples were centrifuged (16,000g for 20 min), and the supernatants containing the nuclear proteins were used for protein determinations, subsequently aliquoted (50 µl), and kept frozen at -70°C for subsequent immunoblot analysis. Lysates were subjected to electrophoresis on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were probed with a PKC
primary antibody and subsequently with peroxidase-conjugated goat anti-rabbit IgG. Signals were visualized by chemiluminescence, using ECL reagent (Amersham Pharmacia, Buckinghamshire, England).
Tritiated thymidine incorporation assay.
A previously described procedure was used (8,26). In brief, INS-1 cells were seeded 2 days before use in 96-well plates (8 x 104 cells/well) and cultured in regular RPMI medium as described above. Cells were then washed with PBS and preincubated for a period of 24 h in minimal RPMI medium, i.e., without serum and glucose but with 0.1% BSA. They were then incubated for 24 h in minimal RPMI medium with various test substances. Proliferation was determined by incorporation of [3H]-thymidine (1 µCi/well) during the final 4 h of the 24-h incubation period. Cells were then harvested with a PHD cell harvester (Cambridge Technology, Watertown, MA), and the radioactivity retained on the dried glass fiber filters was measured.
Adenovirus constructs.
PKC
cDNA was a gift from H. Mischak (Laboratory of Genetics, National Cancer Institute, Bethesda, MD). Dominant-negative (DN) kinase defective PKC
was generated by PCR mutagenesis using the pAlter system (Promega, Madison, WI) to give a K281-W substitution within the ATP-binding site. This mutation results in a DN mutant of PKC
, which has been shown to inhibit PKC
-stimulated nuclear-factor
B (NF
B) reporter gene activity in NIH 3T3 fibroblasts (27) and PKC
-dependent mitogenic activity in oocytes and fibroblasts (28,29). An adenoviral shuttle plasmid (pXCMV) was generated by subcloning the NruI/DraIII digested and blunted expression cassette from pRcCMV (Invitrogen, Carlsbad, CA) into XbaI digested and blunted pXCX3 (pXCX3 was derived from pXCX2). The PKC
constructs (wild-type [WT], DN, and constitutively active [CA]) were then subcloned into the EcoRV site of pXCMV. Recombinant adenoviruses were prepared essentially as described by Graham and Prevec (30). Cesium chloridepurified plasmids that contained the pXCMV PKC
gene cassettes were cotransfected with pJM17 (a circular form of the adenovirus genome) in HEK 293 cells at a ratio of 1:1 using calcium phosphate. Control virus MX17, which does not contain a gene cassette, was constructed by recombination between pXCX2 and pJM17. Once transfected, the HEK 293 cells were maintained in 0.5% agarose and 1x culture medium. Recombinant viruses were isolated 12 weeks later as single plaques and amplified by reinfecting confluent monolayers of HEK 293 cells. Recombinant viruses were prepared from lysates of cytopathic cells 37 days after the second round of infection. Medium from the 35-mm dish was used in further rounds of amplification to generate viral stocks that were purified by CsCl gradient. Plaque assays were performed in HEK 293 cells to determine the titer (pfu/ml) of these stocks, which were then used to infect INS(832/13) cells at the designated multiplicity of infection (MOI). CA PKC
was generated as described previously (31) by an A119E mutation in the pseudosubstrate site of PKC, a substitution that frees the catalytic region from the inhibitory constraint of being bound to the regulatory region.
Infections.
INS(832/13) cells were seeded 2 days before use in six-well plates (4 x 106 cells/well) and cultured in regular RPMI medium as described above. Cells were then incubated with different PKC
adenoviral constructions at an MOI of 10 pfu/cell for 5 h in 0.5 ml of complete RPMI medium. Fresh RPMI medium (1.5 ml) was then added to each well still containing 0.5 ml of media with viruses. Seven hours later, cells were trypsinized and plated in 96-well plates as described above to perform a tritiated thymidine incorporation assay after a 24-h incubation in the absence or presence of GLP-1.
Calculations and statistics.
Data are presented as mean ± SE. Statistical analyses were performed with the SPSS for Windows system. Differences between two conditions were assessed with Students t test for related samples. Differences were deemed to be significant at P < 0.05.
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RESULTS
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Activation of signal transduction pathways downstream of PI-3K by GLP-1.
ERK 1/2, p38 MAPK, and PKB are three potential downstream effectors of PI-3K. We wanted to determine whether their activities in the presence of GLP-1 correlate with cell growth measurements as evaluated with the tritiated thymidine incorporation assay. In vitro ERK 1/2, p38 MAPK, and PKB activities were studied after 1 h of incubation of INS(832/13) cells at 3 or 11 mmol/l glucose with or without 10 nmol/l GLP-1 (Fig. 1). The results indicate that GLP-1 at a maximal effective concentration of 10 nmol/l caused activation of ERK 1/2, p38 MAPK, and PKB at low (3 mmol/l) glucose. Elevated (11 mmol/l) glucose also increased the activity of the same kinases. The action of GLP-1 and glucose was not additive. GLP-1 caused a rise in tritiated thymidine incorporation in INS(832/13) cells to an extent similar to that of 11 mmol/l glucose, and the actions of both agents were not additive. The threshold half-maximum and maximum concentrations of GLP-1 at 3 mmol/l glucose on PKB activation were 0.01 and 10 nmol/l, respectively (data not shown). These values were similar to that obtained for INS(32/13) cell proliferation (data not shown). Overall, the results indicate that both GLP-1 and glucose activate ERK 1/2, p38 MAPK, and PKB and that the action of GLP-1 on the activation of these signaling pathways correlates with the proliferative response of the glucoincretin.

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FIG. 1. Effects of GLP-1 and glucose on ERK 1/2 (A), p38 MAPK (B), PKB (C), and DNA synthesis (D) in INS(832/13) cells. In vitro kinase activities were measured at 3 or 11 mmol/l glucose in the presence or absence of 10 nmol/l GLP-1. DNA synthesis was determined by tritiated thymidine incorporation. Cells were cultured in serum-free RPMI medium containing 3 or 11 mmol/l glucose in the presence or absence of GLP-1 for 24 h. One µCi/well of tritiated thymidine was added during the final 4 h of the 24-h incubation period. Representative immunoblots for each kinase activity measurement are shown. Data are mean ± SE of four independent experiments for kinase activities and four independent experiment each comprising three or four wells for cell proliferation assay. *P < 0.05.
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GLP-1 induces PKC
nuclear translocation.
PKC
, an atypical isoform of PKC, is an additional downstream target of both PI-3K and PDK (19,32). It is translocated from the cytoplasm to the nucleus after its activation by various stimuli (32). We therefore investigated whether GLP-1 causes nuclear translocation of PKC
in INS(832/13) cells as well as in dissociated normal rat ß-cells. Figure 2 shows that a 5-min exposure of INS cells to 10 nmol/l GLP-1 caused PKC
translocation to the nucleus as observed by confocal microscopy in association with immunofluorescence (Fig. 2, top two panels). It is interesting that GLP-1 caused a change in cell shape of INS cells, which appeared less flat and rounder than controls. PKC
nuclear translocation was also observed in dissociated rat ß-cells (identified by insulin staining) after a 2-min treatment with 10 nmol/l GLP-1 (Fig. 2, lower four panels). Figure 3 shows a time-dependent increase in PKC
immunoreactivity in nuclear fractions of INS-cells, with a maximum effect observed at 5 min and a return to basal value after a 30-min incubation period. Consistent with a causal implication of PI-3K in PKC
activation by GLP-1, the PI-3K inhibitor LY294002 (50 µmol/l) suppressed the GLP-1induced PKC
nuclear translocation (data not shown).
Specific inhibitors for PKC
and p38 MAPK suppress GLP-1induced ß-cell proliferation.
To obtain further insight into the signal transduction pathways implicated in the proliferative action of GLP-1, we used specific inhibitors for different kinases known to mediate cell proliferation in response to diverse stimuli. The inhibitors were tested at concentrations at which they are known to be effective without displaying major cytotoxicity in a variety of cell systems (33). Cellular proliferation was evaluated with the tritiated thymidine assay, as described before in INS cells (8,26). Figure 4 shows that the calmodulin-dependent kinase II inhibitor KN-93 and the mitogenic-extracellular signal-regulated kinase (MEK) inhibitor PD98059 did not affect significantly GLP-1induced proliferation, suggesting that calmodulin-dependent kinase II and ERK 1/2 are not involved in the ß-cell growth effect of the glucoincretin. The p38 MAPK inhibitor SB203580 suppressed GLP-1induced proliferation while strongly affecting basal thymidine incorporation as well. Further investigation was performed using isoform-specific pseudosubstrates to inhibit classical (
, ß, and
) or the
atypical isoforms of PKC. The PKC
pseudosubstrate blocked GLP-1induced INS(832/13)-cell proliferation, whereas the inhibitor of cPKC enzymes did not. The p38 MAPK, PKC
, and cPKC inhibitors reduced proliferation observed at basal glucose in the absence of GLP-1 (Fig. 4). However, no apparent cytotoxicity was observed as evaluated by morphologic examination of the cells under the microscope. Although we cannot exclude some cytotoxicity, we favor the view that the corresponding pathways are involved in cell proliferation under nonstimulated conditions. These observations provide pharmacological evidence for the implication of both PKC
and p38 MAPK in the cell growthpromoting action of GLP-1. Because pseudosubstrate peptides are highly specific enzyme inhibitors, the pharmacological evidence is particularly strong for PKC
.
GLP-1 induced ß-cell proliferation is altered by overexpressing PKC
mutants.
A molecular approach was used to document further the implication of PKC
in GLP-1induced proliferation. Thus, adenoviral constructs were used to increase the expression level of various PKC
proteins in INS(832/13) cells. Western blot studies showed that the WT and kinase-dead DN constructs were expressed
20-fold over basal PKC
at an MOI of 10 pfu/cell (data not shown). Overexpressing WT PKC
slightly but significantly enhanced basal proliferation without affecting maximum thymidine incorporation in the presence of GLP-1. The DN construct reduced GLP-1induced proliferation by
60%. CA PKC
increased ß(INS 832/13)-cell proliferation in the absence of GLP-1 to an extent similar to that occurring in the presence of the glucoincretin. In addition, GLP-1 did not further enhance thymidine incorporation under this condition (Fig. 5). Adenoviral infection by itself did not affect INS-cell proliferation because there was no significant difference in tritiated thymidine incorporation between uninfected cells and cells overexpressing ß-gal after infection with a ß-gal adenoviral construct (data not shown).
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DISCUSSION
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GLP-1, a potent glucoincretin hormone and a potentially important drug in the treatment of diabetes (26,34), was described recently as a growth factor in the ß(INS-1)-cell line (8) as well as in mouse islet tissue (9). However, the exact mechanism by which GLP-1 exerts its growth-promoting action remains to be defined.
Pharmacological and biological evidence has suggested that PI-3K plays a central role in the transduction of the GLP-1induced proliferative signal (8). The results of the present study indicate that GLP-1 and glucose activate nonadditively ERK 1/2, p38 MAPK, and PKB, three potential downstream targets of PI-3K, as they do for ß-cell growth (8). PKC
, an atypical isoform of PKC and downstream target of PDK, is also activated by the glucoincretin as evidenced from its translocation from the cytoplasm to the nucleus after GLP-1 treatment of INS(832/13) cells and dissociated rat ß-cells. The transient nuclear translocation of PKC
induced by GLP-1 in ß-cells is similar to that caused by nerve growth factor in PC12 cells in terms of intensity and duration (35). We therefore studied the effect of specific inhibitors of these signaling pathways activated by the glucoincretin on GLP-1induced tritiated thymidine incorporation. On the one hand, the MEK inhibitor PD98059 did not suppress GLP-1induced DNA synthesis. MEK is an upstream kinase and activator of ERK 1/2, thus indicating that the ERK 1/2 pathway is unlikely to be involved in this proliferative process. On the other hand, both the p38 MAPK inhibitor SB203580 and the PKC
pseudosubstrate suppressed the GLP-1induced growth response, thus providing pharmacological evidence for the implication of these particular pathways in this process. Moreover, overexpression of a DN PKC
in INS cells reduced the GLP-1induced tritiated thymidine incorporation, and increasing the expression level of a CA PKC
was sufficient to cause the proliferation of INS cells to the same extent as GLP-1. The combined pharmacological and molecular biology approaches allow the conclusion that PKC
activation is an important step in the proliferative signaling pathway(s) of the glucoincretin.
Recent studies suggested a role for PKC
nuclear translocation in the transduction of proliferative signals in response to various stimuli in different cell types (36,37,38). However, the precise way by which PKC
might activate cell growth is unclear because little is known about the targets of PKC
. An interesting candidate is NF
B, whose activation, which is followed by its nuclear translocation, is known to be modulated by PKC
(39). Thus, NF
B activation is an antiapoptotic signal in some cells (40). It is interesting that another candidate target of PKC
is the transcription factor PDX-1, which is also induced by GLP-1 (8,41). PDX-1 is a key ß-cellspecific transcription factor (42) that regulates the development of the endocrine pancreas (43,44) as well as a number of ß-cell genes, including those encoding insulin, glucokinase, and the glucose transporter GLUT2 (45,46). Moreover, PDX-1 expression has been shown to correlate with the proliferation of ß(INS) cells (8) and pancreatic ß-cell regeneration (47).
GLP-1 was already known to activate p38 MAPK in Chinese hamster ovary cells and rat insulinoma cells (RIN 1046-38) (48). In the present study, we showed that GLP-1 activates p38 MAPK in INS(832/13) cells as well and that p38 MAPK inhibition suppresses the proliferative action of GLP-1. Evidence has been obtained for a role for p38 MAPK along with ERK 1/2 in the mitogenic response of MIN6 ß-cells to serum (49). Because p38 MAPK phosphorylates numerous transcription factors and induces several immediate-to-early response genes involved in cell growth/apoptosis control (50,51), it can be postulated that p38 MAPK is also implicated in the growth-promoting action of GLP-1. The hypothesis that p38 MAPK could cross-talk with the PKB cascade to mediate a proliferative event is a possibility that requires evaluation in the ß-cell in view of a recent publication that documented such an effect in muscle cells (39).
In conclusion, our results indicate that GLP-1 increases ERK 1/2, p38 MAPK, and PKB activities in INS-1 cells nonadditively with glucose. GLP-1 also causes the translocation of PKC
, a downstream target of PI-3K, from the cytoplasm to the nucleus. However, utilization of specific kinase inhibitors reveals that only p38 MAPK and PKC
activation are likely to play a role in the GLP-1induced proliferative response. The use of recombinant adenoviruses to express various PKC
constructs has allowed the demonstration of the implication of PKC
in the GLP-1induced increase in DNA synthesis in INS(832/13) cells. The results are consistent with a model in which GLP-1induced PI-3K activation results in PKC
translocation to the nucleus, which may play a role in the long-term pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.
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ACKNOWLEDGMENTS
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This work was supported by grants from the Canadian Institute of Health Research and the Canadian Diabetes Association (to M.P.).
M.P. is a Canadian Institute of Health Research Scientist.
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FOOTNOTES
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Address correspondence and reprint requests to Dr. Marc Prentki, CR-CHUM, Pavillon de Sève, 4e, 1560 Sherbrooke Est, Montreal, PQ H2L 4M1, Canada. E-mail: marc.prentki{at}umontreal.ca.
Received for publication 16 November 2000 and accepted in revised form 29 June 2001.
aPKC, atypical protein kinase C; BSA, bovine serum albumin; CA, constitutively active; cPKC, classical protein kinase C; DN, dominant-negative; DTT, dithiothreitol; ERK, extracellular signal-related kinases; GLP-1, glucagon-like peptide-1; MAPK, mitogen-activated protein kinase; MEK, mitogenic-extracellular signal-regulated kinase; MOI, multiplicity of infection; NF
B, nuclear-factor
B; PBS, phosphate-buffered saline; PDK, phosphoinositide-dependent kinases; PDX-1, pancreatic and duodenal homeobox gene-1; PI-3K, phosphatidylinositol-3 kinase; PKB, protein kinase B; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type.
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