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Endocrinology Vol. 143, No. 10 3802-3812
Copyright © 2002 by The Endocrine Society

ARTICLE

Activation of Phosphatidylinositol 3-Kinase Contributes to Insulin-Like Growth Factor I-Mediated Inhibition of Pancreatic ß-Cell Death

Wenli Liu, Catherine Chin-Chance, Eun-Jig Lee and William L. Lowe, Jr.

Department of Medicine, Veterans Affairs Chicago Healthcare System, Lakeside Division, and Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15-703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To begin to determine whether IGF-I treatment represents a potential means of enhancing the survival of islet cell grafts after transplantation, the present studies established a model of ß-cell death secondary to loss of trophic support and examined the ability of IGF-I to prevent cell death. The studies were performed using the rat pancreatic ß-cell line, INS-1. Incubating INS-1 cells in RPMI 1640 and 0.25% BSA for 48 h increased cell death, as determined by lactate dehydrogenase release, compared with that of cells maintained in RPMI and 10% fetal calf serum. Addition of 100 ng/ml IGF-I to the serum-free medium decreased lactate dehydrogenase release to a level comparable to that found in cells maintained in fetal calf serum. Similar results were seen using a mouse ß-cell line, MIN6, infected with an adenovirus expressing IGF-I. Examination of IGF-I-stimulated signaling demonstrated that IGF-I increased the phosphorylation of protein kinase B in both cell lines, whereas IGF-I-induced phosphorylation of the MAPKs, ERK1 and -2, was observed only in INS-1 cells. The effect of IGF-I on phosphorylation of substrates of phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase B was also examined in INS-1 cells. IGF-I increased the phosphorylation of glycogen synthase kinase 3ß, BAD, FKHR, and p70S6 kinase. Another pathway that has been shown to mediate the protective of IGF-I in some cell types is activation of cAMP response element-binding protein (CREB). IGF-I increased CREB phosphorylation at a concentration as low as 10 ng/ml, and this effect was inhibited by H89, a PKA inhibitor, and PD98059, a MAPK kinase inhibitor. Consistent with the effect of IGF-I on CREB phosphorylation, IGF-I increased the transcriptional activity of CREB, although it had no effect on CREB binding to DNA. Use of inhibitors of the PI 3-kinase (LY 294002) or ERK (PD98059) pathways or CREB phosphorylation (H89) in the cell death assay demonstrated partial abrogation of the protective effect of IGF-I with LY 294002. These data demonstrate that IGF-I protects pancreatic ß-cells from cell death secondary to loss of trophic support and that, although IGF-I activates several signaling pathways that contribute to its protective effect in other cell types, only activation of PI 3-kinase contributes to this effect in ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I AND -II are homologous mitogenic peptides that mediate their proliferative effect via activation of the IGF-I receptor. Both peptides are expressed in a wide range of tissues throughout development, including pancreatic islets (1, 2, 3, 4). Several lines of investigation have suggested that signaling through the IGF-I receptor modulates ß-cell mass. In vitro studies have demonstrated a proliferative effect of IGF-I on pancreatic ß-cells (5). Moreover, mice with null mutations of the IGF-I receptor and/or signaling molecules important in IGF-I-mediated signal transduction demonstrate decreased ß-cell mass (6, 7). In addition to its role as a mitogen, IGF-I is a potent inhibitor of cell death in pancreatic islets and other cell types (8). Previous studies have demonstrated that IGF-I prevents apoptosis in islets or ß-cells in response to cytokine treatment and during developmentally regulated pancreatic remodeling (9, 10, 11, 12, 13, 14, 15).

Recently, enhanced success using islet cell transplantation to treat type 1 diabetes mellitus has been reported (16, 17). An important limitation of this success is the requirement for two or more transplants to achieve euglycemia. The reason for this limitation is not clear, although previous studies using experimental models of islet transplantation have demonstrated a loss of graft ß-cell mass during the early posttransplant period (18, 19, 20). This loss is thought to be due in part to apoptosis and/or necrosis caused by loss of trophic support secondary to hypoperfusion before revascularization of the transplanted islets (20, 21). This raises the possibility that preventing cell death during the early posttransplant period may augment graft mass and enhance the success of transplantation. Given its ability to inhibit islet cell death in response to various stimuli, IGF-I treatment represents a potential means of enhancing graft survival.

To begin to address that issue, the present studies were designed to establish a model of cell death secondary to loss of trophic support (serum deprivation) using two pancreatic ß-cell lines, INS-1 and MIN-6, and to determine whether IGF-I is able to prevent cell death upon loss of trophic support. In addition, the signaling pathways activated by IGF-I in INS-1 cells were examined to begin to define the signal transduction pathways that mediate the protective effects of IGF-I in pancreatic ß-cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The glucose-sensitive pancreatic ß-cell line, INS-1, were provided by Dr. Claes Wollheim (University Medical Center, Geneva, Switzerland). The cells were maintained as previously described in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS), 10 mM HEPES (pH 7.6), 1 mM sodium pyruvate, 2 mM glutamine, 50 µM ß-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin, and incubated at 37 C in a 5% CO2 and 95% air atmosphere (22). Cells were subcultured at 70–80% confluence. MIN6 cells were the gift of Dr. Jun-ichi Miyazaki (Osaka University Medical School, Osaka, Japan) (23). MIN6 cells were grown as described in DMEM supplemented with 15% FCS, 40 mM sodium bicarbonate, 70 µM ß-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated at 37 C in a 5% CO2 and 95% air atmosphere (23). Cells were subcultured at 70–80% confluence.

Generation of recombinant adenoviral vectors
A cassette containing the human IGF-I-A cDNA (provided by Dr. A. Joseph D’Ercole, University of North Carolina, Chapel Hill, NC) containing the rat somatostatin signal sequence to allow for secretion (24) was subcloned into an adenoviral transfer plasmid (25) based upon pCDNA3 (Invitrogen, Carlsbad, CA) 3' to either the cytomegalovirus (CMV) promoter or a 680-bp fragment of the rat insulin II promoter (RIP) truncated immediately 5' to the initiation codon on exon 1 (RIP was prepared using PCR, and the sequence of the fragment was confirmed by DNA sequence analysis). The resulting plasmid was used to generate recombinant adenovirus. Linearized transfer plasmid containing 393 bp of adenoviral sequence 5' to the CMV-IGF-I or RIP-IGF-I expression cassette was ligated with ClaI-digested Ad5 309/356 DNA representing map units 3.0–100. Ad5 309/356 is a recombinant adenovirus in which the E3 region is deleted. ClaI digestion removes the E1a region, resulting in replication-deficient virus. The ligation products were transfected into 293 cells, in which cellular expression of the E1a protein allows replication of the E1-deleted recombinant viruses. The cloned and purified adenoviral vectors were titrated by plaque assay. Adenoviruses expressing IGF-I were designated Ad-CMV-IGF and Ad-RIP-IGF. Ad-CMV-ß-Gal and Ad-RIP-ß-Gal, which contain ß-galactosidase driven by either the CMV or RIP II promoter, were used as controls.

Cell death assay
INS-1 cells were plated at a density of 2 x 105 cells/well in 12-well plates in normal growth medium (RPMI 1640 and 10% FCS plus the additional supplements as described above). The cells were allowed to attach overnight and then were washed twice and placed back into either normal growth medium or RPMI 1640 and 0.25% BSA in the presence or absence of 100 ng/ml IGF-I. The cells were cultured for an additional 48 h, and then cell death was determined using a cytotoxicity detection kit (Roche, Mannheim, Germany). This assay determines the percentage of cell death, which is measured as lactate dehydrogenase (LDH) released into the medium divided by maximal LDH released per well after addition of Triton X-100 to a final concentration of 1% (100% death). LDH was measured using a colorimetric assay according to the manufacturer’s instructions. Each independent experiment was performed in duplicate.

For MIN6 cells, cells were plated at a density of 5 x 105 cells/well in 12-well plates in normal growth medium (DMEM and 15% FCS plus the additional supplements as described above). The cells were allowed to attach overnight and then were infected with recombinant adenoviral vectors at a multiplicity of infection of 10 plaque-forming units/cell for 16 h in Opti-MEM (Life Technologies, Inc., Grand Island, NY; Invitrogen, San Diego, CA) and 2% FCS. After subsequent incubation for 24 h in normal growth medium, the cells were washed twice and placed into DMEM and 0.25% BSA with 15 mM glucose. The cells were cultured for an additional 48 h, and then cell death was determined using a cytotoxicity detection kit (Roche) as described above. Each independent experiment was performed in duplicate.

Western blot analysis
Antibodies directed against phospho-ERK1/2, protein kinase B (PKB), phospho-PKB (Ser473), BAD, phospho-BAD (Ser112 and Ser136), phospho-glycogen synthase kinase (phospho-GSK-3ß; Ser9), FKHR, phospho-FKHR (Ser256), cAMP response element-binding protein (CREB), phospho-CREB, p70S6 kinase, phospho-p70S6 kinase (Thr389), I{kappa}B, and phospho-I{kappa}B-{alpha} (Ser32; all of the above from Cell Signaling Technology, Beverly, MA) were used at a dilution of 1:1000. Antibodies directed against ERK2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and GSK-3ß (Transduction Laboratories, Inc., Lexington, KY; and BD PharMingen, San Diego, CA) were used at dilutions of 1:7500 and 1:1000, respectively.

Western blot analyses were performed as described previously (26). Briefly, INS-1 cells were plated at a density of 1 x 106 cells/75-cm2 plate, and MIN6 cells were plated at a density of 2.5 x 106 cells/28-cm2 plate. When cells reached about 60% confluence, they were washed and placed into serum-free medium (RPMI 1640 and 0.25% BSA or DMEM and 0.25% BSA) for approximately 14 h before being treated with IGF-I. After treatment with IGF-I, cell lysates were prepared in RIPA cell lysis buffer, and the protein content of the lysate was determined using the Coomassie blue protein assay. Forty micrograms of protein were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane in a semidry apparatus. The membranes were blocked in 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween 20 (TBST) and 4% nonfat dry milk for 60 min at room temperature. Membranes were incubated overnight at 4 C in TBST containing 5% BSA and primary antibody, washed three times for 15 min each time at 22 C in TBST, and incubated for 60 min at room temperature in TBST containing 4% nonfat dry milk and secondary antibody (1:4000 dilution). After three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence detection system from Amersham Pharmacia Biotech (Arlington Heights, IL), according to the manufacturer’s instructions. The intensities of the bands were quantified using scanning densitometry.

EMSA
Nuclear extracts were prepared by collecting cells in 5 ml PBS containing 10 µl 0.5 M EDTA, 5 µl 1 M dithiothreitol, and 25 µl 200 mM phenylmethylsulfonylfluoride. The cells were resuspended in 400 µl cold buffer A (10 mM HEPES (pH 7.9), 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mM p-aminobenzamidine). The cells were incubated on ice for 15 min, followed by the addition of 25 µl 10% Nonidet P-40. The cells were mixed vigorously for 10 sec and centrifuged. The resulting pellet was resuspended in 50 µl cold buffer C (20 mM HEPES, pH 7.9; 0.4 M KCl; 1.0 mM each of EDTA, EGTA, dithiothreitol, and phenylmethylsulfonylfluoride; 20% glycerol; 1 µg/ml pepstain A; 10 µg/ml leupeptin; 10 µg/ml aprotinin; and 0.1 mM p-aminobenzamidine) and incubated at 4 C with shaking for 15 min. The mixture was clarified by centrifugation for 5 min, and the supernatant was collected. The protein concentration was determined using the Coomassie blue protein assay.

For the EMSA, annealed oligonucleotides containing the consensus sequence for CREB (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 3'-TCTCTAACGGACTGCAGTCTCTCGATC-5') were radiolabeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA) and were separated from unincorporated nucleotides over a Sephadex G-25 spin column (Pharmacia Biotech, Piscataway, NJ). Protein binding was performed at 20 C for 20 min in 20 µl 2 mM Tris-Cl, 2 mM KCl, 0.2 mM EDTA, 0.2 mM dithiothreitol, 2.5% glycerol, and 0.01% BSA with 2 µg poly(dI-dC), 5 x 105 cpm 32P-labeled oligonucleotides, and 10 µg nuclear protein. To verify the specificity of the binding reaction, a 100-fold excess of unlabeled oligonucleotide was added to the reaction before adding the labeled probe. For supershift assays, 2 µg antibodies against CREB or activating transcription factor-1 (ATF-1; Santa Cruz Biotechnology, Inc.) were preincubated with nuclear extract for 30 min before the addition of labeled probe. Samples were separated by electrophoresis on a 4% Tris-borate/EDTA polyacrylamide gel at 80 V for 1 h and then at 120 V for 1 h. Gels were dried and exposed for the appropriate period at -70 C with intensifying screens.

Transient transfection and luciferase assays
Plasmids expressing wild-type Gal4-CREB (pGal4-CREB-WT), mutant Gal4-CREB (pGal4-CREB-MUT) in which Ser133 was mutated to alanine and the Gal4 DNA-binding domain alone (pGal4-DBD), and pUAS-TK-Luc, which contains two copies of the Gal4 DNA recognition site cloned 5' to a thymidine kinase promoter and a luciferase reporter gene, were gifts from Dr. J. Larry Jameson (Northwestern University, Chicago, IL). For transfection assays, cells were plated onto 12-well plates at a density of 1.5 x 105 cells/well. Transfections were performed using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instruction. After transfection, the cells were maintained for approximately 16 h in RPMI 1640 and 10% FCS. Cells were placed into RPMI 1640, 0.5% FCS, and 0.25% BSA for 2 h and then transferred to RPMI 1640 and 0.25% BSA with or without 50 ng/ml IGF-I. For experiments using indicated inhibitors, inhibitors were added 30 min before treating the cells with IGF-I. After treatment for 4 h, cells were harvested, and luciferase assays were performed as described previously (27). Light emission was measured with an AutoLumatLB953 luminometer (EG&G Berthold, Bad Wildbad, Germany). The luciferase activity present in each sample was normalized using the protein content of the sample, and all assays were performed in triplicate.

Statistical analyses
Except where noted, values are reported as the mean ± SEM. P values were calculated using one-way repeated-measures ANOVA with Tukey’s pairwise multiple comparison procedure or Kruskal-Wallis one-way ANOVA on ranks with the Dunnett’s pairwise multiple comparison procedure as appropriate, using SigmaStat 2.0 software (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IGF-I on serum deprivation-induced cell death
Initial studies examined the effect of serum deprivation on INS-1 cell death, and the ability of IGF-I to protect the cells from death secondary to serum withdrawal. For these studies, INS-1 cells were placed into RPMI 1640 and 0.25% BSA for 48 h in the presence or absence of 100 ng/ml IGF-I. Cell death was evaluated by measuring LDH release into the medium. As observed in other cell types (8), IGF-I reduced serum deprivation-induced cell death, as reflected by LDH release, by 41% (Fig. 1Go, left panel). This was similar to the level of cell death found in cells maintained in normal growth medium (RPMI 1640 and 10% FCS). To examine the ability of IGF-I to protect another pancreatic ß-cell line from death secondary to serum deprivation, MIN6 cells were used. For these studies, MIN6 cells were infected with an adenoviral vector expressing human IGF-I or, as a control, ß-galactosidase under the control of either the CMV or RIP (Fig. 1Go, right panel). Similar to the finding in INS-1 cells, IGF-I expression reduced serum deprivation-induced LDH release by 67% (Ad-CMV-IGF-I compared with Ad-CMV-ß-Gal) or 52% (Ad-RIP-IGF-I compared with Ad-RIP-ß-Gal).



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  		Figure 1. Effect of serum-free medium on INS-1 and MIN6 cell death. Left panel, INS-1 cells were plated at a density of 2 x 105 cells/well in 12-well plates. After incubation overnight, the cells were washed twice and either returned to normal growth medium (FCS) or placed into RPMI 1640 and 0.25% BSA without (BSA) or with 100 ng/ml IGF-I (IGF-I). After 48-h incubation, the percent LDH activity was determined using a cytotoxicity detection kit as described in Materials and Methods. Values represent percent LDH activity and are the mean ± SEM of 10 independent experiments, each performed in duplicate. *, P <= 0.05 compared with BSA. Right panel, MIN6 cells were plated at a density of 5 x 105 cells/well in 12-well plates. After incubation overnight, the cells were infected for 16 h with the indicated adenoviral vector at a multiplicity of infection of 10 plaque-forming units/cell. After infection, the cells were maintained in normal growth medium for 24 h and were then washed twice and placed into DMEM and 0.25% BSA with 15 mM glucose. The percent LDH activity was determined after 48-h incubation. For experiments with Ad-CMV-IGF and Ad-CMV-ß-Gal, values are the mean ± SEM of three independent experiments, each performed in duplicate. *, P <= 0.05 compared with Ad-CMV-ß-Gal. For experiments with Ad-RIP-IGF and Ad-RIP-ß-Gal, values are the mean ± SD of two independent experiments, each performed in duplicate.

 
IGF-I-induced phosphorylation of ERK and PKB
The ERK pathway has been shown to mediate, at least in part, the protective effect of IGF-I in some cell types (28, 29, 30). As previous studies have demonstrated an effect of the ambient glucose concentration on IGF-I-induced ERK activation in INS-1 cells (31), the ability of IGF-I to increase ERK phosphorylation was examined in INS-1 cells in the presence of 5.5, 15, and 25 mM glucose. Cells were maintained for 48 h in one of the above glucose concentrations, the last 24 h of which were in RPMI 1640 and 0.25% BSA, and then were treated with 50 ng/ml IGF-I (Fig. 2AGo). As shown, IGF-I-induced ERK phosphorylation was evident within 10 min of IGF-I treatment and was maintained for up to 2 h. Moreover, the effect of IGF-I was qualitatively similar regardless of the ambient glucose concentration. In contrast to its effect on ERK phosphorylation, IGF-I had no effect on the phosphorylation of two other MAPKs, p38 kinase and c-Jun N-terminal kinase (data not shown). Another pathway important for IGF-I-mediated cell survival is the phosphatidylinositol 3-kinase (PI 3-kinase) pathway. To determine whether IGF-I was able to activate this pathway, Western blot analyses were performed using an antibody specific for phosphorylated Akt/PKB, a kinase that is downstream of PI 3-kinase. Cells were maintained as described above in different concentrations of glucose and then were treated with 50 ng/ml IGF-I. PKB phosphorylation was apparent 10 min after IGF-I treatment and was sustained for up to 120 min. Again, exposure to different glucose concentrations had no effect on IGF-I-stimulated PKB phosphorylation (Fig. 2BGo).



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  		Figure 2. Time course of the effect of IGF-I on ERK and PKB phosphorylation in INS-1 cells. INS-1 cells were plated at a density of 1 x 106 cells/75-cm2 plate. After overnight incubation, the cells were placed into RPMI 1640 and 10% FCS with a final glucose concentration of 5.5, 15, or 25 mM as indicated for 24 h. The cells were placed into RPMI 1640 and 0.25% BSA with 5.5, 15, or 25 mM glucose for an additional 24 h and then treated with 50 ng/ml IGF-I for the indicated period of time. Cell lysates were prepared, and Western blot analyses were performed as described in Materials and Methods, using antibodies directed against phospho-ERK (A) or phospho-PKB (B). The blots were stripped and reprobed with antibody directed against either ERK (A) or PKB (B).

 
To document the efficacy of different inhibitors on IGF-induced signaling and to determine whether cross-talk between the different signaling pathways is present in INS-1 cells, the effects of PD98059, LY 294002, SB 20358, H-89, and rapamycin, inhibitors of MAPK kinase 1, PI 3-kinase, p38 kinase, protein kinase A (PKA), and p70S6 kinase, respectively, on IGF-I-stimulated signaling was examined (Fig. 3Go, A and B). As expected, IGF-I increased ERK phosphorylation 27.0 ± 8.1-fold (n = 3) compared with phosphorylation in control cells maintained in RPMI 1640 and 0.25% BSA (which was defined as 1.0). In cells treated with the MAPK kinase inhibitor PD98059, the IGF-I induced increase in ERK phosphorylation was markedly reduced (1.9 ± 0.6-fold increase compared with control; n = 3). The other inhibitors had no effect on IGF-I-induced ERK phosphorylation (phosphorylation increased 20- to 28-fold). The PI 3-kinase inhibitor LY 294002 effectively inhibited IGF-I-induced PKB phosphorylation (5.8 ± 0.5- vs. 2.1 ± 0.3-fold increase compared with control cells maintained in RPMI 1640; n = 3). In contrast, no effect of PD98059 on IGF-I-induced PKB phosphorylation was observed (6.6 ± 0.8-fold increase compared with control; n = 3), suggesting that there is little to no cross-talk between the ERK and PI 3-kinase pathways in IGF-I-treated INS-1 cells. Interestingly, H-89 consistently enhanced IGF-I-induced PKB phosphorylation (12.1 ± 2.7-fold increase compared with control; n = 3). This is similar to the effect of H89 on FSH-induced PKB phosphorylation in ovarian granulosa cells (32).



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  		Figure 3. Effect of inhibitors on ERK and PKB phosphorylation. After plating in RPMI 1640 and 10% FCS, INS-1 cells were placed into RPMI 1640 and 0.25% BSA for 24 h. The cells were pretreated for 30 min in the absence or presence of 20 µM PD98059, 20 µM LY 294002, 10 µM SB203580, 10 µM H89, or 10 nM rapamycin and then treated for 15 min with 50 ng/ml IGF-I. Cell lysates were prepared, and Western blot analyses were performed as described in Materials and Methods, using antibodies directed against phospho-ERK (A) or phospho-PKB (B). The blots were stripped and reprobed with antibody directed against either ERK (A) or PKB (B). The results are representative of three independent experiments performed using different cell lysates.

 
The effect of IGF-I on ERK and PKB phosphorylation was also examined in MIN6 cells. For these studies cells were maintained in 15 mM glucose for 64 h, the last 16 h of which were in DMEM and 0.25% BSA, and then were treated with 100 ng/ml IGF-I. Unlike INS-1 cells, IGF-I had no effect on ERK phosphorylation (1.1 ± 0.2-fold increase at 30 min compared with control; n = 3) in MIN6 cells (Fig. 4AGo). In contrast, IGF-I treatment for 30 min stimulated a 5.3 ± 2.1-fold (n = 3) increase in PKB phosphorylation (Fig. 4BGo).



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  		Figure 4. Time course of the effect of IGF-I on ERK and PKB phosphorylation in MIN6 cells. MIN6 cells were plated at a density of 2.5 x 106 cells/28-cm2 plate. After overnight incubation, the cells were placed into DMEM and 15% FCS with a final glucose concentration of 15 mM for 48 h. The cells were then placed into RPMI 1640 and 0.25% BSA with 15 mM glucose for an additional 16 h, followed by treatment with 100 ng/ml IGF-I for the indicated period of time. Cell lysates were prepared, and Western blot analyses were performed as described in Materials and Methods, using antibodies directed against phospho-ERK (A) or phospho-PKB (B). The blots were stripped and reprobed with antibody directed against either ERK (A) or PKB (B).

 
IGF-I-induced phosphorylation of targets of the PI 3-kinase and PKB pathways
Several targets of the PI 3-kinase and/or PKB pathways have been identified that may contribute to the ability of this signaling pathway to promote cell survival. These substrates include p70S6 kinase; BAD, which is a proapoptotic member of the Bcl-2 family; members of the forkhead (FKHR) family of transcription factors; GSK-3ß; and I{kappa}B kinase, which phosphorylates I{kappa}B, resulting in activation of nuclear factor-{kappa}B (33, 34, 35). Treatment of INS-1 cells with 50 ng/ml IGF-I resulted in phosphorylation of p70S6 kinase, which was evident 30 min after IGF-I treatment (3.3 ± 0.9-fold increase compared with control; n = 3) and sustained for up to 2 h (Fig. 5AGo). To examine IGF-I-induced BAD phosphorylation, antibodies specific for BAD phosphorylated at either Ser112 or Ser136 were used in Western blot analyses. IGF-I stimulated phosphorylation of Ser112, an effect that was evident in 10 min (6.8 ± 2.2-fold increase compared with control; n = 3) and was sustained for up to 2 h (Fig. 5BGo). In contrast, basal phosphorylation of Ser136 was observed, but was not further increased by IGF-I treatment (data not shown). Interestingly, Ser112 is phosphorylated by kinases activated by the MAPK pathway, whereas Ser136 is the substrate of the PI 3-kinase pathway (34, 36). The FKHR family of transcription factors includes FKHR, FKHRL1, and AFX (34), phosphorylation of which results in cytoplasmic retention of the transcription factors and inhibition of their transcriptional activity (37). IGF-I stimulated phosphorylation of FKHR (4.2 ± 0.5-fold increase compared with control at 10 min; n = 3) with kinetics similar to those observed for other signaling molecules and kinases (Fig. 5CGo). GSK-3ß is a kinase that plays a significant role in multiple cellular processes, including cellular metabolism, and has recently been shown to affect cell survival, in that transfection of cells with constitutively active forms of GSK-3ß induces apoptosis (34). Phosphorylation of GSK-3ß inhibits its kinase activity. In INS-1 cells, IGF-I stimulated phosphorylation of GSK-3ß, with phosphorylation again evident within 10 min of IGF-I treatment (3.4 ± 0.9-fold compared with control; n = 3), and this phosphorylation was sustained after 2 h of IGF-I treatment (Fig. 5DGo). A final substrate of PKB is I{kappa}B kinase, which, upon activation, phosphorylates I{kappa}B. This targets I{kappa}B for ubiquitination and degradation, thus activating nuclear factor-{kappa}B (38). In contrast to BAD, FKHR, and GSK-3ß, treatment of INS-1 cells with 50 ng/ml IGF-I had no effect on phosphorylation of I{kappa}B (Fig. 5EGo).



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  		Figure 5. Time course of IGF-I-induced phosphorylation of signaling molecules. After plating in RPMI 1640 and 10% FCS, INS-1 cells were placed into RPMI 1640 and 0.25% BSA for 24 h. The cells were then treated for the indicated period of time with 50 ng/ml IGF-I. Cell lysates or nuclear lysates (for FKHR; C) were prepared, and Western blot analyses were performed as described in Materials and Methods, using antibodies directed against phospho-p70S6 kinase (A), phospho-BAD (Ser112; B), phospho-FKHR (C), phospho-GSK-3ß (D), or phospho-I{kappa}B (E). The blots were then stripped and reprobed with antibody directed against p70S6 kinase (A), BAD (B), FKHR (C), or GSK-3ß (D). The results are representative of three independent experiments performed using different cell lysates.

 
IGF-I-induced activation of CREB
Another molecule shown previously to contribute to the antiapoptotic effect of IGF-I in neurons and other cell types is CREB (36, 39, 40, 41). To determine whether IGF-I is able to activate CREB in pancreatic ß-cells, INS-1 cells were treated with 50 ng/ml IGF-I, and Western blot analyses were performed using antibodies specific for phospho-CREB. As shown, IGF-I stimulated the transient phosphorylation of CREB, with phosphorylation apparent 10 min after IGF-I treatment (2.9 ± 0.4-fold increase compared with control; n = 3), with a decrease toward basal phosphorylation levels 30 min after IGF-I treatment (Fig. 6AGo). Also evident is another member of the CREB family, phospho-ATF-1, with which the anti-phospho-CREB antibody cross-reacts. It was phosphorylated with kinetics similar to those of CREB. Subsequent studies demonstrated that IGF-I at a concentration of 10 ng/ml stimulated maximal phosphorylation of CREB (Fig. 6BGo). Finally, the effects of different kinase inhibitors on IGF-I-mediated CREB phosphorylation were examined (Fig. 6CGo). IGF-I-induced CREB phosphorylation (2.4 ± 0.3-fold increase compared with control; n = 3) was decreased to basal levels (0.9 ± 0.3 compared with control; n = 3) by H89, an inhibitor of PKA. Interestingly, in cells treated with PD98059, IGF-I-induced CREB phosphorylation was also reduced (1.2 ± 0.2-fold increase compared with control; n = 3). In contrast, rapamycin, LY 294002, and SB 20358 had no effect on CREB phosphorylation (1.8- to 2.9-fold increases compared with control).



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  		Figure 6. A, Time course of IGF-I-induced CREB phosphorylation. After plating in RPMI 1640 and 10% FCS, INS-1 cells were placed into RPMI 1640 and 0.25% BSA for 24 h. The cells were then treated for the indicated period of time with 50 ng/ml IGF-I. Nuclear extracts were prepared, and Western blot analyses were performed as described in Materials and Methods, using antibodies directed against phospho-CREB. Anti-phospho-CREB also detects phospho-ATF-1, which is indicated on the blot. B, Dose-response effect of IGF-I on CREB phosphorylation. INS-1 cells were treated as described above, except that they were treated for 10 min with the indicated concentration of IGF-I. Western blot analyses were performed as described above. C, Effect of inhibitors on IGF-I-induced phosphorylation. INS-1 cells were treated as described above, except that 30 min before IGF-I treatment cells were treated with RPMI 1640 and 0.25% BSA in the absence or presence of 20 µM PD98059, 20 µM LY 294002, 10 µM SB203580, 10 µM H89, or 10 nM rapamycin. Cells were then treated for 10 min with 50 ng/ml IGF-I, and Western blot analyses were performed as described above. For all experiments the blots were stripped and reprobed with anti-CREB antibody. The results in each panel are representative of three independent experiments performed using different cell lysates.

 
The effect of CREB phosphorylation on DNA binding appears to be cell type and stimulus specific, as varying effects of CREB phosphorylation on DNA binding have been demonstrated (42, 43, 44). To determine whether IGF-I-induced phosphorylation of CREB altered its DNA binding, gel shift analyses were performed using an oligonucleotide that contained a consensus CREB DNA-binding site (Fig. 7Go). In INS-1 cells, two protein-DNA binding complexes were present. Supershift analyses using antibodies directed against CREB and ATF-1 led to either supershift or abrogation, respectively, of the more rapidly migrating complex (Fig. 7Go, lanes e and f), suggesting that it may represent a heterodimer of CREB and ATF-1. The identity of the more slowly migrating complex is unclear, although formation of this DNA-protein complex was abrogated by preincubating the nuclear extract with a 100-fold excess of cold oligonucleotide (Fig. 7Go, lane d). Interestingly, treatment of INS-1 cells for 2 h with either 50 ng/ml IGF-I or 20 µM forskolin had no effect on CREB binding (Fig. 7Go, lanes a–c). Similarly, treatment of INS-1 cells with 50 ng/ml IGF-I for periods ranging from 30 min to 12 h had no effect on CREB phosphorylation (data not shown).



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  		Figure 7. Autoradiogram of a gel shift analysis demonstrating nuclear protein binding to a CREB consensus DNA binding sequence. Experiments were performed as described in Materials and Methods, using 5 x 105 cpm 32P-labeled oligonucleotide and 10 µg nuclear protein prepared from INS-1 cells maintained in RPMI 1640 and 0.25% BSA (lane a), treated for 2 h with either 50 ng/ml IGF-I (lane b) or 20 µM forskolin (lane c). The specificity of protein binding was demonstrated by preincubating nuclear extracts for 30 min in a 100-fold excess of unlabeled oligonucleotide (lane d). Two protein-DNA complexes are indicated. Supershift analyses were performed by preincubating the nuclear extracts for 30 min with 2 µg antibody directed against either CREB (lane e) or ATF-1 (lane f).

 
Phosphorylation of CREB on Ser133 increases its transcriptional activity. To assess the ability of IGF-I to enhance CREB activity, INS-1 cells were transfected with pGal/CREB-WT, an expression vector that expresses a Gal4/CREB fusion protein that contains the DNA-binding domain of Gal4 and the trans-activation domain of CREB, which includes Ser133. To measure activation of the fusion protein by phosphorylation of the CREB trans-activation domain, the cells were cotransfected with a reporter plasmid, pUAS-TK-Luc, that contains two copies of the Gal4 DNA recognition site cloned 5' to a thymidine kinase promoter and a luciferase reporter gene. Cotransfection of INS-1 cells with pGal/CREB-WT and pUAS-TK-Luc, followed by treatment with 50 ng/ml IGF-I for 6 h, resulted in a 2-fold increase in luciferase activity (Fig. 8Go). In contrast, in cells cotransfected with pUAS-TK-Luc and a vector expressing the Gal4 DNA-binding domain alone (pGal4/DBD), IGF-I treatment had no effect on luciferase activity. To further define the role of Ser133 phosphorylation in IGF-I-stimulated transcription, similar experiments were performed using pGal4/CREB-MUT in which Ser133 was replaced with alanine. Basal luciferase activity was decreased by about 50% compared with that in cells transfected with pGal/CREB-WT, and IGF-I treatment had no effect on luciferase activity, suggesting that the effect of IGF-I on CREB activity was mediated by phosphorylation of Ser133.



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  		Figure 8. Effect of IGF-I on Gal4/CREB activity. INS-1 cells were cotransfected with pUAS-TK-Luc and pGal4-CREB-WT, pGal4-CREB-MUT, or pGal4-DBD as described in Materials and Methods. After transfection, cells were maintained in normal growth medium for 16 h and then transferred to RPMI 1640, 0.5% FCS , and 0.25% BSA for 2 h. Cells were then treated with RPMI 1640 and 0.25% BSA without (serum free) or with 50 ng/ml IGF-I for 6 h. The cells were harvested, and luciferase activity was determined. The values represent the relative luciferase activity compared with the level in cells that had been transfected with pGal4-CREB-WT and pUAS-TK-Luc and maintained in serum-free medium, which was defined as 1.0. The values are the mean ± SEM of three independent experiments performed in triplicate. *, P <= 0.05 compared with Gal4-CREB-WT maintained in serum-free medium.

 
Effect of inhibitors on inhibition of cell death by IGF-I in INS-1 cells
Having established that IGF-I is able to activate multiple signaling pathways in INS-1 cells that contribute to its protective effect in other cell types, a final series of studies used the previously described model of cell death secondary to serum withdrawal to begin to define the signaling pathways that mediate the protective effect of IGF-I on loss of trophic support in INS-1 cells. Initial studies examined the effect of LY 294002 and PD98059 on the ability of IGF-I and 10% FCS to prevent cell death (Fig. 9AGo). Treatment with LY 294002 partially abrogated the protective effect of IGF-I and had a somewhat greater effect on the protection mediated by 10% FCS. Moreover, LY 294002 also enhanced cell death in the cells maintained in 0.25% BSA. In contrast, treatment with PD98059 had no effect on the protective effect of either IGF-I or 10% FCS. Finally, a potential role for IGF-I-induced CREB phosphorylation was examined using the PKA inhibitor H89, which, as shown in Fig. 5CGo, inhibits IGF-I-induced CREB phosphorylation. Although 10 µM H89 increased cell death in cells maintained in 0.25% BSA, it had no effect on the protection mediated by either IGF-I or 10% FCS (Fig. 9BGo). The above data suggest that IGF-I-induced activation of the PI 3-kinase pathway contributes to the protective effect of IGF-I, whereas ERK activation and CREB phosphorylation appear to play only a small role.



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  		Figure 9. Effects of inhibitors on prevention of cell death by IGF-I. A, INS-1 cells were plated at a density of 2 x 105 cells/well in 12-well plates. After incubation overnight, the cells were washed twice and placed into either normal growth medium (FCS) or RPMI 1640 and 0.25% BSA without (BSA) or with 100 ng/ml IGF-I (IGF-I) in the presence or absence of either 20 µM LY 294002 or 20 µM PD98059. After 48-h incubation, the percent cell death was determined using a cytotoxicity detection kit as described in Materials and Methods. Values represent the percent LDH activity and are the mean ± SEM of six independent experiments, each performed in quadruplicate. +, P <= 0.05 compared with BSA without inhibitor; *, P <= 0.05 compared with cells maintained in the same condition in the absence of inhibitor. B, INS-1 cells were treated as described above, except that they were maintained in the presence or absence of 10 µM H89. Cell death was determined as described above, and the values represent the percent LDH activity and are the mean ± SEM of four independent experiments, each performed in duplicate. +, P <= 0.05 compared with BSA without H89; *, P <= 0.05 compared with BSA without H-89.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have examined the role of IGF-I and -II as antiapoptotic factors in pancreatic ß-cells. These studies have generally focused on the role of the IGFs during development or the ability of IGF-I to protect islet/ß-cells from cytokine-induced cell death. Specifically, decreased endogenous IGF-II production in pancreatic islets appears to account in part for the physiological apoptosis that occurs during pancreatic remodeling in the early postnatal period in rats (9, 45). A variety of studies have also demonstrated an ability of IGF-I to prevent cytokine-induced death of ß-cells (10, 11, 12, 13, 14, 15), but only limited information on the mechanism for this effect of IGF-I is available. The protective effect is mediated in part via an inhibition of cytokine-induced nitric oxide production and, as would be expected, decreased induction of caspases (11, 12, 15). Moreover, IGF-I prevented cytokine-induced alterations in the levels of members of the Bcl-2 family, a decline in Bcl-2 levels, which is antiapoptotic, and an increase in levels of Bax, which is proapoptotic (11). More limited information on signaling pathways that mediate the protective effect of IGF-I is available. Activation of PI 3-kinase mediated the protective effect of IGF-I against cytokine-induced death in INS-1 cells and canine islets (10, 11). The ability of IGF-I to activate signaling molecules distal to PKB and the roles of other signaling pathways, e.g. CREB or the ERKs, have not been reported.

One of the goals of the present study was to examine in more detail signaling pathways activated by IGF-I in pancreatic ß-cells, with an emphasis placed upon pathways that may prevent cell death. As has been demonstrated in many cell types, IGF-I treatment of INS-1 cells increased the phosphorylation of PKB, reflecting activation of the PI 3-kinase pathway as well as ERK-1 and -2. Interestingly, a previous study reported that ERK activation by IGF-I in INS-1 cells was glucose dependent at glucose concentrations between 0 and 9 mM and was no longer evident at 18 mM glucose (31). In contrast, the present study demonstrated that ERK activation by IGF-I was evident in cells maintained in 5.5, 15, or 25 mM glucose. This discrepancy is probably secondary to differences in study design. In the previous study INS-1 cells were maintained in the absence of glucose and then treated acutely with glucose with or without IGF-I, whereas in the present study cells were maintained in the ambient glucose concentration for 48 h before treatment with IGF-I. Interestingly, in MIN6 cells, a mouse ß-cell line, IGF-I increased PKB phosphorylation, but had no effect on ERK phosphorylation. Given the consistent activation of the PI 3-kinase pathway by IGF-I in INS-1 and MIN6 cells, phosphorylation of kinases distal to PI 3-kinase was further examined. Similar to findings in studies focusing upon the proliferative effect of IGF-I in INS-1 cells (31, 46, 47), increased phosphorylation of p70S6 kinase and GSK-3ß was demonstrated in IGF-I-treated INS-1 cells in the present study.

In addition to confirming and expanding upon the results of previous studies, the present study examined IGF-I-induced phosphorylation of a number of signaling molecules that appear to contribute to the protective effect of IGF-I in other cell types but have not been previously examined in ß-cells. These include two substrates of PKB, BAD and FKHR, as well as CREB. FKHR is a member of the forkhead family of transcription factors and induces expression of proapoptotic genes, e.g. Fas ligand and Bim (37, 48). Phosphorylation of forkhead family members appears to inhibit their import into the nucleus, thus decreasing their transcriptional capability (49). Consistent with the findings in a limited number of other cell types (37, 50, 51, 52), IGF-I stimulated FKHR phosphorylation in ß-cells. BAD is another proapoptotic member of the Bcl-2 family whose phosphorylation promotes its dissociation from other proapoptotic members of the Bcl-2 family and binding to 14-3-3 proteins, the net effect of which is antiapoptotic (53). The present study demonstrates that in ß-cells, IGF-I induces phosphorylation of BAD on Ser112, but not Ser136. Ser112 is a substrate of the MAPK-activated Rsks, whereas Ser136 is phosphorylated by PKB (34, 36, 54, 55). Thus, despite marked activation of PKB by IGF-I in ß-cells, Ser136 is not phosphorylated. A similar dissociation between IGF-I-induced PKB activation and Ser136 phosphorylation has been demonstrated in 293 cells (55). Rather, BAD phosphorylation in response to IGF-I treatment of INS-1 cells appears to be mediated by an ERK-stimulated pathway. Finally, as CREB phosphorylation and activation have been shown to mediate a protective effect of IGF-I in neural cells and cardiomyocytes (37, 39, 40, 41), the ability of IGF-I to phosphorylate CREB was examined in the present study. IGF-I both phosphorylated and increased the transcriptional activation of CREB in ß-cells.

Having confirmed that IGF-I activates multiple signaling pathways in INS-1 cells, many of which are thought to attenuate cell death, a second goal of the present study was to determine whether IGF-I was able to protect ß-cells from cell death upon loss of trophic support and to begin to define the mechanism for that effect. Previous studies that have examined this issue in other cell types have shown that IGF-I is effective in preventing cell death due to loss of trophic support, but the mechanism for this effect is, in general, cell type specific. In Schwann cells the protective effect of IGF-I was dependent upon activation of PI 3-kinase and inhibition of c-Jun N-terminal kinase (56, 57, 58). In PC-12 cells multiple pathways accounted for the effect of IGF-I. PI 3-kinase and ERK activation both contributed, as did p38 kinase- and CREB-mediated transcription of the bcl-2 gene (29, 41). Finally, in cerebellar granule neurons, activation of PI 3-kinase, but not the ERKs or CREB, accounted for the protective effect of IGF-I (36). To date, the ability of IGF-I to protect ß-cells from death due to loss of trophic support has not been addressed. In the present study IGF-I effectively prevented cell death upon loss of trophic support and was equipotent with 10% FCS in mediating protection in INS-1 cells. More detailed analyses using inhibitors of the MAPK, PI 3-kinase, and PKA pathways demonstrated that the protective effect of IGF-I appeared to be mediated in part through activation of PI 3-kinase with essentially no contribution of either the MAPK or PKA pathway.

Different mechanisms of cell death have been described, including programmed cell death (apoptosis) and necrosis. Previous studies demonstrating loss of graft ß-cell mass after islet transplantation have demonstrated evidence for both apoptosis and necrosis in the islets (20, 21). To date, we have been unable to demonstrate evidence for apoptosis in INS-1 cells deprived of serum, suggesting that cell death is occurring by necrosis (Chin-Chance, C., and W. L. Lowe, Jr., unpublished observations). In addition to the well defined ability of IGF-I to prevent apoptotic cell death (8), IGF-I has been shown to prevent necrotic cell death in a variety of cell types, including cardiac myocytes, skeletal muscle, and cortical neurons (59, 60, 61). Moreover, a possible role for IGF-II in the prevention of necrotic cell death of ß-cells has been suggested (62). Unlike IGF-I-mediated protection from apoptotic cell death, little is known about the cell signaling pathways responsible for the effect of IGF-I on necrotic cell death. Future studies examining the role of the PI 3-kinase pathway in more detail as well as the more specific roles of the substrates of PKB that are phosphorylated in response to IGF-I treatment, including GSK-3ß, BAD, and FKHR, will better define the mechanism for the protective effect of IGF-I.

In summary, the present study has established a new model of pancreatic ß-cell death secondary to loss of trophic support and has demonstrated the ability of IGF-I to prevent cell death using this model. It also has provided new insight into potential pathways that may contribute to the effect of IGF-I by examining IGF-I-stimulated signaling pathways in INS-1 cells and documenting the phosphorylation of several molecules that have not been previously examined in pancreatic ß-cells. Future studies will allow us to determine whether this protective effect of IGF-I is able to prevent loss of transplanted islets and to better define the specific pathways that mediate the protective effect of IGF-I.


   Acknowledgments
 
The authors thank Drs. Larry Jameson, Joe D’Ercole, Claes Wolheim, and Jun-ichi Miyazaki for providing cells and reagents, and Drs. Xiaojuan Chen, Dixon Kaufman, and Peter Kopp for helpful discussions.


   Footnotes
 
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and the Northwestern Memorial Foundation.

C.C.-C. is supported by a fellowship from Abbott Laboratories.

Abbreviations: ATF-1, Activating transcription factor-1; CMV, cytomegalovirus; CREB, cAMP response element-binding protein; FCS, fetal calf serum; GSK, glycogen synthase kinase; LDH, lactate dehydrogenase; PI 3-kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; RIP, rat insulin II; TBST, 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween 20; WT, wild-type.

Received January 17, 2002.

Accepted for publication June 13, 2002.


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Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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