Glucose-Dependent Insulinotropic Polypeptide Is a Growth Factor for ß (INS-1) Cells by Pleiotropic Signaling

Andrea Trümper, Katja Trümper, Heidi Trusheim, Rudolf Arnold, Burkhard Göke and Dieter Hörsch

Department of Internal Medicine (A.T., K.T., H.T., R.A., D.H.), Division of Gastroenterology and Metabolism, Philipps-University, Marburg, Germany D-35033; and Department of Medicine II (B.G.), Ludwig Maximilians University, Munich, Germany D-81377

Address all correspondence and requests for reprints to: Dieter Hörsch, M.D., Department of Internal Medicine, Division of Gastroenterology and Metabolism, Philipps-University, Baldingerstrasse, D-35033 Marburg, Germany. E-mail: hoerschd{at}post.med.uni-marburg.de


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the G-protein-coupled receptor for glucose-dependent insulinotropic polypeptide facilitates insulin-release from pancreatic ß-cells. In the present study, we examined whether glucose-dependent insulinotropic polypeptide also acts as a growth factor for the ß-cell line INS-1. Here, we show that glucose-dependent insulinotropic polypeptide induced cellular proliferation synergistically with glucose between 2.5 mM and 15 mM by pleiotropic activation of signaling pathways. Glucose-dependent insulinotropic polypeptide stimulated the signaling modules of PKA/cAMP regulatory element binder, MAPK, and PI3K/protein kinase B in a glucose- and dose-dependent manner. Janus kinase 2 and signal transducer and activators of transcription 5/6 pathways were not stimulated by glucose-dependent insulinotropic polypeptide. Activation of PI3K by glucose-dependent insulinotropic polypeptide and glucose was associated with insulin receptor substrate isoforms insulin receptor substrate-2 and growth factor bound-2 associated binder-1 and PI3K isoforms p85{alpha}, p110{alpha}, p110ß, and p110{gamma}. Downstream of PI3K, glucose-dependent insulinotropic polypeptide-stimulated protein kinase B{alpha} and protein kinase Bß isoforms and phosphorylated glycogen synthase kinase-3, forkhead transcription factor FKHR, and p70S6K. These data indicate that glucose-dependent insulinotropic polypeptide functions synergistically with glucose as a pleiotropic growth factor for insulin-producing ß-cells, which may play a role for metabolic adaptations of insulin-producing cells during type II diabetes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GLUCO-INCRETIN EFFECT results in higher insulin response and consecutive smaller increase in blood sugar after an oral glucose load compared with intravenous administration. Two major insulinotropic incretin hormones have been characterized: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (1, 2).

GLP-1 is synthesized in entero-endocrine L-cells of the ileum and colon and released into circulation as bioactive truncated GLP-1 (7–36 amide). GLP-1 receptors are expressed on pancreatic ß-cells, and activation of GLP-1 receptors at high glucose levels results in potentiation of glucose-dependent insulin secretion and activation of insulin gene transcription (1, 2). The incretin effect of GLP-1 is preserved in type II diabetes mellitus, which is currently exploited in clinical studies for the therapy of type II diabetes mellitus (2). In addition to its insulinotropic characteristics, GLP-1 also functions as a growth and differentiation factor for pancreatic ß-cells by pleiotropic activation of mitogenic signaling modules (3, 4, 5, 6, 7, 8).

GIP is synthesized in duodenal K cells as a 42-amino acid peptide and secreted as a hormone by the stimulation of the duodenum with nutrients, mainly by fat and glucose. The GIP receptor belongs the family of G protein-linked seven-transmembrane receptors and is highly expressed on ß-cells. Stimulation of GIP receptors induces a rise in cAMP and intracellular calcium, which facilitates glucose-dependent insulin release from pancreatic ß-cells physiologically (1, 9, 10, 11). However, the incretin effect of GIP is not preserved in type II diabetes or in relatives of type II diabetic subjects (1). Thus, defective insulinotropic signaling by the GIP receptor has been implicated in the pathogenesis of type II diabetes (1, 12). In addition to its role as an insulinotropin, GIP is involved in metabolic regulation of insulin-sensitive tissues by sensitizing adipose cells to insulin (12). Furthermore, several lines of evidence indicate a function of GIP as a growth and metabolic factor for ß-cells. In Chinese hamster ovary (CHO) cells stably expressing the GIP receptor, GIP activates MAPK, which could be partially blocked by wortmannin, an inhibitor of PI3K (13). Wortmannin also partially inhibits GIP-mediated insulin secretion in ß-cells providing indirect evidence that GIP signaling in ß cells may involve activation of mitogenic lipid kinase PI3K (14). A knockout of the GIP receptor demonstrated not only a defect in entero-insular axis but also the failure of ß cells to adapt metabolically to insulin resistance (15). Although it was not reported whether islet size is decreased in GIP receptor knockout mice, a recent study showed that dominant negative overexpression of the GIP receptor in pancreatic ß-cells leads to diminished islet size (Göke, B., and A. Volz, personal communication).

In the light of these studies, we examined whether GIP acts as a ß-cell growth factor using the differentiated ß-cell line INS-1 (16). In addition, we elucidated patterns of signaling by GIP on major mitogenic signaling modules in ß- cells, namely the pathways of PKA/cAMP regulatory element binder (CREB), MAPK, PI3K/protein kinase B (PKB), and janus kinase 2 (JAK2)/signal transducers and activators of transcription 5/6 (STATs5/6).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitogenic Effects of GIP and Glucose
To determine whether GIP and glucose act as growth factors for ß-cells, proliferation of INS-1 cells was determined by rate of DNA synthesis by enzyme-linked detection of 5-bromo-deoxyuridine (BrdU) incorporation (17). BrdU incorporation at 2.5 mM glucose without GIP served as control and was set at 1. The addition of glucose at rising concentrations in the absence of GIP induced a dose-dependent increase in proliferation, which was maximal at 15 mM glucose (2.8 ± 0.14 mean ± SD of control; n = 12; Fig. 1AGo) and stagnated at higher concentrations of glucose up to 25 mM (data not shown). Addition of 10-7 M GIP instigated a further 1.5- to 1.9-fold rise in INS-1 cell proliferation at glucose concentrations between 2.5 and 15 mM (Fig. 1AGo). GIP-induced INS-1 cell proliferation was absent at 0 mM glucose and stagnated at glucose concentrations higher than 15 mM (data not shown). Statistical analysis by ANOVA revealed that the increase of proliferation at basal levels and after stimulation with GIP were always highly significant when compared with control levels at 2.5 mM glucose. Furthermore, the GIP-induced rise in proliferation was highly significant when compared with respective basal levels (Fig. 1AGo).



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Figure 1. Mitogenic Effects of GIP and Glucose

A, Proliferation of INS-1 cells stimulated with a glucose gradient and GIP. INS-1 cells were starved overnight and stimulated for 24 h with 10-7 M GIP at 2.5 mM, 7.5 mM, and 15 mM glucose. DNA synthesis was measured by adding BrdU for the last 6 h of the stimulation period and subsequent detection by ELISA. Each bar represents the mean ± SD of 12 independent experiments. They are expressed as relative to control assigning a value of 1 to cells stimulated with 2.5 mM glucose in the absence of GIP. B, Dose-response of INS-1 cell proliferation by GIP at 15 mM glucose. INS-1 cells were stimulated with 15 mM glucose and a GIP gradient between 10-10 and 10-6 M at 15 mM glucose for 24 h. Cellular proliferation was measured by BrdU incorporation. Each bar represents the mean ± SD of six independent experiments. They are expressed as relative to control assigning a value of 1 to cells stimulated with 15 mM glucose. Statistical analysis was performed by ANOVA. n.s., Nonsignificant, P = 0.08 for 10-10 and 10-9 M GIP; *, P < 0.05; **, P < 0.005.

 
To elucidate whether mitogenic effects by GIP were dose-dependent, INS-1 cells were stimulated by a rising concentration of GIP between 10-10 M and 10-6 M at 15 mM glucose. At low concentrations of 10-10 M and 10-9 M, GIP induced only a minor rise in BrdU-incorporation, which was not statistically significant in the ANOVA analysis. However at higher concentrations of GIP between 10-8 M and 10-6 M, INS-1 cell proliferation increased 1.4- to 1.5 fold, which was highly statistically significant (Fig. 1BGo).

Activation of PKA/CREB by GIP and Glucose
Activation of the PKA/CREB signaling module was measured by phosphorylation of CREB in a transactivation assay using luciferase as a reporter gene (Fig. 2AGo) and by immunoblotting with an antibody specific for phosphorylated CREB at serine133 (Fig. 2Go, B–D). Dose-response of GIP-induced CREB phosphorylation was examined at 2.5 mM and 15 mM glucose (Fig. 2AGo). CREB phosphorylation at 2.5 mM glucose served as control and was set at 1. Elevation of glucose to 15 mM initiated a 4.1-fold rise in CREB phosphorylation (Fig. 2AGo). At 2.5 mM glucose, GIP instigated a dose-dependent increase in CREB phosphorylation, which was maximal at 10-7 M by 21-fold with an EC50 of approximately 10-8 M. At 15 mM glucose, GIP stimulated CREB phosphorylation in a dose-dependent manner in a similar pattern as at 2.5 mM, although at a much higher level of activation (Fig. 2AGo). Here, maximal activation was 38-fold at 10-6 M with an EC50 between 10- 9 M and 10-8 M GIP. At both glucose concentrations, a sharp rise in CREB phosphorylation was observed between 10-9 M and 10-8 M and a slower elevation at higher concentrations of GIP (Fig. 2AGo). Additional elevation of glucose concentrations above 15 mM did not further increase basal and GIP-stimulated CREB phosphorylation (data not shown). ANOVA analysis revealed that glucose-induced rise of basal CREB phosphorylation was highly statistically significant. Furthermore, GIP-induced elevation of CREB phosphorylation was significant (P < 0.05) or highly significant (P < 0.005) at all GIP and glucose concentrations compared with basal levels and also when GIP-induced CREB phosphorylation was compared between low (2.5 mM) and high (15 mM) glucose concentrations (Fig. 2AGo).



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Figure 2. Activation of the PKA/CREB Signaling Module by GIP and Glucose

A, Dose response of GIP-induced CREB phosphorylation at low (2.5 mM) and high (15 mM) glucose concentrations. INS-1 cells were transfected with CREB transactivator plasmid as described in Materials and Methods. INS-1 cells were stimulated for 16 h with different concentrations of GIP and glucose. CREB phosphorylation was determined by the luciferase activity of a cotransfected reporter plasmid. The luciferase activity of INS-1 cells stimulated with 2.5 mM glucose without GIP served as control and was set at 1. Each bar represents the mean ± SD of four to five independent experiments. Statistical analysis was performed by ANOVA. *, P < 0.05; **, P < 0.005. B, Time course of CREB phosphorylation by GIP. INS-1 cells were starved overnight and stimulated with 10-7 M GIP at indicated times. Cells were lysed and 100 µg of proteins were separated by SDS-PAGE and immunoblotted by Western analysis. The degree of CREB phosphorylation was determined using activation-specific antibody for pCREB Ser133. Proteins were detected using enhanced chemiluminescence, and band densities were quantified by densiometry. Data represent a typical blot of n = 3. They are expressed as relative to control assigning a value of 1 to nonstimulated cells. C, Dose response of CREB phosphorylation by GIP. Experiment was performed as in panel B except that cells were stimulated with indicated concentrations of GIP for 60 min. D, Glucose dependency of CREB phosphorylation by GIP. INS-1 cells were stimulated with 10-7 M GIP at 2.5 mM, 7.5 mM, and 15 mM glucose for 60 min. CREB phosphorylation of INS-1 cells stimulated with 2.5 mM glucose only was set at 1.

 
The kinetics of GIP-induced CREB phosphorylation was examined by immunoblotting. CREB phosphorylation was rapid with an almost 4-fold rise in phosphorylation at 10 min. Maximal phosphorylation was increased 7-fold at 60 min. After 60 min, CREB phosphorylation decreased down to almost control levels at 120 min (Fig. 2BGo). The dose response of CREB phosphorylation demonstrated by immunoblotting (Fig. 2CGo) was similar to that shown by transactivating luciferase assays (Fig. 2AGo). In the immunoblot, maximal phosphorylation of CREB was observed at 10-7 M GIP with an EC50 at about 10-8 M GIP. Again, there was a sharp elevation in CREB phosphorylation between 10-9 M and 10-8 M GIP (Fig. 2CGo) as in the transactivating luciferase assay (Fig. 2AGo). Glucose dependency of GIP-stimulated CREB phosphorylation was examined by immunoblotting using a glucose gradient of 2.5 mM, 7.5 mM, and 15 mM glucose and by stimulation with 10-7 M GIP (Fig. 2DGo). Elevation of glucose from 2.5 mM to 15 mM instigated a rise in CREB phosphorylation to 3.2-fold compared with the control, which was further stimulated 2- to 3-fold above basal levels by the addition of GIP (Fig. 2DGo). Since elevation of glucose without GIP also stimulated CREB phosphorylation, we examined the kinetic glucose-stimulated CREB phosphorylation. When 7.5 or 15 mM glucose was added after glucose starvation of INS-1 cells of 24 h, increased phosphorylation was only noted after 6 h and was maximal by 12 h (data not shown), implicating a much slower kinetics of glucose-induced CREB phosphorylation compared with the CREB activation by GIP. Therefore, to determine the glucose dependency of CREB phosphorylation by GIP in the immunoblot analysis, we equilibrated INS-1 cells in 7.5 and 15 mM glucose concentration overnight. Equal loading of protein lysates was verified by immunoblotting for nonphosphorylated CREB (data not shown).

Activation of MAPK by GIP and Glucose
Activation of the MAPK pathway signaling module was examined by phosphorylation of transcription factor Elk-1 in a transactivation assay using luciferase as a reporter gene (Fig. 3AGo) and by immunoblotting with an antibody specific for phosphorylated MAPK [extracellular signal-regulated kinases 1 and 2 (ERK 1/2)] (Fig. 3Go, B–D). The experimental design was as described for CREB phosphorylation. Glucose at 15 mM stimulated Elk-1 phosphorylation 4.6-fold. At low and high glucose, GIP induced a maximal Elk-1 phosphorylation at 10-6 M (10-fold at 2.5 mM and 23-fold at 15 mM). However, the EC50 was slightly shifted to the left at 15 mM glucose (2.5 mM glucose: EC50 at 10-8 M GIP; 15 mM glucose: EC50 between 10-9 M and 10-8 M GIP; Fig. 3AGo). As in the case of CREB phosphorylation, there was a sudden approximately 2-fold rise in Elk-1 phosphorylation between 10-9 M and 10-8 M GIP (compare Figs. 2Go and 3Go). ANOVA analysis revealed that glucose-induced Elk-1 phosphorylation was highly statistically significant. Elk-1 phosphorylation by GIP became significant at 10-10 at 15 mM glucose and at 10-9 at 2.5 mM glucose and was highly statistically significant at higher concentrations of GIP. The difference between GIP-induced CREB activation at low (2.5 mM) and high (15 mM) glucose concentrations was always highly significant (Fig. 3AGo).



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Figure 3. Activation of the MAPK Signaling Module by GIP and Glucose at the Level of Transcription Factor Elk-1 (A) and the MAPK ERK-1 and ERK-2 (B–D)

A, Dose response of GIP-induced Elk-1 phosphorylation at low (2.5 mM) and high (15 mM) glucose concentrations. INS-1 cells were transfected with Elk-1 transactivator plasmid as described in Materials and Methods. INS-1 cells were stimulated for 16 h with different concentrations of GIP and glucose. Elk-1 phosphorylation was determined by the luciferase activity of a cotransfected reporter plasmid. The luciferase activity of INS-1 cells stimulated with 2.5 mM glucose without GIP was set at 1. Each bar represents the mean ± SD of four to five independent experiments. Statistical analysis was performed by ANOVA. *, P < 0.05; **, P < 0.005. B, Time course of ERK phosphorylation by GIP. INS-1 cells were starved overnight and stimulated with 10-7 M GIP at indicated time points. Cells were lysed and 100 µg of proteins were separated by SDS-PAGE and immunoblotted by Western analysis. The degree of ERK phosphorylation was determined using activation-specific antibody for pTyr (204) of ERK-1 and ERK-2. Proteins were detected using enhanced chemiluminescence, and band densities were quantified by densiometry. Data represent a typical blot of n = 3. They are expressed as relative to control, assigning a value of 1 to nonstimulated cells. C, Dose-response of ERK phosphorylation by GIP. Experiment was performed as in panel B except that cells were stimulated with indicated concentrations of GIP for 60 min. D, Glucose dependency of ERK phosphorylation by GIP. INS-1 cells were stimulated with 10-7 M GIP at 2.5 mM, 7.5 mM, and 15 mM glucose for 60 min. ERK phosphorylation of INS-1 cells stimulated with 2.5 mM glucose only was set at 1.

 
The kinetics, dose response, and glucose response of MAPK activation were examined by immunoblotting for activated MAPK kinases ERK 1/2 (Fig. 3Go, B–D). GIP at 10-7 M instigated a fast rise in ERK phosphorylation at 10 min, which was maximal between 30 and 90 min and decreased at 120 min (Fig. 3BGo). A dose response similar to that for Elk-1 phosphorylation was demonstrated for ERK with a maximal phosphorylation at 10-7 M and 10-6 M GIP with an EC50 between 10-9 M and 10-8 M (Fig. 3CGo). Elevation of glucose to 7.5 mM and 15 mM stimulated basal ERK phosphorylation 2.4-fold, which was further increased by the addition of GIP (Fig. 3DGo). Kinetics and dose responses of glucose-induced Elk-1 and ERK phosphorylation were similar to that described for CREB phosphorylation. Equal loading of protein lysates was verified by immunoblotting for nonphosphorylated ERK (data not shown).

Activation of PI3K/PKB by GIP and Glucose
Stimulation of PI 3K by GIP and glucose was examined at the level of signaling molecules known to activate PI3K using antibodies for phosphorylated tyrosine (pY), insulin receptor substrate (IRS) isoforms IRS-1, IRS-2, and growth factor bound-2 associated binder-1 (Gab-1) (18, 19). In addition, PI3K activation was elucidated at the level of PI3K applying antibodies for PI3K regulatory subunit p85{alpha} and catalytic subunits p110{alpha}, p110ß, and p110{gamma} (18, 19, 20, 21). INS-1 cells were stimulated at 2.5 mM, 7.5 mM, and 15 mM glucose overnight and subsequently with 10-7 M GIP for 60 min, since we could show that GIP-induced PI3K activation was maximal at this time point (data not shown). Cell lysates were subjected to immunoprecipitation, and PI3K activity was determined as described in Materials and Methods. PI3K activation by GIP was difficult to reproduce at 2.5 mM glucose. Thus, only stimulation of PI3K by GIP at 7.5 mM and 15 mM glucose was included in the analysis. Basal levels of PI3K were elevated by increasing glucose concentration to 15 mM, which was most pronounced for Gab-1- and p110ß-associated PI3K activity (Fig. 4Go). The addition of GIP instigated modest amplifications in PI3K activity at 7.5 mM glucose except for p110{gamma}- associated PI3K activity, which was stimulated 4.1-fold (Fig. 4GGo). At 15 mM glucose, a more robust amplification of PI3K activity in all immunoprecipitates was observed, most notably in anti-Gab-1 and anti-p110{gamma} associated PI3K activity (Fig. 4Go). No stimulation of GIP-induced PI3K activity was associated with anti-IRS-1, anti-insulin receptor ß-chain, anti-JAK2, and p85ß immunoprecipitates (data not shown).



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Figure 4. PI3K Activation by GIP and Glucose

Cells were starved overnight and stimulated with 10-7 M GIP for 60 min at 7.5 and 15 mM glucose. Equal amounts of cell lysates were subjected to immunoprecipitation with antibodies to pY (A), IRS-2 (B), Gab-1 (C), p85{alpha} (D), p110{alpha} (E), p110ß (F), and p110{gamma} (G). PI3K assays were performed as described in Materials and Methods. 32P-incorporation into phosphatidylinositol 3-P was quantified after separation by TLC using densiometry. The band shown represents phosphatidylinositol 3-P. Shown are representative autoradiographs of n = 3. They are expressed as relative to control assigning a value of 1 to nonstimulated cells at 7.5 mM glucose.

 
The serine-threonine kinase PKB is activated by PI3K. To elucidate whether PKB is stimulated by GIP in ß-cells, PKB activation was detected by immunoblotting INS-1 cell lysates with an activation-specific antibody for pPKBSer473. This method was used to examine the kinetic (Fig. 5AGo), dose response (Fig. 5BGo), and glucose dependency (Fig. 5CGo) of PKB stimulation by GIP. Compared with PKA/CREB and MAPK, PKB activation by GIP exhibited slower kinetics with a maximal 2.6- to 3.5-fold phosphorylation between 30 and 90 min and a decline to almost control levels at 360 min (Fig. 5AGo). ANOVA revealed a highly significant elevation in PKB phosphorylation between 30 and 120 min (Fig. 5AGo). The dose-response curve of PKB phosphorylation by GIP demonstrated maximal 2.6-fold phosphorylation between 10-8 M and 10-6 M with a sudden rise of activation between 10-9 M and 10-8 M, which also corresponded to the EC50 (Fig. 5BGo). At 10-9 M, GIP-induced PKB phosphorylation became highly statistically significant. Using a glucose gradient of 2.5 mM, 7.5 mM, and 15 mM, we could show that basal and GIP-stimulated PKB phosphorylation increased with rising glucose concentrations (Fig. 5CGo) similar to the glucose response of CREB and ERK phosphorylation (Figs. 2DGo, 3DGo, and 5CGo). Statistical analysis by ANOVA revealed that the increase in basal levels and after stimulation with GIP by elevation of glucose was statistically significant as well as the difference between basal levels and GIP-induced PKB phosphorylation at 2.5 mM, 7.5 mM, and 15 mM glucose (Fig. 5CGo). Equal loading of protein lysates was verified by immunoblotting for nonphosphorylated PKB{alpha} (data not shown).



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Figure 5. Activation of PKB by GIP and Glucose

A, Time course of PKB phosphorylation by GIP. INS-1 cells were starved overnight and were stimulated with 10-7 M GIP at 11 mM glucose. Cells were lysed and 100 µg of proteins were separated by SDS-PAGE and immunoblotted by Western analysis. The degree of PKB phosphorylation was determined using activation-specific antibody for pSer473 PKB{alpha} as described in Materials and Methods. Immunoblots using an antiserum recognizing total PKB{alpha} served as a control for equal loading. Proteins were detected using enhanced chemiluminescence, and band densities were quantified by densiometry. Data are the mean ± SEM of 6–10 independent experiments. They are expressed as relative to control, assigning a value of 1 to nonstimulated cells at 360 min. B, Dose-response of GIP-induced PKB phosphorylation. Experiment was performed as in panel A except that cells were stimulated with indicated concentrations of GIP for 60 min. C, Glucose dependency of PKB phosphorylation by GIP. INS-1 cells were stimulated with 10-7 M GIP at 2.5 mM, 7.5 mM, and 15 mM glucose for 60 min. PKB phosphorylation of INS-1 cells stimulated with 2.5 mM glucose only was set at 1. Statistical analysis was performed by ANOVA. *, P < 0.05; **, P < 0.005.

 
Multiple isoforms of PKB are expressed in pancreatic ß-cells (21, 22) and may be differentially activated by glucose and GIP. We therefore examined the isoform-specific stimulation of PKB using specific antisera for PKB{alpha}, PKBß, and PKB{gamma} to perform isoform-specific PKB assays in immunoprecipitates of glucose and GIP-stimulated INS-1 cell lysates as described in Materials and Methods. GIP induced activation of PKB serine kinase activity glucose dependently between 7.5 mM and 15 mM glucose in PKB{alpha}- and PKBß- (Fig. 6AGo and B) but not in PKB{gamma}-immunoprecipitates (not shown). The transcription factor FKHR and cytoplasmatic kinases, glycogen-synthase 3{alpha}/ß (GSK-3) and p70S6K (21) are downstream targets of PKB. To find out whether FKHR, GSK-3, and p70S6K are stimulated by glucose and GIP, we used phosphorylation-specific antibodies for activated FKHR, GSK, and p70S6K. All targets of PKB were phosphorylated by stimulation by glucose and GIP at 7.5 and 15 mM glucose (Fig. 6Go, C–F). We noticed in the PKB assays that native GSK-3 was coimmunoprecipitated by PKB{alpha} and PKBß antibodies (data not shown) in a similar time course as the phosphorylation in straight cell lysates (Fig. 6DGo), indicating that GSK is associated with stimulated PKB and that PKB is a GSK kinase. Equal loading of protein lysates was verified by immunoblotting for nonphosphorylated GSK-3, FKHR, and p70S6K (data not shown).



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Figure 6. Activation of PKB Isoforms PKB{alpha} and PKBß, the Transcription Factor FKHR, and Cytoplasmatic Kinases GSK-3 and p70S6K by GIP and Glucose

Cells were starved overnight and stimulated with 10-7 M GIP for 60 min at 7.5 and 15 mM glucose. A and B, Equal amounts of cell lysates were subjected to immunoprecipitation with antibodies to PKB{alpha} (A) and PKBß (B). PKB assays were performed as described in Materials and Methods. Shown are representative autoradiographs of n = 3. They are expressed as relative to control assigning a value of 1 to nonstimulated cells at 7.5 mM glucose. Phosphorylation of FKHR (C), GSK-3 (D), and p70S6K (E and F) was determined by immunoblotting using phosphorylation-specific antibodies. Shown are representative autoradiographs of n = 3.

 
JAK/STAT Pathways
GH is a major mitogen of ß-cells by the activation of JAK/STAT signaling module, especially JAK2 and STAT5 (23, 24). To determine whether GIP shares this mitogenic pathway with GH, we stimulated INS-1 cells with 10-8 M human recombinant GH and 10-7 M GIP and immunoblotted cell lysates with phosphorylation-specific antibodies for tyrosine701 of STAT1, serine727 of STAT3, tyrosine705 of STAT3, tyrosine694 of STAT5, and tyrosine641 of STAT6. Neither human GH nor GIP induced phosphorylation of STAT1 and STAT3. In contrast, GIP failed to phosphorylate STAT5 and STAT6, which were phosphorylated by stimulation with GH in INS-1 cells, indicating that GIP does not activate the mitogenic JAK2/STATs5/6 pathway in INS-1 cells (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-secreting ß-cells of the pancreas are highly specialized cells with a low mitogenic index. The ß-cell pool may be replenished by neogenesis of ß-cells from precursor cells in pancreatic ducts or by islet cell replication (25, 26, 27). IGF-I, GH, and PRL have been characterized as growth factors for ß-cells. Elevated glucose levels also induce cellular proliferation in ß-cells (23, 24, 28, 29, 30, 31, 32). Recently, it could be shown that the gluco-incretin hormone GLP-1 acts as a growth, antiapoptotic, and differentiation factor for ß-cells in animal models, in isolated islets, and clonal ß-cells (4, 5, 6, 7, 8). These results prompted us to examine whether and how the other gluco-incretin hormone GIP acts as growth factor for ß-cells. Here, we show that GIP induced ß-cell proliferation in the ß-cell line INS-1 by pleiotropic signaling. Our results indicate that GIP may be added to the growing list of ß-cell growth factors. Recent animals studies corroborate the hypothesis of GIP as a ß-cell growth factor. Selective expression of a dominant negative mutant of the human GIP receptor in ß-cells as a transgene yielded diminished islet size and overt diabetes in mice (Göke, B., and A. Volz, personal communication). In addition, the failure of GIP receptor knockouts to respond to high fat diet with hyperinsulinemia like wild-type mice points to a role of GIP signaling in the regulation of appropriate ß-cell mass in insulin resistance (15).

The GIP receptor belongs to the family of G protein-coupled seven-transmembrane receptors and is expressed not only in pancreatic ß-cells but also in brain, adipose tissue, intestine, and heart (9, 10). Stimulation of the GIP receptor leads to rise of cytosolic cAMP by the activation of membrane-bound adenylate cyclase and a rise in intracellular Ca++ (9, 10, 11). We examined whether GIP activates major mitogenic signaling modules and demonstrated pleiotropic stimulation of PKA/CREB, p44/p42 MAPK, and PI3K/PKB signaling pathways by the GIP receptor in a similar dose response. These effects were synergistic with glucose between 2.5 mM and 15 mM. In contrast, there was no evidence of JAK2/STATs5/6 pathway activation by GIP in INS-1 cells (Fig. 7Go). These results indicate that signaling of ß-cell growth factors is transduced by pleiotropic, yet specific, pathways. Receptor tyrosine kinases such as insulin and IGF-I receptors activate PI3K/PKB and MAPK (32, 33, 34). Cytokine-like receptors such as the GH receptor stimulate JAK2/STAT5 and PI3K (23, 24, 30, 35). Finally, G protein-coupled receptors such as GIP- and GLP-1 receptors induce activation of PKA/CREB, MAPK, and PI3K/PKB (3, 31, 36), but not JAK/STAT pathways (Fig. 7Go). In this context, it is interesting to note that mitogenic signaling pathways activated by both receptors for GIP and GLP-1 are similar in ß-cells (3, 31, 35, 36), indicating a mitogenic signaling redundancy of both incretin hormones (37).



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Figure 7. Schematic Presentations of Signaling Cascades Stimulated by GIP in Pancreatic ß-Cells

AC, Membrane-coupled adenylate cyclase; AFX, forkhead transcription factor AFX; Elk, transcription factor Elk; FKHR, Forkhead in rhabdomyosarcoma; G{alpha}s: {alpha}-subunit of stimulatory G protein; Gß{gamma}: ß{gamma}-subunits of G proteins; MEK, MAPK/ERK kinase; B-Raf, serine/threonine kinase B-Raf; RAS, small GTPase, cellular protein homolog to v-ras; ?TK: unidentified tyrosine kinase.

 
Glucose concentration in the stimulation medium was essential for the activation of all signaling modules examined. Synergistic effects of glucose and GIP were additive in the immunoblotting analysis of CREB, ERK1/2, and PKB phosphorylation with a relative short stimulation of 60 min whereas transactivating luciferase assays for CREB and Elk-1 demonstrated superadditive effects at a stimulation period of 16 h. Thus, the superadditive effects seen in the transactivating luciferase assays may be caused by the accumulation of reporter luciferase. On the other hand, detailed analysis of PI3K signal transmission by glucose and GIP revealed additive and superadditive effects, implying differential substrate specificity of glucose and GIP induced signal transduction by PI3K. Blockage of glucokinase by alloxan (38) and prevention of glucose phosphorylation by 2-deoxy-D-glucose (39) indicated signal transduction by derivatives of glucose metabolism in ß-cells (Ref. 40 and Trümper, A., and D. Hörsch; unpublished results). Clearly, ATP generated from pyruvate in mitochondria and glutamate (40) are favorite candidates as mitogenic and antiapoptotic second messengers of glucose-induced signaling as well as for glucose-induced synergism in GIP signaling. However, it has to kept in mind that activation of signal transduction pathways by glucose may not be the result of a second messenger-like ATP but reflect the altered ADP:ATP ratio in ß-cells at high glucose levels (40). ATP is a necessary cofactor for kinase reactions (41). The increased availability of ATP for kinase reactions at high glucose levels may facilitate kinase reactions and thus mimic a second messenger effect.

Growth factors signal to the nucleus by cytoplasmatic signaling cascades inducing the transcription of cell-specific sets of genes by the activation of transcription factors (23, 24, 25, 28, 29, 42, 43). Here, we showed that GIP and glucose phosphorylate transcription factors CREB, Elk-1, and FKHR. In addition, glucose and GIP phosphorylated p70S6K, a crucial regulator of translation (44). These data indicate that mitogenic effects of glucose and GIP in ß-cells may not only be caused by activation of transcription but also by initiation of protein translation. Recently, it has been shown that a major part of the mitogenic response of ß-cells to glucose may be attributed to the activation of p70S6K (45). Furthermore, a knockout of p70S6K1 leads to diminished ß-cell size and glucose resistance in mice (44), indicating that stimulation of p70S6K is essential for regulation of ß-cell growth.

Altered ß-cell function is pivotal for the development of type 2 diabetes. As the disease develops, the ability of ß-cells to secrete compensatory amounts of insulin decreases, thereby increasing hyperglycemia and insulin resistance (27, 46, 47, 48). Thus, mitogenic and antiapoptotic activation of G protein-coupled receptors, strongly expressed on ß-cells, may prove to be a therapeutic approach for late stages of type II diabetes. Although, short-term antidiabetic effects of GIP are not preserved in type II diabetes and glucose intolerance due to lack of insulinotropic action (1), long-term application of GIP may prove to be an therapeutic approach for type II diabetes. We have shown that GIP is a growth and antiapoptotic factor for ß-cells by pleiotropic activation of PKA/CREB, MAPK, and PI3K/PKB pathways. Thus, long-term application of GIP may be beneficial in the treatment of type II diabetes as a ß-cell growth factor by improving ß-cell function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Silica-gel TLC plates were obtained from Merck & Co., Inc. (Darmstadt, Germany), protein G-agarose was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Nitrocellulose paper (Optitran BA-S85) was from Schleicher & Schuell, Inc. (Keene, NH) and [{gamma}-32P]ATP was from Amersham Pharmacia Biotech (Arlington Heights, IL). GIP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) and recombinant human GH were obtained from Bachem (Bubendorf, Switzerland). 5-Bromo-deoxyuridine (BrdU) incorporation and cell death detection ELISAs were from Roche (Mannheim, Germany). New England Biolabs, Inc. (Beverly, MA) supplied the PKB{alpha} antiserum, phospho-specific antibodies for serine473 and threonine308 of PKB{alpha}, serine133 of CREB, serine411 of p70S6K, threonine421/serine424 of p70S6K, tyrosine701 of STAT1, serine727 of STAT3, tyrosine705 of STAT3, tyrosine694 of STAT5, tyrosine641 of STAT6, and serine21/9 of glycogen-synthase kinase 3{alpha}/ß (GSK-3), and GSK-3{alpha}/ß cross-tide protein, along with respective control antibodies for nonphosphorylated kinases as well as enhanced chemiluminescence reagents. Antibodies for Gab-1, insulin receptor substrate (IRS) IRS-1, IRS-2, p110{alpha}, p110ß, p85{alpha}, pY, and PKB-isoforms {alpha}, ß, and {gamma} were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies for phosphorylated ERK-1 and ERK-2 (pERK Tyr204), control ERK-antibodies, and p110{gamma} antibodies were from Santa Cruz Biotechnology, Inc.. Reagents for SDS-PAGE were from Bio-Rad Laboratories, Inc. (Hercules, CA), cell culture reagents were from Life Technologies, Inc.(Karlsruhe, Germany), and all other chemicals were from Sigma (St. Louis, MO).

Cell Culture
INS-1 cells (passage 80–120) were grown as previously described (16) in regular RPMI-1640 medium supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM ß-mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified (5% CO2, 95% air) atmosphere. Before stimulation, INS-1 cells were starved in medium without serum, glucose, and sodium pyruvate.

BrdU Incorporation
Cells were seeded at a density of 3 x 103 in 96-well plates, grown for 24 h in regular medium, washed once with 10 mM PBS (pH 7.4), and subsequently starved for 24 h. They were then incubated for 24 h in RPMI medium with different glucose concentrations and test substances. During the last 6 h of stimulation, 20 µl of a BrdU solution was added and ELISA (17) was performed according to guidelines provided by the manufacturer.

Transreporting System for Elk1 and CREB Phosphorylation
INS-1 cells were grown for 48 h in normal medium in six-well plates until they reached 60–80% confluency. Cells were then washed twice with PBS, transfected with luciferase reporter gene (pFR-Luc) and either Elk1 (pFA-2-Elk1) or CERB (pFA2-CREB) transactivator domains (all from Stratagene, La Jolla, CA) by lipid-based transfection (Pfx-6; Invitrogen, Groningen, The Netherlands) for 8 h in INS-1 medium without serum. Subsequently, cells were grown in INS-1 medium with 5 mM glucose and 5% FBS and then stimulated for 16 h in INS-1 medium containing 1% FBS with GIP at different glucose concentrations.

Immunoprecipitation and Immunoblotting
INS-1 cells were starved for 12 h and were then equilibrated for another 12 h in the indicated glucose concentrations. One hour before stimulation, the stimulation medium was changed. Cells were lysed after stimulation in ice-cold lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 5 mM sodium vanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 1.5 mg/ml benzamidine, and 34 µg/ml phenylmethylsulfonyl fluoride) and sonicated for 15 sec, and insoluble material was removed by centrifugation at 15,000 rpm in a microfuge for 10 min. For immunoblotting, 100 µg of protein per lane were separated by 10% SDS-PAGE, Western-transferred on nitrocellulose membranes, and immunoblotted as previously described (18). For immunoprecipitation experiments, 500 µg of protein lysate were immunoprecipitated with indicated antibodies and 60 µl of protein G- or protein A-agarose, respectively, for 2–4 h at 4 C. Beads were washed twice with protein lysis buffer and either used immediately for SDS-PAGE or frozen at -80 C. Protein bands were visualized with enhanced chemiluminescence. Autoradiographs were scanned, and band density was determined using Gelscan 3D software (BioSciTec, Marburg, Germany). Statistical analysis was performed by ANOVA.

PI3K Assays
Immune-complexed PI3K activity was determined as previously described (18). Immune complexes were incubated in a 55 µl reaction mixture containing 200 µM ATP, 5 µCi [{gamma}-32P]ATP, 200 mM MgCl2, and 5 µg phosphatidylinositol for 20 min at room temperature. Reactions were stopped by the addition of 150 µl of CHCl3/CH3OH/11.6 N HCl (33:66:0.6) and subsequently of 120 µl of CHCL3. The organic phase was washed once with 150 µl of CH3OH/1 N HCl (1:1), 20 µl 8 N HCl, and 160 µl CHCl3/methanol 1:1. The organic phase was removed by centrifugation and applied to silica gel TLC plates, developed in CHCl3/CH3OH/H2O/NH4OH (60:47:11.3:2), dried, and visualized by autoradiography. The band representing phosphatidylinositol 3-P was quantified as described.

PKB Assays
Immunocomplexed PKB activity was determined by incubating washed antibodies with 200 µmol/liter ATP and 1 µg GSK3{alpha} cross-tide fusion protein. Phosphorylation of the substrate was determined by immunoblotting with a phosphorylation-specific antibody for serine21/9 of GSK3{alpha}/ß at 30 kDa (New England Biolabs, Inc.).


    ACKNOWLEDGMENTS
 
We thank A. Volz and R. Göke for critical reading of the manuscript and H. Schmidt for valuable technical support.


    FOOTNOTES
 
Parts of this study are contained in the medical thesis of A. Trümper.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to D.H. (Ho 1762/2-1).

Abbreviations: BrdU, 5-bromo-deoxyuridine; CREB, cAMP regulatory element binder; ERK, extracellular signal-regulated kinase; Gab-1, growth factor bound-2 associated binder-1; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; GSK, glycogen-synthase kinase; IRS, insulin receptor substrate; JAK, janus kinase; PKB, protein kinase B; pY, phosphorylated tyrosine; STAT, signal transducer and activator of transcription.

Received for publication January 29, 2001. Accepted for publication May 17, 2001.


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