The glucoincretin hormone glucagon-like peptide-1 (GLP-1) increases pancreatic β-cell proliferation and survival through sequential activation of the epidermal growth factor receptor (EGFR), phosphatidylinositol-3 kinase (PI 3-kinase), and Akt. We investigated the role of transcription factor FoxO1 in the proliferative and antiapoptotic actions of GLP-1 in β-cells. GLP-1 inhibited FoxO1 through phosphorylation-dependent nuclear exclusion in pancreatic β (INS832/13) cells. The effect of GLP-1 was suppressed by inhibitors of EGFR (AG1478) and PI 3-kinase (LY294002). In contrast, LY294002 but not AG1478 suppressed insulin-induced FoxO1 phosphorylation. Expression of constitutively nuclear FoxO1 in β-cells prevented the proliferative and antiapoptotic actions of GLP-1 in cultured β-cells and the increase in pancreatic β-cell mass in response to Exendin4 in transgenic mice. Gene expression and chromatin immunoprecipitation assays demonstrated that GLP-1 increases pancreatic and duodenal homeobox gene-1 and Foxa2 expression and inhibits FoxO1 binding to both promoters. We propose that FoxO1 mediates the pleiotropic effects of the glucoincretin hormone on cell proliferation and survival.

The glucoincretin hormone glucagon-like peptide-1 (GLP-1) (13) improves insulin secretion in subjects with impaired glucose tolerance and type 2 diabetes (46). GLP-1 signals via cAMP/PKA and cAMP-regulated guanine nucleotide exchange factors of the Epac (7). The biological actions of GLP-1 include stimulation of Insulin expression and insulin biosynthesis (8), presumably via increased expression and activity of the β-cell–specific transcription factor pancreatic and duodenal homeobox gene-1 (PDX1) (9,10). In experimental animal models, GLP-1 increases islet mass (10,11). In cultured β (INS)-cells, GLP-1 acts as a growth factor (9,12,13) via proteolytic maturation of betacellulin (BTC), leading to transactivation of the epidermal growth factor receptor (EGFR), phosphatidylinositol-3 kinase (PI 3-kinase), protein kinase B/Akt, and protein kinase C ζ (9,12,13). This signaling cascade mediates the glucoincretin action of the hormone as well (14). Finally, GLP-1 has recently been shown to prevent β-cell apoptosis in animal models of diabetes (11,15) and glucolipotoxicity (16).

Forkhead transcription factors (Fox) of the O subclass are transcriptional effectors of insulin and IGF signaling. Their activity is inhibited by PI 3-kinase/Akt signaling via phosphorylation-dependent nuclear exclusion (17). We have shown that FoxO1 is a prominent mediator of growth factor signaling in β-cells and that FoxO1 controls β-cell mass through Pdx1 (1821). Thus, the potential exists for a role of FoxO1 in GLP-1 action in the β-cell.

We report that GLP-1 inhibits the forkhead transcription factor FoxO1 through phosphorylation-dependent nuclear exclusion. Moreover, FoxO1 inhibition plays a role in the proliferative and antiapoptotic actions of GLP-1 in the β-cell. Finally, quantitative real-time PCR and chromatin immunoprecipitation assays reveal that GLP-1 alters the transcriptional profile of the β-cell through FoxO1 inhibition.

Reagents.

LY294002 and AG1478 were purchased from Biomol (Plymouth Meeting, PA). Human GLP-1 fragment 7–36 amide and Exendin4 were obtained from Sigma (St. Louis, MO). RPMI 1640, FCS, and other culture media were purchased from Gibco BRL (Burlington, ON, Canada).

Cell culture and incubation.

INS832/13 (22) cells (passage 36–70) were grown in RPMI 1640 medium supplemented with 10 mmol/l HEPES, 10% heat-inactivated FCS, 2 mmol/l l-glutamine, 1 mmol/l sodium pyruvate, 50 μmol/l β-mercaptoethanol, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. Cells at 70% confluence were washed with PBS and preincubated in serum-free RPMI supplemented with 5 mmol/l glucose and 0.1% BSA (Fraction V; Sigma) for at least 4 h before treatment. LY294002 and AG1478 were applied 30 min before growth factor treatment.

Animal studies.

Eight-week-old wild-type and FoxO1 transgenic mice were treated with daily intraperitoneal injections of Exendin4 (10 nmol/kg) or saline for 7 days (n = 4 each). Animals were killed and pancreas sections processed for insulin and Ki67 immunohistochemistry to assess β-cell proliferation. Cross-sectional islet area was measured using Image Pro Plus software (Media Cybernetics, Silver Spring, MD), and small β-cell clusters (<6 cells) were counted for each section. For each animal, at least three sections spaced 80 μm apart were studied (18).

Western blot.

Proteins were extracted and quantified by BCA (bicinchoninic acid) assay (Roche, Rockford, IL) before fractionation on 8 or 10% polyacrylamide gels. Primary antibodies (anti–forkhead transcription factor Foxo1 [anti-FKHR], anti–phospho-FKHR, anti–extracellular signal–related kinase 1/2 [anti-ERK1/2], and anti–phospho-ERK1/2) were purchased from Cell Signaling (Beverly, MA). Western blotting was performed as described previously (19).

Transfection and confocal microscopy.

Cells were transfected with FoxO1-GFP (23) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in LabTek chambered slides (Nalgene). In brief, 4 ×105 cells were transduced with 1 μg plasmid DNA and 2 μl Lipofectamine 2000 in 0.5 ml RPMI medium. The following day, cells were treated for the indicated time periods and fixed in 4% paraformaldehyde in PBS for 15 min. Image acquisition was performed using a Zeiss confocal microscope (23).

Recombinant adenovirus transduction.

The constitutively nuclear mutant FoxO1 carries single amino acid substitutions replacing the three main phosphorylation sites, Thr24→Ala, Ser253→Asp, and Ser316→Ala24. Constitutively nuclear FoxO1 transduction was carried out as previously described (24). INS832/13 cells were seeded 2 days before use in 100-mm Petri dishes and cultured in RPMI. Cells were then infected with constitutively nuclear FoxO1 or β-galactosidase (β-Gal) adenoviral constructs at a multiplicity of infection of 50 plaque-forming units/cell for 1 h in 1 ml complete medium. The viral solution was then replaced with complete medium, and cells were allowed to recover for 24 h before being used.

Cell proliferation.

Proliferation of INS832/13 cells was evaluated using an enzyme-linked immunosorbent assay–based bromodeoxyuridine (BrdU) incorporation kit (Roche). In brief, INS832/13 cells were transduced for 1 h in 100-mm Petri dishes, trypsinized, seeded in 96-well plates at 70% confluence, and incubated overnight in minimal RPMI medium before the experiment. BrdU was added to the culture medium for 1 h before harvesting cells. Cells were then fixed and incubated with a peroxidase-conjugated anti-BrdU antibody, and the immune complexes were quantified using a spectrophotometer to measure absorbance (Bio-Rad, Hercules, CA).

Measurements of caspase activity and mitochondrial membrane potential.

Cells were plated on chambers slide (Nalgene Nunc, Naperville, IL) and incubated for 24 h in complete RPMI. The following day, cells were washed and treated under different conditions in minimal RPMI medium for 4 h. Caspase activation was measured with the sulforhodamine multi-caspase activity kit and mitochondrial membrane potential with Mit-E-Ψ mitochondria permeability detection kit (Biomol). Briefly, SR-VAD-FMK, a sulforhodamine derivative of a potent caspase inhibitor, was added during the last 1 h of a 4-h incubation. SR-VAD-FMK is cell permeable and covalently binds to active caspases, allowing for detection by fluorescence microscopy. The lipophilic Mit-E-Ψ reagent was added for the last 15 min of a 90-min incubation. Mit-E-Ψ enters the mitochondria and aggregates to emit red fluorescence. In the presence of a loss of mitochondrial membrane potential (ΔΨ), red fluorescence is replaced by cytoplasmic green fluorescence.

Quantitative real-time PCR.

RNA was isolated using Qiagen kits (Valencia, CA), and quantitative real-time PCR was performed as described previously (25).

Chromatin immunoprecipitation.

Cells were fixed to isolate intact chromatin and treated as described previously. FoxO1 was immunoprecipitated with anti-FKHR (H-128) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and bound DNA was amplified by PCR. Primers sequences are available on request (18,19).

Calculations and statistics.

Data are presented as means ± SE. Statistical analyses were performed with SPSS using ANOVA.

GLP-1 inhibits FoxO1 in β (INS832/13) cells.

FoxO1 is a prominent effector of PI 3-kinase/Akt, a signaling cascade that is activated by GLP-1 in β-cells. We investigated whether GLP-1 affects FoxO1 phosphorylation and subcellular localization in pancreatic β (INS832/13) cells. In cultured β-cells, FoxO1 is constitutively phosphorylated and cytoplasmic, probably because of endogenously produced insulin (26). To measure the effects of GLP-1 on FoxO1 phosphorylation, we cultured INS832/13 cells in serum-free RPMI medium supplemented with 5 mmol/l glucose for 24 h. At the end of the incubation period, we added 10 nmol/l GLP-1 for various lengths of time (Fig. 1A). Under these conditions, GLP-1 induced FoxO1 phosphorylation in a time-dependent manner, with a maximal effect observed at 30 min. The action of GLP-1 was mimicked by the EGFR ligand BTC (not shown). The effect of GLP-1 was blocked by inhibitors of EGFR (AG1478, 250 nmol/l) and PI 3-kinase (LY294002, 50 μmol/l) signaling (Fig. 1B). Insulin also increased FoxO1 phosphorylation. Unlike the effect of GLP-1, that of insulin could be blocked by the PI 3-kinase inhibitor LY294002 but not by the EGFR inhibitor AG1478 (Fig. 1C).

We next studied the GLP-1 effect on the subcellular localization of FoxO1 by fluorescence microscopy. To this end, we transiently expressed a FoxO1-GFP fusion protein in INS832/13 cells (23). GLP-1 caused a time-dependent translocation of FoxO1 from the nucleus to the cytoplasm, consistent with FoxO1 phosphorylation (Fig. 2, first row). Insulin, BTC, and serum had comparable effects (Fig. 2, second row). Inhibition of PI 3-kinase by LY294002 preempted translocation induced by all ligands tested (Fig. 2, third row). In contrast, AG1478 was able to prevent translocation induced by GLP-1 and BTC but not that induced by insulin (Fig. 2, fourth row).

These data indicate that GLP-1 inhibits FoxO1 through phosphorylation-dependent nuclear exclusion. Given that GLP-1 has been shown to activate sequentially EGFR and PI 3-kinase (9,12,13,16), the observed changes in FoxO1 phosphorylation/subcellular localization suggest that FoxO1 mediates GLP-1 action on β-cells.

Proliferative and antiapoptotic actions of GLP-1 require FoxO1 inactivation.

Because FoxO1 controls cell cycle progression (27), we examined whether its inhibition is required for GLP1-dependent β-cell proliferation. Incubation of serum-deprived cells with high glucose increased BrdU incorporation by ∼50%, as did GLP-1 and BTC. Addition of 10% serum to the medium resulted in a 3.5-fold increase in BrdU incorporation (Fig. 3A). We next transduced cells with a constitutively nuclear FoxO1 mutant (20) or with control β-gal adenovirus. Overexpression of constitutively nuclear FoxO1 inhibited β-cell proliferation induced by glucose, GLP-1, BTC, and serum, without significantly affecting basal proliferation, whereas control virus encoding β-gal had no effect (Fig. 3A). This result indicates that FoxO1 nuclear exclusion is necessary for GLP-1–and BTC-induced proliferation.

To evaluate whether FoxO1 inactivation was required for the antiapoptotic effect of GLP-1, we examined caspase activation in response to H2O2, an inducer of oxidative stress (28), using a fluorescent probe in cells transduced with constitutively nuclear FoxO1 or β-Gal. Addition of H2O2 increased the number of cells expressing activated caspase by ∼threefold (Fig. 3B). GLP-1 reversed the effects of H2O2. Transduction with constitutively nuclear FoxO1 blocked the ability of GLP-1 to decrease the number of caspase-positive cells (Fig. 3B). These data indicate that GLP-1 action on β-cell survival requires FoxO1 inhibition.

Our results indicate that constitutively nuclear FoxO1 inhibits GLP-1 action on proliferation and survival. To determine the mechanism by which FoxO mediates its effects in β-cells, we examined whether constitutively nuclear FoxO1 affects GLP-1–dependent increase in cAMP levels, preserves mitochondrial membrane potential, and prevents GLP-1–induced ERK1/2 activation, three metabolic actions of the hormone that do not require gene transcription. GLP-1 induced a rise in cAMP levels in β-Gal–transduced cells, as did treatment with 10 mmol/l nicotinamide (29). Basal cAMP levels were ∼50% lower in cell transduced with constitutively nuclear FoxO1. This decrease is similar to that observed in growth-arrested cells and is likely to reflect the FoxO1-induced growth arrest. However, the magnitude of the GLP-1–induced rise was unaffected by constitutively nuclear FoxO1 (Fig. 4A). Next, we used a fluorescent probe (Mit-E-Ψ) to evaluate changes in mitochondrial membrane potential in cells transduced with constitutively nuclear FoxO1 after H2O2 treatment in the absence or presence of GLP-1. H2O2 caused a disruption of mitochondrial membrane potential, indicated by the reduction in red fluorescence and the appearance of green fluorescence. GLP-1 preserved the integrity of mitochondrial membrane potential in the presence of H2O2 and constitutively nuclear FoxO1 (Fig. 4B). Identical observations were made in untransduced cells (not shown). These data indicate that FoxO1 does not affect GLP-1 action on mitochondrial membrane potential. The mechanism by which GLP-1 acts on the mitochondria to preserve the membrane potential remains elusive but may be related to stimulation of mitochondrial ATP synthesis (30). Next, we investigated whether constitutively nuclear FoxO1 interferes with the ability of GLP-1 to activate ERK1/2. In untransduced cells, GLP-1 caused a moderate increase in ERK1/2 phosphorylation by 177 ± 34% (P < 0.05), as previously reported (12). Transduction of INS cells with constitutively nuclear FoxO1 did not prevent the GLP-1–induced rise in phospho-ERK (Fig. 4C). These results indicate that constitutively nuclear FoxO1 does not affect actions of GLP-1 that do not require gene transcription.

To investigate the role of FoxO1 in the proliferative actions of GLP-1 in vivo, we determined whether GLP1 affects β-cell mass in transgenic mice expressing constitutively nuclear FoxO1 in β-cells. The mutant transgene carries a single amino acid substitution replacing the main Akt phosphorylation site and is therefore constitutively nuclear (line 305) (19). We administered either saline or the long-acting GLP-1 analog Exendin4 (10 nmol/kg) daily for 7 days to either wild-type or 305 transgenic mice. We then measured β-cell proliferation by Ki67 immunostaining and islet mass by morphometry of pancreatic sections stained with anti-insulin antiserum (representative images are shown in Fig. 5A). Exendin4 administration resulted in a fourfold increase in Ki67-positive β-cells in control animals (Fig. 5B) and a twofold increase in cross-sectional islet area (Fig. 5C). We also observed a small increase in the number of small clusters of insulin-positive cells (<6 cells) in wild-type mice treated with Exendin4 compared with saline-treated littermates (Fig. 5D). In contrast, 305 transgenic mice failed to increase the number of Ki67-positive cells, islet size, and small β-cell clusters (Fig. 5B–D). These observations indicate that FoxO1 inactivation is required for the proliferative action of GLP-1 in vivo.

Constitutively nuclear FoxO1 prevents GLP-1 effect on gene expression.

We next evaluated the contribution of FoxO1 nuclear exclusion to the GLP-1 transcriptional response in β-cells. GLP-1 increased the levels of mRNAs encoding Pdx1, Foxa2, and Insulin2 and decreased the levels of Igfbp1 (Fig. 6A). In all instances, constitutively nuclear FoxO1 prevented the effect of GLP-1. To determine whether this effect was due to a direct inhibition of FoxO1-dependent transcription, we measured occupancy of the forkhead binding site in the Igfbp1 and Foxa2 promoters using chromatin immunoprecipitation assays. Under basal conditions, we detected FoxO1 bound to both promoters. GLP-1 inhibited FoxO1 binding. GLP-1 was similarly able to decrease FoxO1 binding in H2O2-treated cells, consistent with its effect to inhibit H2O2-induced caspase activation (Fig. 3). In contrast, GLP-1 had no effect in cells expressing constitutively nuclear FoxO1 (Fig. 6B). It should be pointed out that GLP-1 inhibits Igfbp1 and promotes Foxa2 expression. Because FoxO1 binds to both promoters, the data are consistent with the notion that it acts as a transcriptional activator for some genes and as a repressor for others (18).

GLP-1 is a potent glucoincretin hormone with valuable properties for diabetes treatment. Although its proliferative and antiapoptotic properties have been demonstrated in the β-cell, the molecular mechanism by which GLP-1 exerts its effect on β-cell mass is not fully understood. Pharmacological and biochemical evidence indicates that PI 3-kinase/Akt signaling plays a central role in GLP-1 action on β-cell growth and survival. In the present study, we investigated the role of the transcription factor FoxO1 in the action of GLP-1 on β-cell proliferation and survival. As recently hypothesized (31), we show that GLP-1 inhibits FoxO1 transcriptional activity through phosphorylation-dependent nuclear exclusion in INS832/13 cells. Moreover, the ability of the long-acting GLP-1 derivative Exendin4 to increase β-cell mass was blunted in transgenic mice expressing constitutively nuclear FoxO1 in β-cells. Finally, we demonstrate that FoxO1 inactivation plays an important role in the effect of GLP-1 on the expression of important β-cell transcription factors, such as Pdx1 and Foxa2. Our data indicate that FoxO1 inhibition is required for GLP1-dependent proliferation (Fig. 7).

Restoration of insulin secretion and β-cell mass is central to the treatment of diabetes (3234). Through its pivotal role in PI 3-kinase/Akt signaling, FoxO1 has the potential to integrate the proliferative and antiapoptotic signals of β-cell growth factors. We have previously shown that FoxO1 plays an important role in metabolic control, but because of the complex interactions among different tissues, it remained unclear whether this effect is due to intrinsic actions of FoxO1 in the β-cell or to its regulation of peripheral insulin sensitivity (19). In this study, we show that expression of constitutively nuclear FoxO1 inhibits β-cell proliferation induced by glucose, GLP-1, BTC, and serum. Moreover, a recent publication demonstrates that FoxO1 mediates the action of glucose-dependent inhibitory peptide on β-cell survival by regulating Bax expression (35). Based on those results and on the present study, it appears that FoxO1 is a general regulator of β-cell mass in response to incretins.

The mechanism by which FoxO1 inhibition by GLP-1 increases β-cell mass is complex. We have previously shown that FoxO1 inhibits Pdx1 expression, thereby providing a mechanism by which FoxO1 controls β-cell proliferation and insulin secretion (18). Several publications indicate that Pdx1 plays a role in β-cell differentiation, replication, and regeneration. Thus, Pdx1 haploinsufficient mice fail to increase β-cell mass in response to insulin resistance (36), and increased Pdx1 expression is associated with an early burst of β-cell proliferation after subtotal pancreatectomy (37). It has also been shown that GLP-1 increases Pdx1 expression, but the mechanism is unclear (9). The present findings indicate that GLP1 increases Pdx1 expression via inhibition of FoxO1. Moreover, we show that GLP-1 increases Foxa2 expression, and this is associated with decreased FoxO1 binding to the Foxa2 promoter. Thus, it appears that the mechanism by which FoxO1 controls Foxa2 expression is similar to the mechanism by which it controls Pdx1. Further work will be required to understand the molecular mechanism of transcriptional inhibition by FoxO1.

In conclusion, we show that FoxO1 is in the GLP-1 signal transduction pathway. As GLP receptor agonists enter the clinical arena, FoxO1 represents a potential target to modulate its beneficial effects in diabetes.

FIG. 1.

GLP-1 increases FoxO1 phosphorylation via EGFR and PI 3-kinase signaling. A: INS832/13 cells were incubated overnight in serum-free medium and then treated with 10 nmol/l GLP-1 for the indicated time periods. B: Cells were treated with 10 nmol/l GLP-1 with or without 250 nmol/l AG1478 (AG) or 50 μmol/l LY294002 (LY) for 30 min. C: Cells were treated with 10 nmol/l insulin with or without 250 nmol/l AG1478 (AG) or 50 μmol/l LY294002 (LY) for 30 min. In both B and C, AG and LY were added 30 min before stimulation. FoxO1 phosphorylation was analyzed by immunoblotting using anti–phospho-FoxO1 and anti-FoxO1 antisera.

FIG. 1.

GLP-1 increases FoxO1 phosphorylation via EGFR and PI 3-kinase signaling. A: INS832/13 cells were incubated overnight in serum-free medium and then treated with 10 nmol/l GLP-1 for the indicated time periods. B: Cells were treated with 10 nmol/l GLP-1 with or without 250 nmol/l AG1478 (AG) or 50 μmol/l LY294002 (LY) for 30 min. C: Cells were treated with 10 nmol/l insulin with or without 250 nmol/l AG1478 (AG) or 50 μmol/l LY294002 (LY) for 30 min. In both B and C, AG and LY were added 30 min before stimulation. FoxO1 phosphorylation was analyzed by immunoblotting using anti–phospho-FoxO1 and anti-FoxO1 antisera.

FIG. 2.

GLP-1 induces FoxO1 nuclear exclusion. Immunocytochemical analysis of FoxO1 localization in INS832/13 cells after transient transfection with FoxO1-GFP. Representative images are shown. Bar = 10 μm.

FIG. 2.

GLP-1 induces FoxO1 nuclear exclusion. Immunocytochemical analysis of FoxO1 localization in INS832/13 cells after transient transfection with FoxO1-GFP. Representative images are shown. Bar = 10 μm.

FIG. 3.

GLP-1 effects on β-cell proliferation and survival require FoxO1 inactivation. A: Proliferation was evaluated by measuring BrdU incorporation in control cells or after transduction with constitutively nuclear FoxO1 or β-Gal. INS832/13 cells were incubated overnight in serum-free medium containing 5 mmol/l glucose (G5) and treated for 24 h with 25 mmol/l glucose (G25), GLP-1, BTC, or medium supplemented with 10% serum (log phase of growth [Log]). Results represent means ± SE of three separate experiments carried out at least in triplicate. *P < 0.05 by ANOVA. B: Apoptosis was evaluated in cells transduced with β-gal or constitutively nuclear FoxO1 after a 4-h incubation with or without 25 μmol/l H2O2 and in the absence or presence of 10 nmol/l GLP-1. The percentage of apoptotic nuclei detected in microscopic fields in different conditions is shown. *P < 0.05 by ANOVA.

FIG. 3.

GLP-1 effects on β-cell proliferation and survival require FoxO1 inactivation. A: Proliferation was evaluated by measuring BrdU incorporation in control cells or after transduction with constitutively nuclear FoxO1 or β-Gal. INS832/13 cells were incubated overnight in serum-free medium containing 5 mmol/l glucose (G5) and treated for 24 h with 25 mmol/l glucose (G25), GLP-1, BTC, or medium supplemented with 10% serum (log phase of growth [Log]). Results represent means ± SE of three separate experiments carried out at least in triplicate. *P < 0.05 by ANOVA. B: Apoptosis was evaluated in cells transduced with β-gal or constitutively nuclear FoxO1 after a 4-h incubation with or without 25 μmol/l H2O2 and in the absence or presence of 10 nmol/l GLP-1. The percentage of apoptotic nuclei detected in microscopic fields in different conditions is shown. *P < 0.05 by ANOVA.

FIG. 4.

Constitutively nuclear FoxO1 does not affect the ability of GLP-1 to increase cAMP and preserve mitochondrial membrane potential. A: INS832/13 cells were transduced with either β-Gal or constitutively nuclear FoxO1 in complete RPMI medium or incubated overnight in serum-free medium. The following day, cells were preincubated for 30 min in Krebs-Ringer bicarbonate HEPES buffer medium containing 5 mmol/l glucose and subsequently treated for 20 min with GLP-1 or 10 mmol/l nicotinamide. *P < 0.05 by ANOVA. B: Mitochondrial membrane potential was evaluated in INS832/13 cells transduced with constitutively nuclear FoxO1. The fluorescent compound Mit-E-Ψ enters the mitochondria and emits red fluorescence. A loss of mitochondrial membrane potential (ΔΨ) leads to Mit-E-Ψ leakage from the mitochondria and results in green fluorescence. C: INS cells, transduced with either β-Gal or constitutively nuclear FoxO1, were treated as described in Fig. 1B and C. FoxO1 phosphorylation was analyzed by immunoblotting using anti–phospho-ERK1/2 and anti-ERK1/2 antisera.

FIG. 4.

Constitutively nuclear FoxO1 does not affect the ability of GLP-1 to increase cAMP and preserve mitochondrial membrane potential. A: INS832/13 cells were transduced with either β-Gal or constitutively nuclear FoxO1 in complete RPMI medium or incubated overnight in serum-free medium. The following day, cells were preincubated for 30 min in Krebs-Ringer bicarbonate HEPES buffer medium containing 5 mmol/l glucose and subsequently treated for 20 min with GLP-1 or 10 mmol/l nicotinamide. *P < 0.05 by ANOVA. B: Mitochondrial membrane potential was evaluated in INS832/13 cells transduced with constitutively nuclear FoxO1. The fluorescent compound Mit-E-Ψ enters the mitochondria and emits red fluorescence. A loss of mitochondrial membrane potential (ΔΨ) leads to Mit-E-Ψ leakage from the mitochondria and results in green fluorescence. C: INS cells, transduced with either β-Gal or constitutively nuclear FoxO1, were treated as described in Fig. 1B and C. FoxO1 phosphorylation was analyzed by immunoblotting using anti–phospho-ERK1/2 and anti-ERK1/2 antisera.

FIG. 5.

Exendin4 action on pancreatic β-cell area is blunted in transgenic mice with β-cell–specific expression of constitutively nuclear FoxO1. Wild-type or 305 transgenic mice received daily injections of Exendin4 (10 nmol/kg body wt) or saline for 7 days. Pancreatic β-cell mass and β-cell replication were evaluated by morphometry and immunohistochemistry with anti-insulin (brown) and anti-Ki67 antisera (blue). Representative images of pancreas sections are shown in A. The arrow indicates a Ki67-positive nucleus. Bar graphs show the percentage of Ki67-positive β-cells (B), the relative cross-sectional islet area (C), and the number of small β-cell clusters, defined as groups consisting of less than six insulin-positive cells (D). *P < 0.05 by ANOVA.

FIG. 5.

Exendin4 action on pancreatic β-cell area is blunted in transgenic mice with β-cell–specific expression of constitutively nuclear FoxO1. Wild-type or 305 transgenic mice received daily injections of Exendin4 (10 nmol/kg body wt) or saline for 7 days. Pancreatic β-cell mass and β-cell replication were evaluated by morphometry and immunohistochemistry with anti-insulin (brown) and anti-Ki67 antisera (blue). Representative images of pancreas sections are shown in A. The arrow indicates a Ki67-positive nucleus. Bar graphs show the percentage of Ki67-positive β-cells (B), the relative cross-sectional islet area (C), and the number of small β-cell clusters, defined as groups consisting of less than six insulin-positive cells (D). *P < 0.05 by ANOVA.

FIG. 6.

FoxO1 plays a role in the transcriptional response of the β-cell to GLP-1. A: Total RNA was extracted from INS832/13 cells transduced with constitutively nuclear FoxO1 or β-Gal after a 3-h incubation period in the presence or absence of 10 nmol/l GLP-1. Gene expression was evaluated by quantitative real-time PCR and is plotted as fold change from basal levels. *P < 0.05 by ANOVA. B: Chromatin immunoprecipitation assays. FoxO1 was immunoprecipitated from intact chromatin extracted from INS832/13 cells using anti-FoxO1 antiserum. An isotype-matched IgG was used as control. Eluted DNA was amplified using oligonucleotides flanking the forkhead binding site in the Foxa2 and Igfbp1 promoters. Cells were treated with GLP-1 or H2O2. Total input DNA is shown as control. A representative result from three separate experiments is shown except for the effect of GLP-1 in constitutively nuclear FoxO1–expressing cells, which was performed twice.

FIG. 6.

FoxO1 plays a role in the transcriptional response of the β-cell to GLP-1. A: Total RNA was extracted from INS832/13 cells transduced with constitutively nuclear FoxO1 or β-Gal after a 3-h incubation period in the presence or absence of 10 nmol/l GLP-1. Gene expression was evaluated by quantitative real-time PCR and is plotted as fold change from basal levels. *P < 0.05 by ANOVA. B: Chromatin immunoprecipitation assays. FoxO1 was immunoprecipitated from intact chromatin extracted from INS832/13 cells using anti-FoxO1 antiserum. An isotype-matched IgG was used as control. Eluted DNA was amplified using oligonucleotides flanking the forkhead binding site in the Foxa2 and Igfbp1 promoters. Cells were treated with GLP-1 or H2O2. Total input DNA is shown as control. A representative result from three separate experiments is shown except for the effect of GLP-1 in constitutively nuclear FoxO1–expressing cells, which was performed twice.

FIG. 7.

Model of GLP-1 action on β-cell proliferation and survival. Glp-1 promotes EGFR trans-phosphorylation (dashed line), which in turn links GLP-1 signaling to activation of the PI 3-kinase→Akt pathway, leading to FoxoO1 phosphorylation and nuclear exclusion. The biochemical pathway linking GLP-1 receptors to EGFR remains to be elucidated.

FIG. 7.

Model of GLP-1 action on β-cell proliferation and survival. Glp-1 promotes EGFR trans-phosphorylation (dashed line), which in turn links GLP-1 signaling to activation of the PI 3-kinase→Akt pathway, leading to FoxoO1 phosphorylation and nuclear exclusion. The biochemical pathway linking GLP-1 receptors to EGFR remains to be elucidated.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J.B. is a Juvenile Diabetes Research Foundation Postdoctoral Fellow. D.A. received National Institutes of Health Grant DK64819.

1.
Fehmann HC, Goke R, Goke B: Cell and molecular biology of the incretin hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide.
Endocr Rev
16
:
390
–410,
1995
2.
Holz GG, Kuhtreiber WM, Habener JF: Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7–37).
Nature
361
:
362
–365,
1993
3.
Thorens B, Waeber G: Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.
Diabetes
42
:
1219
–1225,
1993
4.
Drucker DJ: Minireview: the glucagon-like peptides.
Endocrinology
142
:
521
–527,
2001
5.
Gutniak M, Orskov C, Holst JJ, Ahren B, Efendic S: Antidiabetogenic effect of glucagon-like peptide-1 (7–36)amide in normal subjects and patients with diabetes mellitus.
N Engl J Med
326
:
1316
–1322,
1992
6.
Holst JJ: Therapy of type 2 diabetes mellitus based on the actions of glucagon-like peptide-1.
Diabetes Metab Res Rev
18
:
430
–441,
2002
7.
Kang G, Chepurny OG, Holz GG: cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic beta-cells.
J Physiol
536
:
375
–385,
2001
8.
Fehmann HC, Habener JF: Insulinotropic hormone glucagon-like peptide-I(7–37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.
Endocrinology
130
:
159
–166,
1992
9.
Buteau J, Roduit R, Susini S, Prentki M: Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells.
Diabetologia
42
:
856
–864,
1999
10.
Stoffers DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, Habener JF, Egan JM: Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas.
Diabetes
49
:
741
–748,
2000
11.
Farilla L, Hui H, Bertolotto C, Kang E, Bulotta A, Di Mario U, Perfetti R: Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.
Endocrinology
143
:
4397
–4408,
2002
12.
Buteau J, Foisy S, Rhodes CJ, Carpenter L, Biden TJ, Prentki M: Protein kinase Cζ activation mediates glucagon-like peptide-1–induced pancreatic β-cell proliferation.
Diabetes
50
:
2237
–2243,
2001
13.
Buteau J, Foisy S, Joly E, Prentki M: Glucagon-like peptide 1 induces pancreatic β-cell proliferation via transactivation of the epidermal growth factor receptor.
Diabetes
52
:
124
–132,
2003
14.
MacDonald PE, Wang X, Xia F, El-kholy W, Targonsky ED, Tsushima RG, Wheeler MB: Antagonism of rat beta-cell voltage-dependent K+ currents by exendin 4 requires dual activation of the cAMP/protein kinase A and phosphatidylinositol 3-kinase signaling pathways.
J Biol Chem
278
:
52446
–52453,
2003
15.
Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ: Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis.
J Biol Chem
278
:
471
–478,
2003
16.
Buteau J, El-Assaad W, Rhodes CJ, Rosenberg L, Joly E, Prentki M: Glucagon-like peptide-1 prevents beta cell glucolipotoxicity.
Diabetologia
47
:
806
–815,
2004
17.
Kops GJ, Burgering BM: Forkhead transcription factors: new insights into protein kinase B (c-akt) signaling.
J Mol Med
77
:
656
–665,
1999
18.
Kitamura T, Nakae J, Kitamura Y, Kido Y, Biggs WH III, Wright CV, White MF, Arden KC, Accili D: The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth.
J Clin Invest
110
:
1839
–1847,
2002
19.
Nakae J, Biggs WH III, Kitamura T, Cavenee WK, Wright CV, Arden KC, Accili D: Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1.
Nat Genet
32
:
245
–253,
2002
20.
Nakae J, Kitamura T, Silver DL, Accili D: The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression.
J Clin Invest
108
:
1359
–1367,
2001
21.
Nakae J, Kitamura T, Ogawa W, Kasuga M, Accili D: Insulin regulation of gene expression through the forkhead transcription factor Foxo1 (Fkhr) requires kinases distinct from Akt.
Biochemistry
40
:
11768
–11776,
2001
22.
Hohmeier HE, Mulder H, Chen G, Henkel-Rieger R, Prentki M, Newgard CB: Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.
Diabetes
49
:
424
–430,
2000
23.
Frescas D, Valenti L, Accili D: Nuclear trapping of the forkhead transcription factor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogenetic genes.
J Biol Chem
280
:
20589
–20595,
2005
24.
Wrede CE, Dickson LM, Lingohr MK, Briaud I, Rhodes CJ: Protein kinase B/Akt prevents fatty acid-induced apoptosis in pancreatic beta-cells (INS-1).
J Biol Chem
277
:
49676
–49684,
2002
25.
Okamoto H, Nakae J, Kitamura T, Park BC, Dragatsis I, Accili D: Transgenic rescue of insulin receptor-deficient mice.
J Clin Invest
114
:
214
–223,
2004
26.
Kitamura YI, Kitamura T, Kruse JP, Raum JC, Stein R, Gu W, Accili D: FoxO1 protects against pancreatic beta cell failure through NeuroD and MafA induction.
Cell Metab
2
:
153
–163,
2005
27.
Accili D, Arden KC: FoxOs at the crossroads of cellular metabolism, differentiation, and transformation.
Cell
117
:
421
–426,
2004
28.
Robertson RP, Harmon J, Tran PO, Tanaka Y, Takahashi H: Glucose toxicity in β-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection.
Diabetes
52
:
581
–587,
2003
29.
Sato A, Wang PC, Ohgawara H: Effect of stimulators such as GLP-1, PACAP, and nicotinamide on glucose-stimulated insulin secretion from porcine pancreatic endocrine cells in long-term culture.
Pancreas
25
:
86
–93,
2002
30.
Tsuboi T, da Silva Xavier G, Holz GG, Jouaville LS, Thomas AP, Rutter GA: Glucagon-like peptide-1 mobilizes intracellular Ca2+ and stimulates mitochondrial ATP synthesis in pancreatic MIN6 beta-cells.
Biochem J
369
:
287
–299,
2003
31.
Holz GG, Chepurny OG: Diabetes outfoxed by GLP-1?
Sci STKE
2005
:
pe2
,
2005
32.
Bonner-Weir S: Perspective: postnatal pancreatic beta cell growth.
Endocrinology
141
:
1926
–1929,
2000
33.
Bonner-Weir S, Sharma A: Pancreatic stem cells.
J Pathol
197
:
519
–526,
2002
34.
Edlund H: Pancreatic organogenesis: developmental mechanisms and implications for therapy.
Nat Rev Genet
3
:
524
–532,
2002
35.
Kim SJ, Winter K, Nian C, Tsuneoka M, Koda Y, McIntosh CH: Glucose-dependent insulinotropic polypeptide (GIP) stimulation of pancreatic {beta}-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxo1, and down-regulation of bax expression.
J Biol Chem
280
:
22297
–22307,
2005
36.
Kulkarni RN, Jhala US, Winnay JN, Krajewski S, Montminy M, Kahn CR: PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance.
J Clin Invest
114
:
828
–836,
2004
37.
Sharma A, Zangen DH, Reitz P, Taneja M, Lissauer ME, Miller CP, Weir GC, Habener JF, Bonner-Weir S: The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration.
Diabetes
48
:
507
–513,
1999