Glucagon-like Peptide-1 Receptor Signaling Modulates beta  Cell Apoptosis*

Yazhou LiDagger §, Tanya HansotiaDagger §||, Bernardo YustaDagger , Frederic Ris**, Philippe A. Halban**, and Daniel J. DruckerDaggerDagger

From the Dagger  Department of Medicine, Banting and Best Diabetes Centre, Toronto General Hospital, University of Toronto, Ontario M5G 2C4, Canada, and the ** Louis-Jeantet Research Laboratories, University Medical Centre, 1211 Geneva 4, Switzerland

Received for publication, September 13, 2002, and in revised form, October 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments beta  cell mass via activation of beta  cell proliferation and islet neogenesis. We examined whether GLP-1 receptor signaling modifies the cellular susceptibility to apoptosis. Mice administered streptozotocin (STZ), an agent known to induce beta  cell apoptosis, exhibit sustained improvement in glycemic control and increased levels of plasma insulin with concomitant administration of the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained significantly lower for weeks after cessation of exendin-4. STZ induced beta  cell apoptosis, which was significantly reduced by co-administration of Ex-4. Conversely, mice with a targeted disruption of the GLP-1 receptor gene exhibited increased beta  cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat beta  cells exposed to interleukin 1beta , tumor necrosis fator alpha , and interferon gamma  in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of beta -catenin after treatment with cycloheximide in vitro. These findings demonstrate that GLP-1 receptor signaling directly modifies the susceptibility to apoptotic injury, and provides a new potential mechanism linking GLP-1 receptor activation to preservation or enhancement of beta  cell mass in vivo.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucagon-like peptide-1 (GLP-1)1 is derived from posttranslational processing of proglucagon in enteroendocrine L cells (1) and is secreted from the distal gut after nutrient ingestion (2). The termination of GLP-1 action by the enzyme dipeptidyl peptidase IV occurs within minutes following GLP-1 secretion (3-5), yet GLP-1 exerts several rapid metabolic actions including stimulation and inhibition of insulin and glucagon secretion, respectively (6-10). GLP-1 action is essential for glucose homeostasis, because GLP-1 receptor blockade with the antagonist exendin (9-39) increases blood glucose and decreases levels of circulating insulin in human and rodent studies (11-14).

Activation of GLP-1 receptor signaling leads to enhanced expression of mRNA transcripts for glucokinase, GLUT-2, Pdx-1, and insulin in beta  cell lines (15-17) and in both normal and diabetic rodents (18-20). Furthermore, GLP-1 and exendin-4 promote differentiation of exocrine cell lines toward a beta  cell phenotype (21), a process that appears to depend on the expression of Pdx-1 (22, 23).

GLP-1 receptor signaling is also coupled to formation of new beta  cells through enhanced proliferation of existing beta  cells (24) and via induction of islet neogenesis (25). The mitogenic actions of GLP-1 are detectable in normal rodents (20, 24) and in the setting of experimental diabetes (19, 25). Administration of GLP-1 or exendin-4 to newborn rats treated with the beta  cell toxin streptozotocin (STZ) leads to increased beta  cell mass at postnatal day 7, which persists and remains increased at 2 months of age. The increased beta  cell mass in the GLP-1/exendin-4 treated rats was attributed to both enhanced beta  cell proliferation and increased numbers of small budding islets (26). Because STZ is known to induce beta  cell destruction in part through activation of apoptotic pathways (27-29), we examined whether GLP-1 receptor activation influences beta  cell mass via regulation of cellular susceptibility to apoptotic cell death.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Materials-- Tissue culture medium, serum, flasks, plates, and antibiotics, including G418, were from Invitrogen. Cycloheximide, forskolin, and protease inhibitor mixture were purchased from Sigma. Exendin-4 was from California Peptide Research (Napa, CA).

Animal Experiments-- Male C57BL/6 mice, 8 weeks of age, were used for experiments shown in Figs. 1-3. Age- and sex-matched CD-1 GLP-1R+/+ control mice housed in the same animal facility were used for studies of GLP-1R-/- mice in the CD1 background (8-week-old male mice). All animals were maintained on standard laboratory chow under a 12 h:12 h light-dark schedule, and experiments were conducted according to protocols and guidelines approved by the Toronto General Hospital Animal Care Committee. STZ (Sigma) (50 mg/kg body weight, intraperitoneal injection once daily for 5 days) was administered as a freshly prepared solution in 0.1 mM sodium citrate pH 5.5. Exendin-4 (Ex-4; 24 nmol/kg body weight, a dose selected based on therapeutic efficacy in previous mouse experiments (30)) was administered as a single daily intraperitoneal injection. For studies depicted in Figs. 1 and 2, morning blood glucose was measured periodically throughout the experimental period and an oral glucose tolerance tests was done at day 30. For histological studies of islet apoptosis, Ex-4 administration was commenced either 2 or 7 days before STZ in separate experiments and continued until the last injection of STZ; C57BL/6 mice were sacrificed within ~24 h after the last STZ injection. For studies of apoptosis in GLP-1R-/- mice, wild-type GLP-1R+/+ and GLP-1R-/- mice were divided into separate groups (n = 4-6) and administered a slightly lower dose of STZ (40 mg/kg body weight) because of the different sensitivities of CD-1 versus C57BL/6 mice to STZ as delineated in preliminary dose-response studies caused by the known species-specific sensitivity to streptozotocin-induced apoptosis (31). After completion of the experiments (~48 h after the last dose of STZ), mice were euthanized by CO2 anesthesia, blood was collected by cardiac puncture for plasma insulin determinations, and pancreases were removed, fixed in 10% formalin overnight, and embedded in paraffin for histological analyses.

Oral Glucose Tolerance Test and Measurement of Plasma and Pancreatic Insulin Levels-- Oral glucose tolerance tests were carried out after an overnight fast as described (14, 32). A blood sample was collected from the tail vein during the 10-20 min time period for measurement of plasma insulin using a rat insulin enzyme-linked immunoassay kit (Crystal Chem. Inc., Chicago, IL) with mouse insulin as a standard (14).

Histological Assessment of Islet Apoptosis and Proliferation-- To detect apoptosis, TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end labeling) staining was performed using ApopTag Peroxidase in situ Apoptosis detection kit (S7100) (Intergen Company, Purchase, NY), according to the manufacturer's instructions as described (33). Slides were analyzed with a Leica microscope, and apoptotic rates were calculated as the number of TUNEL-positive cells per islet, n = 6-7 pancreases for each experimental group of C57Bl/6 +/+ mice, or n = 4-6 pancreases for each group of CD1 GLP-1R+/+ or GLP-1R-/- mice. Analysis of serial consecutive islet sections stained with either insulin or the ApopTag reagent demonstrated that the apoptotic nuclei were localized to insulin-immunopositive beta  cells.

Islet cell proliferation was assessed by counting the number of 5'-bromo-2'-deoxyuridine-positve (BrdUrd+) islet cells in multiple pancreatic sections from both wild-type C57BL/6 and CD-1 and GLP-1R-/- CD1 mice administered BrdUrd (Roche) by intraperitoneal injection, 50 mg/kg body weight, ~ 5 h prior to removal of the pancreas. Immunohistochemical detection of BrdUrd+ cells was carried out using an anti-BrdUrd antibody (CalTag Laboratories, Burlingame, CA). Serial sections were stained for either insulin or BrdUrd, and islet and pancreatic areas were measured using a Leica microscope and Q500MC software. The vast majority of BrdUrd+ cells were immunopositive for insulin, and hence represented beta  cells. The relative cross-sectional beta  cell area, as a percentage of total pancreatic area, was assessed quantitatively as previously described (34-36). The number of BrdUrd-positive cells are expressed both per islet and per 105 µm2 beta -cell area.

Rat Islets and Sorted beta  Cells-- Islets were isolated from the pancreas of adult male Wistar rats (180-220 g) by collagenase digestion and purified on a gradient of Ficoll (37). The islets were further dissociated into single cells by trypsinization, and beta  cells were sorted on the basis of their autofluorescence using a FACStar Plus (BD Biosciences) as described (37). The sorted cell population comprises 95% beta  cells (37). Cells were allowed to recover from the isolation/sorting procedures by culture overnight in Dulbecco's modified Eagle's medium, 10% fetal calf serum, 11.2 mM glucose using plastic dishes to which they did not attach. For measurement of apoptosis by ELISA (see below), cells were seeded (5-8 × 105 cells/ml, Dulbecco's modified Eagle's medium, 11.2 mM glucose, 10% fetal calf serum, 50 µl/well) in 96-well plates pre-coated with extracellular matrix from 804G rat bladder carcinoma cells (Desmos, San Diego, CA) (38). For TUNEL labeling (see below) and labeling with BrdUrd, the sorted beta  cells were seeded at the same density and in the same medium as 50-µl microdroplets placed at the center of 35-mm-diameter plastic Petri dishes coated with 804G matrix. This allowed for use of an inverted-stage fluorescent microscope to examine the cells (under a coverslip) after fixation. Sorted rat beta  cells maintained in monolayer culture were exposed to a mixture of three cytokines for 18 h at 8.3 mM glucose. These conditions were established on the basis of preliminary experiments (data not shown) designed to obtain marked augmentation of apoptosis without significant cell necrosis or detachment of cells from the culture vessel. All incubations of cells were in a humidified atmosphere of 5% CO2 at 37 °C. Exendin-4 was present throughout the 18-h incubation with or without cytokines at a final concentration of 100 nM.

Quantification of Apoptosis and Cell Division in Isolated beta  Cells-- Apoptosis of purified beta  cells was estimated using Cell Death Detection ElisaPLUS (Roche Biochemicals, Mannheim, Germany) for determination of cytoplasmic histone-associated-DNA-fragments (mono- and oligonucleosomes) in cell lysates, a method that correlates well with apoptosis quantification by annexin V staining (39). Alternatively, cells seeded in microdroplets on Petri dishes were processed for estimation of apoptosis using the TUNEL technique according to the manufacturer's instructions ("in situ cell death detection kit" from Roche Biochemicals) following fixation for 20 min in 4% paraformaldehyde and permeabilization using 0.5% Triton X-100 for 4 min at room temperature. Cell replication was assessed by incorporation of BrdUrd (Sigma). For this purpose, BrdUrd (10 µM) was included throughout the 18-h incubation with cytokines or exendin. Cells were then fixed, and BrdUrd+ cells were visualized by immunoflueorescence.

Cell Culture and Apoptosis Experiments-- BHK fibroblasts were grown in Dulbecco's modified Eagle's medium, 4.5g/l glucose supplemented with 5% calf serum. Cells were transfected with cDNAs encoding the rat GLP-1 receptor cloned in the pcDNA3.1 eukaryotic expression vector (Invitrogen, San Diego, CA). Stably transfected cell populations were selected by growth in G418 (Invitrogen) at 0.8 mg/ml for 2 weeks and studies of apoptosis in BHK-GLP-1R cells were done using pools of G418-resistant clones. For apoptosis experiments, cells were replated in culture medium lacking G418, serum-starved overnight, and treated with cycloheximide in the presence or absence of the indicated peptides or drugs as described (40).

Cell Viability Assay-- Cells were exposed to either vehicle or cycloheximide in the presence or absence of the indicated drugs, and the number of viable cells was assessed by measuring the bioreduction of a methane thiosulforate tetrazolium salt at 490 nm using the CellTiter 96 aqueous assay (Promega, Madison, WI.)

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- Cell pellets were lysed at 4 °C in radioimmune precipitation assay buffer containing a protease inhibitor mixture, and cleared lysates were boiled in sample buffer containing beta -mercaptoethanol and stored at -70 °C. Protein concentration was determined using bovine serum albumin as a standard and equal amounts of cell lysates were separated by discontinuous SDS-polyacrylamide gel electrophoresis under reducing conditions and electrotransferred onto Hybond-C nitrocellulose membrane (Amersham Biosciences). The resultant blot was blocked with 5% skim milk in phosphate-buffered saline containing 0.2% Tween 20 and incubated with the indicated primary antibody overnight at room temperature. Proteins were detected with a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence commercial kit (Amersham Biosciences). Western blot analyses were carried out using primary antibodies reactive to active caspase-3 p17 subunit (1:1000 dilution; PerkinElmer Life Sciences), cytochrome c (1:250 dilution; BIOSOURCE International), porin/VDAC 31HL (1:500 dilution; Calbiochem), Akt (1:1000; Cell Signaling Technology (Beverly, MA)) and actin (1:5,000 dilution; Sigma).

Statistical Analysis-- All values are presented as means ± S.E. Statistical significance between groups was evaluated by student's t test or Bonferroni-corrected analysis of variance.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As treatment of rodents with GLP-1 agonists leads to increased islet mass in association with beta  cell proliferation and islet neogenesis (19, 25), we hypothesized that GLP-1 might also enhance beta  cell mass via protection from cellular apoptosis. To test this hypothesis, wild-type C57BL/6 mice were treated with low-dose streptozotocin, a chemical known to induce beta -cell apoptosis (29), in the presence or absence of the GLP-1 analog Ex-4, administered for 2 days before STZ, during, and 3 days after STZ (Fig. 1). The pretreatment regimen was selected in part because of observations that pretreatment of mice with the related glucagon-like peptide GLP-2 significantly reduced apoptosis in experimental models of intestinal injury (33, 41). Mice treated with STZ developed progressive hyperglycemia, with levels of blood glucose rising steadily several days after STZ administration. In contrast, mice that received both STZ (5 days) and Ex-4 (10 days) exhibited a significantly delayed onset of hyperglycemia (compare day 9-12 glucose in STZ versus STZ+Ex-4 mice, Fig. 1) and blood glucose remained significantly lower even 2 weeks after the last dose of Ex-4 (Fig. 1, p < 0.05 for STZ alone versus STZ+Ex-4 glucose between day 9-29). Furthermore, levels of circulating insulin at day 30 were significantly greater in STZ+Ex-4 mice, 20 days after the last Ex-4 injection (Fig 1).


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Fig. 1.   Morning-fed blood glucose and plasma insulin in wild-type C57BL/6 mice treated with saline, exendin-4 (Ex-4), STZ, or STZ+Ex-4. Blood glucose was significantly lower from day 9 to day 29 in STZ+Ex-4 mice compared with glucose in mice receiving STZ alone (p < 0.05), whereas fed plasma insulin level measured at day 30 was significantly higher in STZ+Ex-4 mice versus STZ alone (*, p < 0.05); n = 10 mice per each experimental group. C, control (saline).

A separate experiment was carried out using a different pre-treatment period starting exendin-4 administration 7 days before STZ, and continuing exendin-4 administration for a total of 28 days, with assessment of oral glucose tolerance and glucose-stimulated insulin at day 30. A similar protective response to Ex-4 was observed in this longer experiment shown in Fig 2. Although hyperglycemia developed in all mice treated with STZ, levels of blood glucose were significantly lower in the STZ+Ex-4 group, even more than 3 weeks after cessation of Ex-4 (Fig. 2a; p < 0.05 for day 15-52 glucose, STZ versus STZ+Ex-4). Oral glucose tolerance testing on day 30, 2 days after the last dose of Ex-4, revealed significantly lower glucose excursion specifically at early time points following oral glucose loading, in association with significantly increased levels of plasma insulin in STZ+Ex-4 mice (Fig. 2b; p < 0.002). Furthermore, the levels of fed plasma insulin remained significantly greater in the STZ+Ex-4-treated mice and were comparable to levels detected in Ex-4-alone mice that did not received streptozotocin (p < 0.05) at day 55, 27 days after the last dose of exendin-4 (Fig. 2a).


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Fig. 2.   Ex-4 reduces hyperglycemia in ST2-treated mice. a, morning-fed blood glucose and plasma insulin levels in wild-type C57BL/6 mice treated with saline (C, control), exendin-4 (Ex-4), STZ, or STZ + Ex-4. Morning-fed blood glucose was significantly lower from day 15 to day 52 in Ex-4+STZ mice versus STZ-treated mice alone (p < 0.05). The levels of fed plasma insulin were significantly increased at day 55 in STZ mice treated with Ex-4 (*, p < 0.05 for insulin in STZ versus STZ+Ex-4 mice). n = 10 mice per group; *, p < 0.05. b, difference in blood glucose from fasting baseline (Delta G) during oral glucose tolerance tests carried out at day 30. n = 8 mice per group; *, p < 0.05 for differences with and without Ex-4. The area under the curve (AUC) for glucose levels from 0 to 20 min was significantly lower in STZ+Ex-4 mice compared with mice receiving STZ alone (p < 0.05). Plasma insulin concentrations (inset) were measured in blood obtained during the oral glucose tolerance tests between the 10- and 20-min time points. OGTT, oral glucose tolerance test.

To ascertain the mechanisms underlying the sustained improvement in levels of glucose and insulin in STZ+Ex-4 mice, we assessed pancreatic histological sections for the presence of apoptotic beta  cells in separate groups of mice treated with STZ, with or without Ex-4. Only a rare apoptotic beta  cell was detectable in histological sections from pancreases of control or Ex-4-treated mice in the absence of STZ (Fig 3a). In contrast, morphological features of apoptosis, including pyknotic nuclei, were readily detectable in pancreatic sections from STZ-treated mice. The numbers of TUNEL-positive apoptotic beta  cells were markedly increased in STZ-treated mice, and significantly reduced (4.5-fold) in mice administered both STZ and Ex-4, whether expressed as the number of apoptotic cells per islet or normalized to relative beta  cell area (Fig. 3, b and d; p < 0.001, STZ versus STZ+Ex-4).


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Fig. 3.   Ex-4 reduces islet apoptosis in ST2-treated mice. a, photomicrograph of islet apoptosis (arrows denote TUNEL-positive cells) in wild-type control (C), Ex-4, STZ, and STZ+Ex-4 C57BL/6 mice ~24 h after 5 daily injections of STZ. b and d, number of apoptotic cells detected on day 8 at the end (E) of the experiment, ~24 h after the last dose of STZ normalized per islet (b) or per relative islet area (d) was quantified as described under "Materials and Methods." *, p < 0.05, STZ versus STZ+Ex-4 mice, n = 6-7 mice per group, 2 slides analyzed per mouse. The relative extent of basal beta -cell apoptosis observed after STZ varies in the different genetic strains of mice in the experiments shown in Fig. 3 (C57Bl/6) versus Fig. 4 (CD1), as previously described (31, 66). Approximately 20 islets per slide were assessed, with a minimum of 2 slides analyzed per mouse. c and e, number of BrdUrd+ islet cells was quantified in multiple histological sections from the four different experimental groups, and expressed relative to the number of islets (c) or normalized to relative islet area (e). *, p < 0.05 for number of BrdUrd+ cells in exendin-4 (Ex-4) versus control (C) mice.

Because GLP-1 agonists have been shown to induce beta -cell proliferation (19), we assessed the extent of beta -cell proliferation and expansion of islet mass in the same experiment. Wild-type mice treated with Ex-4 alone for 7 days in the absence of STZ exhibited a greater than 2-fold increase in the number of BrdUrd+ beta  cells, whether expressed as BrdUrd+ cells per islet, or normalized to beta  cell area (Fig. 3, c and e, respectively; p < 0.05 for control versus Ex-4-treated mice). In contrast, we did not detect a significant increase in the number of BrdUrd+ cells in Ex-4-treated mice treated for 5 consecutive days with STZ (Fig. 3, c and e).

These findings demonstrate that exogenous activation of GLP-1 receptor signaling reduced STZ-associated islet apoptosis in wild-type mice in vivo. To ascertain whether basal levels of endogenous GLP-1 receptor signaling protected beta  cells from external injury, we administered STZ to mice with a targeted disruption of the Glp-1R gene (GLP-1R-/- mice (32)). Blood glucose increased more rapidly in GLP-1R-/- versus GLP-1R+/+ mice after STZ administration (Fig. 4, c and e; p < .05 for glucose at day 7 in STZ-treated GLP-1R+/+ versus GLP-1R-/- mice) and remained significantly greater in STZ-treated GLP-1R-/- from day 7-16 (Fig. 4, c and e; p < 0.05; GLP-1R-/- versus control GLP-1R+/+ mice treated with STZ). In contrast, after day 16, the levels of blood glucose in STZ-treated mice remained elevated however no significant differences in glucose (Fig. 4, c) were detected in GLP-1R-/- versus GLP-1R+/+ by day 28, 23 days after the last dose of STZ. Similarly, levels of glucose-stimulated insulin at day 7 were not significantly different. In contrast, the number of apoptotic beta  cells detected ~48 h after the last dose of STZ was increased in both GLP-1R+/+ and GLP-1R-/- mice and was significantly greater (2.7-fold) in GLP-1R-/- mice treated with identical doses of STZ (Fig. 4, a and b; p < 0.002). Taken together, the data presented in Figs. 1-4 demonstrate that activation or abrogation of GLP-1 receptor signaling regulates the extent of murine beta  cell apoptosis in vivo.


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Fig. 4.   Islet apoptosis, glucose, and plasma insulin in GLP-1R+/+ and GLP-1R-/- mice following vehicle (0.1 mM sodium citrate buffered saline) or STZ administration. Equal volumes of saline (vehicle) or STZ were administered by intraperitoneal injection to groups of mice for 5 days, after which mice were euthanized ~48 h later for histological assessment of islet apoptosis (a and b) (arrows denote apoptotic beta  cells detected by TUNEL assay) as described under "Materials and Methods." Approximately 30 islets per slide were assessed, with a minimum of 2 slides analyzed per mouse. *, p < 0.03, GLP-R+/+ vehicle versus STZ; **, p < 0.0003 GLP-1R+/+ versus GLP-1R-/- both treated with STZ; ***, p < 0.0001 GLP-1R-/- vehicle versus STZ for rates of islet apoptosis in GLP-1R+/+ versus GLP-1R-/- mice with and without STZ. The entire pancreas was cut into multiple pieces and embedded, and two representatives slides from each mouse were analyzed for apoptosis (n = 4-6 mice per group). No changes in beta -cell mass or beta -cell proliferation were detected in GLP-1R+/+ versus GLP-1R-/- mice at the end of this experiment (data not shown). c-e, fed blood glucose in CD1 control GLP-1R+/+ and CD1GLP-1R-/- mice treated for 5 days with STZ. The area under the curve for blood glucose between days 0 and 16 is shown in e. *, p < 0.05 for glucose in GLP-1R+/+ versus GLP-1R-/- mice treated with STZ in c and for AUC glucose (e) between days 0 and 16 in wild-type (WT) versus GLP-1R-/- (KO) mice treated with streptozotocin (STZ). No significant differences were observed in levels of glucose-stimulated insulin assessed at day 28 (d).

To determine whether GLP-1 agonists exert direct anti-apoptotic effects on islet beta  cells in vitro using a different approach for generation of cytotoxic injury, we induced apoptosis in purified populations of sorted rat beta  cells using a combination of cytokines (1 ng/ml interleukin 1beta , 5 ng/ml tumor necrosis factor alpha , 5 ng/ml interferon gamma ) as previously described (42). Incubation with cytokines alone for 18 h produced a 4.9-fold increase in apoptosis, however co-incubation with cytokines and exendin-4 significantly reduced the extent of apoptosis (Fig 5a) by 44.0 ± 5.2% compared with the extent of apoptosis with cytokines alone (p < 0.001). To assess directly the percentage of apoptotic cells under these various conditions, individual rat beta  cells were examined for TUNEL staining (Fig. 5b). Cytokines increased the percentage of TUNEL-positive beta  cells from 0.6 to 4.2%. Consistent with results seen by ELISA, exendin-4 significantly reduced the percentage of TUNEL-positive beta  cells compared with values seen with cytokines alone (Fig. 5b; p < 0.001). Because GLP-1 agonists are known to stimulate beta  cell proliferation, it was possible that the observed quantitative effects on apoptosis were to some degree modified by increased cell replication. To test this possibility, cell division was estimated by incorporation of BrdUrd (present throughout the 18 h incubation period). As expected for primary adult beta  cells, the number of dividing cells was extremely low under control conditions. Although there did appear to be a modest increase with exendin-4 treatment, no more than 10 BrdUrd-fluorescent cells were visible in each monolayer culture of 25,000 cells (amounting to less than 0.05%) even under these conditions. In the presence of cytokines, the cells change their morphology making it difficult to observe BrdUrd-positive cells with any accuracy, but there was no evidence for any increase in cell division. Similarly, glucose-stimulated insulin secretion was abolished by treatment with the cytokines and exendin-4 was not able to reverse this impairment (data not shown), in keeping with the known and severe effects of this combination of cytokines on beta -cell function (43).


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Fig. 5.   The GLP-1 receptor agonist exendin-4 reduces cytokine-induced apoptosis of rat beta  cells. beta  cells were sorted (from adult rat islets) and established in monolayers. The cells were incubated for 18 h with cytokines (1 ng/ml interleukin 1beta , 5 ng/ml tumor necrosis factor alpha , 5 ng/ml interferon gamma ) with or without exendin 4 (100 nM) as indicated. a, apoptosis was quantified by ELISA and normalized to values measured in untreated cells. Data are mean ± S.E. (n = 5 per experiment) for one of a total of 3 independent experiments. p < 0.001 for differences between all 3 groups. b, TUNEL-positive cells were assessed directly by immunohistochemistry. Data (TUNEL-positive cells as percentage of all cells in microscopic field) are mean ± S.E., n = 8 fields from 2 independent experiments (4 fields per experiment). The number of cells per field (mean ± S.E., n = 8) was 126 ± 6, 120 ± 6, 110 ± 8, and 111 ± 6 for controls, exendin-4 alone, cytokines alone, or exendin-4 plus cytokines, respectively (differences between groups not statistically significant). *, p < 0.001 for cytokines alone versus cytokines plus exendin-4.

These findings demonstrate that direct activation of the beta -cell GLP-1 receptor is coupled to reduction of apoptosis in wild-type mice in vivo and in primary rat beta -cell cultures in vitro. To determine whether GLP-1 receptor activation is sufficient for direct engagement of antiapoptotic pathways in heterologous cells, we introduced the rat GLP-1 receptor into BHK fibroblasts. BHK-GLP-1R cells responded to GLP-1 with a dose-dependent increase in cAMP accumulation, whereas GLP-1 had no effect on wild-type BHK cells (data not shown). After treatment with cycloheximide, BHK cells exhibit morphological features associated with apoptosis including membrane blebbing, cell shrinkage and detachment, and cell fragmentation into apoptotic bodies (40). BHK-GLP-1R cells treated with 80 µM cycloheximide exhibited progressively reduced cell viability, whereas co-incubation of cells with Ex-4 significantly increased cell viability at multiple time points (Fig. 6a). The relative activities of caspase-3, caspase-8, and caspase-9 were markedly induced after exposure to cycloheximide and significantly reduced after treatment with Ex-4 in the same experiments (data not shown). Cycloheximide-treated BHK-GLP-1R cells exhibited increased levels of the active p17 caspase 3 subunit that were markedly diminished by treatment with Ex-4 (Fig. 6b). Similarly, Ex-4 reduced the translocation of cytochrome c from the mitochondrial compartment to the cytosol in the setting of cycloheximide-induced apoptosis (Fig. 6c). Furthermore, the levels of intact beta -catenin, a caspase-3 substrate, and the prosurvival kinase Akt, were markedly reduced following exposure to cycloheximide, whereas Ex-4 treatment clearly preserved levels of both intact beta -catenin and Akt (Fig. 6d).


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Fig. 6.   Ex-4 protects BHK-GLP-1R cells from cycloheximide-induced apoptosis. For assessment of cell viability, cells were cultured as described under "Materials and Methods" and treated with 80 µM CHX in the presence or absence of 20 nM Ex-4 or 20 µM forskolin, a concentration previously shown to attenuate cycloheximide- or LY294002-induced apoptosis in BHK cells (40, 67). Dose-response studies demonstrated significant cAMP accumulation in BHK-GLP-1R cells with 10-a, 100 nM exendin-4 (data not shown). a, cell viability was quantified using a tetrazolium salt bioreduction assay and expressed as a percentage of the values obtained from analysis of vehicle alone-treated cultures. No effect on cell survival or cAMP accumulation was detected in wild-type BHK cells treated with Ex-4 (data not shown). Data shown are the means ± S.E. from 4 independent experiments, each one performed in triplicate. ***, p < 0.001; **, p < 0.01, CHX plus either Ex-4 or forskolin versus CHX alone. b, effect of Ex-4 or forskolin on CHX-induced caspase-3 activation in BHK-GLP-1R cells. BHK-GLP-1R cells were serum-starved for 24 h and then treated with CHX in the presence or absence of Ex-4 or forskolin for 15 h. Cell extracts were analyzed by immunoblotting for active caspase-3 p17 subunit as described under "Materials and Methods." Equal loading was verified by reprobing the blots with an anti-actin antibody. Results are representative of three independent experiments. The relative densitometric units for the intensity of the caspase-3 p17 subunit normalized to the values obtained for actin in multiple experiments (n = 4) is shown to the left of the Western blot. *, p < 0.05 for CHX versus CHX+FK or CHX+Ex-4. c, Ex-4 prevents cycloheximide-induced release of cytochrome c into the cytosol in BHK-GLP-1R cells. BHK-GLP-1R cells were exposed to CHX in the presence or absence of Ex-4 or forskolin. After 15 h, mitochondrial pellets and cytosolic supernatants were prepared, and Western blot analysis was performed to detect cytochrome c in both subcellular fractions. The quality of the subcellular fractionation and the equivalent protein loading per lane were monitored by probing the blots for porin and actin, respectively. d, exendin-4 reduces CHX-induced degradation of beta -catenin and Akt in BHK-GLP-1R cells. BHK-GLP-1R cells were serum-starved for 24 h and then treated with CHX in the presence or absence of Ex-4 or forskolin for 15 h. Cell extracts were then analyzed by immunoblotting for beta  -catenin cleavage and Akt as described under "Materials and Methods." Equal loading was verified by reprobing the blots with an anti-hsp90 antibody. The relative densitometric units (RDU) of the beta -catenin cleavage products derived from scanning the blots of several independent experiments (n = 4) are shown to the left of the Western blot. FK, forskolin; *, p < .05; **, p < .001, CHX alone versus CHX plus Ex-4, or CHX plus forskolin, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Original concepts of GLP-1 action focused on its role as a gut-derived incretin acting on islet beta  cells to augment nutrient-stimulated insulin release in the postprandial state (44, 45). The finding that GLP-1 agonists also promote beta  cell proliferation and islet neogenesis (19, 25), taken together with the defects in islet topography observed in the GLP-1R-/- mouse (35) has broadened the physiological actions of incretin peptides to encompass the regulation of beta  cell mass. Indeed, recent evidence suggests a potential role for the structurally related incretin, glucose-dependent insulinotropic polypeptide as a second gut-derived regulator of beta -cell proliferation (46).

The concept that circulating gut peptides exert cytoprotective actions on distal target tissues is exemplified by the actions of GLP-2, a proglucagan-derived peptide co-secreted from the L cell together with GLP-1 (47, 48). GLP-2 promotes cell proliferation indirectly via activation of a distinct G-protein-coupled receptor expressed in human enteroendocrine cells and murine enteric neurons, leading to expansion of the mucosal epithelium in the small and large bowel (49-53). Although the anti-apoptotic actions of GLP-2 are not readily apparent in the normal epithelial mucosa, induction of experimental intestinal injury is associated with increased crypt apoptosis that is markedly suppressed by exogenous GLP-2 administration (33, 41).

Given the low basal rate of apoptosis in the normal islet (54), induction of islet injury is necessary to unmask the anti-apoptotic effects of GLP-1 on the beta  cell in vivo. Rats treated with intraperitoneal STZ for 5 days exhibited increased pancreatic levels of prohormone convertase-1, the primary enzyme responsible for liberation of GLP-1 from proglucagon (55). Furthermore, the levels of bioactive GLP-1(7-36 amide) were significantly elevated in the circulation and pancreas of STZ-treated diabetic rats (55). These findings suggest that at least some types of experimental diabetes associated with beta -cell injury may be characterized by enhanced liberation of both pancreatic and intestinal GLP-1, which may have physiological implications for protection of vulnerable beta  cells from further cell death.

A recent report suggests that GLP-1 may exert cytoprotective effects in rat islets incubated with cytokines in vitro (56). Because primary cultures of rat islets generally contain a mixture of both islet and contaminating non-islet cell types (57), it is not possible to determine whether the antiapoptotic effects of GLP-1 in mixed islet cultures are exerted through direct or indirect actions on the islet beta  cell. Furthermore, the GLP-1 receptor has been localized to not only beta  but also to islet alpha  and delta  cells (58); hence even mixed islet endocrine cultures free of exocrine contamination will contain several distinct cell types capable of responding to exogenous GLP-1 in vitro.

To determine whether GLP-1 exerts antiapoptotic effects via direct or indirect mechanisms, we used two independent experimental models, highly purified sorted rat beta  cells and heterologous cells transfected with the GLP-1 receptor. The finding that GLP-1 directly inhibits apoptosis both in populations of islet beta  cells exposed to cytokines and in BHK-GLP-1R fibroblasts treated with cycloheximide strongly implicates a direct antiapoptotic effect of GLP-1 agonists transduced via the GLP-1 receptor. Similarly, heterologous cells stably transfected with the GLP-2 receptor exhibit enhanced survival after CHX treatment and reduced activation of proapoptotic effector caspases after treatment of cells with GLP-2 (33, 40). Because both GLP-1 and GLP-2 increase the levels of cAMP, and forskolin mimics the antiapoptotic actions of these peptides in vitro, activation of downstream cAMP-dependent prosurvival pathways may be an important feature of several G-protein-coupled receptors that regulate cellular apoptosis. Indeed, the vasoactive intestinal peptide and pituitary adenylate cyclose-activating peptide receptors are also expressed on islet beta  cells (59) and coupled to adenylate cyclase activation and these insulinotropic peptides exert antiapoptotic and cytoprotective actions in vitro (60-63). Nevertheless, elevated levels of intracellular cAMP are not always associated with enhanced beta -cell survival (64), hence the precise signal-transduction mechanisms linking G-protein-coupled receptor activation to beta -cell injury require further clarification.

The majority of actions ascribed to GLP-1 have been deduced following exogenous GLP-1 administration and some, but not all of these effects are physiologically important for metabolic regulation and beta -cell function. GLP-1R-/- beta  cells exhibit reduced levels of cAMP, defects in glucose-stimulated insulin secretion (32), and abnormalities in glucose-stimulated calcium signaling (65). Nevertheless, ob/ob:GLP-1R-/- mice exhibit enhanced islet proliferation and up-regulation of islet mass despite the complete absence of GLP-1R signaling (34). In contrast, the finding that GLP-1R-/- beta  cells exhibit enhanced susceptibility to STZ-induced apoptosis demonstrates that GLP-1 receptor signaling is an important physiological determinant of beta -cell survival following external injury. Because GLP-1 analogues are currently being developed for the treatment of type 2 diabetes, a disease characterized by progressive deterioration and ultimate loss of beta  cell function, understanding the cytoprotective and proliferative mechanisms activated by GLP-1 in the islet beta  cell is potentially relevant to the therapy of type 2 diabetes.

    FOOTNOTES

* This work was partially supported by grants from the Juvenile Diabetes Research Foundation (JDRF 2000-559 to D. J. D. and JDRF 4-1999-844 to P. A. H.), the Canadian Diabetes Association and Ontario Research and Development Challenge Fund (to D. J. D.), and by the Swiss National Science Foundation (Grant 3200-061776.00 to P. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Supported by a postdoctoral fellowship Award from the Canadian Diabetes Association.

|| Supported by a Novo Nordisk-Banting and Best Diabetes Centre studentship.

Dagger Dagger Supported by a Senior Scientist Award (Canadian Institutes of Health Research). To whom correspondence should be addressed: Toronto General Hospital, 200 Elizabeth St. MBRW4R-402, Toronto, Ontario M5G 2C4, Canada. Tel.:416-340-4125; Fax: 416-978-4108; E-mail: d.drucker@utoronto.ca.

Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M209423200

    ABBREVIATIONS

The abbreviations used are: GLP-1, glucagon-like peptide-1; STZ, streptozotocin; Ex-4, exendin-4; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling; BrdUrd, 5'-bromo-2'-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; BHK, baby hamster kidney; CHX, cycloheximide.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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