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• W2149502431 abstract "Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments β cell mass via activation of β 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 β 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 β 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 β cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat β cells exposed to interleukin 1β, tumor necrosis fator α, and interferon γ in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of β-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 β cell mass in vivo. Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments β cell mass via activation of β 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 β 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 β 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 β cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat β cells exposed to interleukin 1β, tumor necrosis fator α, and interferon γ in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of β-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 β cell mass in vivo. glucagon-like peptide-1 streptozotocin exendin-4 terminal deoxynucleotide transferase-mediated dUTP nick end labeling 5′-bromo-2′-deoxyuridine enzyme-linked immunosorbent assay baby hamster kidney cycloheximide Glucagon-like peptide-1 (GLP-1)1 is derived from posttranslational processing of proglucagon in enteroendocrine L cells (1Mojsov S. Heinrich G. Wilson I.B. Ravazzola M. Orci L. Habener J.F. J. Biol. Chem. 1986; 261: 11880-11889Abstract Full Text PDF PubMed Google Scholar) and is secreted from the distal gut after nutrient ingestion (2Orskov C. Holst J.J. Knuhtsen S. Baldissera F.G.A. Poulsen S.S. Nielsen O.V. Endocrinology. 1986; 119: 1467-1475Crossref PubMed Scopus (409) Google Scholar). The termination of GLP-1 action by the enzyme dipeptidyl peptidase IV occurs within minutes following GLP-1 secretion (3Kieffer T.J. McIntosh C.H. Pederson R.A. Endocrinology. 1995; 136: 3585-3596Crossref PubMed Scopus (0) Google Scholar, 4Deacon C.F. Nauck M.A. Toft-Nielsen M. Pridal L. Willms B. Holst J.J. Diabetes. 1995; 44: 1126-1131Crossref PubMed Google Scholar, 5Hansen L. Deacon C.F. Orskov C. Holst J.J. Endocrinology. 1999; 140: 5356-5363Crossref PubMed Google Scholar), yet GLP-1 exerts several rapid metabolic actions including stimulation and inhibition of insulin and glucagon secretion, respectively (6Kreymann B. Ghatei M.A. Williams G. Bloom S.R. Lancet. 1987; ii: 1300-1304Abstract Scopus (1500) Google Scholar, 7Mojsov S. Weir G.C. Habener J.F. J. Clin. Invest. 1987; 79: 616-619Crossref PubMed Scopus (681) Google Scholar, 8Orskov C. Nielsen J.H. FEBS Lett. 1988; 229: 175-178Crossref PubMed Scopus (42) Google Scholar, 9Komatsu R. Matsuyama T. Namba M. Watanabe N. Itoh H. Kono N. Tarui S. Diabetes. 1989; 38: 902-905Crossref PubMed Google Scholar, 10Wettergren A. Schjoldager B. Mortensen P.E. Myhre J. Christiansen J. Holst J.J. Dig. Dis. Sci. 1993; 38: 665-673Crossref PubMed Scopus (587) Google Scholar). 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 (11Edwards C.M. Todd J.F. Mahmoudi M. Wang Z. Wang R.M. Ghatei M.A. Bloom S.R. Diabetes. 1999; 48: 86-93Crossref PubMed Scopus (304) Google Scholar, 12Schirra J. Sturm K. Leicht P. Arnold R. Goke B. Katschinski M. J. Clin. Invest. 1998; 101: 1421-1430Crossref PubMed Google Scholar, 13Kolligs F. Fehmann H.-C. Goke R. Goke B. Diabetes. 1995; 44: 16-19Crossref PubMed Google Scholar, 14Baggio L. Kieffer T.J. Drucker D.J. Endocrinology. 2000; 141: 3703-3709Crossref PubMed Google Scholar). Activation of GLP-1 receptor signaling leads to enhanced expression of mRNA transcripts for glucokinase, GLUT-2, Pdx-1, and insulin in β cell lines (15Drucker D.J. Philippe J. Mojsov S. Chick W.L. Habener J.F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3434-3438Crossref PubMed Scopus (683) Google Scholar, 16Wang Y.H. Egan J.M. Raygada M. Nadiv O. Roth J. Montrose-Rafizadeh C. Endocrinology. 1995; 136: 4910-4917Crossref PubMed Google Scholar, 17Wang X. Cahill C.M. Pineyro M.A. Zhou J. Doyle M.E. Egan J.M. Endocrinology. 1999; 140: 4904-4907Crossref PubMed Google Scholar) and in both normal and diabetic rodents (18Wang Y. Perfetti R. Greig N.H. Holloway H.W. DeOre K.A. Montrose-Rafizadeh C. Elahi D. Egan J.M. J. Clin. Invest. 1997; 99: 2883-2889Crossref PubMed Scopus (155) Google Scholar, 19Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar, 20Perfetti R. Zhou J. Doyle M.E. Egan J.M. Endocrinology. 2000; 141: 4600-4605Crossref PubMed Scopus (352) Google Scholar). Furthermore, GLP-1 and exendin-4 promote differentiation of exocrine cell lines toward a β cell phenotype (21Zhou J. Wang X. Pineyro M.A. Egan J.M. Diabetes. 1999; 48: 2358-2366Crossref PubMed Scopus (301) Google Scholar), a process that appears to depend on the expression of Pdx-1 (22Hui H. Wright C. Perfetti R. Diabetes. 2001; 50: 785-796Crossref PubMed Scopus (208) Google Scholar, 23de la Tour D. Halvorsen T. Demeterco C. Tyrberg B. Itkin-Ansari P. Loy M. Yoo S.J. Hao E. Bossie S. Levine F. Mol. Endocrinol. 2001; 15: 476-483PubMed Google Scholar). GLP-1 receptor signaling is also coupled to formation of new β cells through enhanced proliferation of existing β cells (24Edvell A. Lindstrom P. Endocrinology. 1999; 140: 778-783Crossref PubMed Scopus (132) Google Scholar) and via induction of islet neogenesis (25Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1074) Google Scholar). The mitogenic actions of GLP-1 are detectable in normal rodents (20Perfetti R. Zhou J. Doyle M.E. Egan J.M. Endocrinology. 2000; 141: 4600-4605Crossref PubMed Scopus (352) Google Scholar, 24Edvell A. Lindstrom P. Endocrinology. 1999; 140: 778-783Crossref PubMed Scopus (132) Google Scholar) and in the setting of experimental diabetes (19Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar, 25Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1074) Google Scholar). Administration of GLP-1 or exendin-4 to newborn rats treated with the β cell toxin streptozotocin (STZ) leads to increased β cell mass at postnatal day 7, which persists and remains increased at 2 months of age. The increased β cell mass in the GLP-1/exendin-4 treated rats was attributed to both enhanced β cell proliferation and increased numbers of small budding islets (26Tourrel C. Bailbe D. Meile M.-J. Kergoat M. Portha B. Diabetes. 2001; 50: 1562-1570Crossref PubMed Scopus (341) Google Scholar). Because STZ is known to induce β cell destruction in part through activation of apoptotic pathways (27Morgan N.G. Cable H.C. Newcombe N.R. Williams G.T. Biosci. Rep. 1994; 14: 243-250Crossref PubMed Scopus (54) Google Scholar, 28Kaneto H. Fujii J. Seo H.G. Suzuki K. Matsuoka T. Nakamura M. Tatsumi H. Yamasaki Y. Kamada T. Taniguchi N. Diabetes. 1995; 44: 733-738Crossref PubMed Scopus (372) Google Scholar, 29O'Brien B.A. Harmon B.V. Cameron D.P. Allan D.J. J. Pathol. 1996; 178: 176-181Crossref PubMed Scopus (168) Google Scholar), we examined whether GLP-1 receptor activation influences β cell mass via regulation of cellular susceptibility to apoptotic cell death. 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). Male C57BL/6 mice, 8 weeks of age, were used for experiments shown in Figs. Figure 1, Figure 2, Figure 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 (30Greig N.H. Holloway H.W., De Ore K.A. Jani D. Wang Y. Zhou J. Garant M.J. Egan J.M. Diabetologia. 1999; 42: 45-50Crossref PubMed Scopus (195) Google Scholar)) 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 (31Rossini A.A. Appel M.C. Williams R.M. Like A.A. Diabetes. 1977; 26: 916-920Crossref PubMed Scopus (133) Google Scholar). 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.Figure 2Ex-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 miceversus 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 (ΔG) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Ex-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 β-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 (31Rossini A.A. Appel M.C. Williams R.M. Like A.A. Diabetes. 1977; 26: 916-920Crossref PubMed Scopus (133) Google Scholar, 66). Approximately 20 islets per slide were assessed, with a minimum of 2 slides analyzed per mouse. cand 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oral glucose tolerance tests were carried out after an overnight fast as described (14Baggio L. Kieffer T.J. Drucker D.J. Endocrinology. 2000; 141: 3703-3709Crossref PubMed Google Scholar, 32Scrocchi L.A. Brown T.J. MacLusky N. Brubaker P.L. Auerbach A.B. Joyner A.L. Drucker D.J. Nature Med. 1996; 2: 1254-1258Crossref PubMed Scopus (653) Google Scholar). 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 (14Baggio L. Kieffer T.J. Drucker D.J. Endocrinology. 2000; 141: 3703-3709Crossref PubMed Google Scholar). 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 (33Boushey R.P. Yusta B. Drucker D.J. Cancer Res. 2001; 61: 687-693PubMed Google Scholar). 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 β 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 β cells. The relative cross-sectional β cell area, as a percentage of total pancreatic area, was assessed quantitatively as previously described (34Scrocchi L.A. Hill M.E. Saleh J. Perkins B. Drucker D.J. Diabetes. 2000; 49: 1552-1560Crossref PubMed Scopus (52) Google Scholar, 35Ling Z., Wu, D. Zambre Y. Flamez D. Drucker D.J. Pipeleers D.G. Schuit F.C. Virchows Arch. 2001; 438: 382-387Crossref PubMed Scopus (74) Google Scholar, 36Finegood D.T. McArthur M.D. Kojwang D. Thomas M.J. Topp B.G. Leonard T. Buckingham R.E. Diabetes. 2001; 50: 1021-1029Crossref PubMed Scopus (338) Google Scholar). The number of BrdUrd-positive cells are expressed both per islet and per 105 μm2 β-cell area. Islets were isolated from the pancreas of adult male Wistar rats (180–220 g) by collagenase digestion and purified on a gradient of Ficoll (37Rouiller D.G. Cirulli V. Halban P.A. Exp. Cell Res. 1990; 191: 305-312Crossref PubMed Scopus (87) Google Scholar). The islets were further dissociated into single cells by trypsinization, and β cells were sorted on the basis of their autofluorescence using a FACStar Plus (BD Biosciences) as described (37Rouiller D.G. Cirulli V. Halban P.A. Exp. Cell Res. 1990; 191: 305-312Crossref PubMed Scopus (87) Google Scholar). The sorted cell population comprises 95% β cells (37Rouiller D.G. Cirulli V. Halban P.A. Exp. Cell Res. 1990; 191: 305-312Crossref PubMed Scopus (87) Google Scholar). 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) (38Bosco D. Meda P. Halban P.A. Rouiller D.G. Diabetes. 2000; 49: 233-243Crossref PubMed Scopus (202) Google Scholar). For TUNEL labeling (see below) and labeling with BrdUrd, the sorted β 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 β 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. Apoptosis of purified β 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 (39Barsig J. Kaufmann S.H. Infect. Immun. 1997; 65: 4075-4081Crossref PubMed Google Scholar). 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. 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 (40Yusta B. Boushey R.P. Drucker D.J. J. Biol. Chem. 2000; 275: 35345-35352Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). 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.) 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 β-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; BIOSOURCEInternational), porin/VDAC 31HL (1:500 dilution; Calbiochem), Akt (1:1000; Cell Signaling Technology (Beverly, MA)) and actin (1:5,000 dilution; Sigma). 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. As treatment of rodents with GLP-1 agonists leads to increased islet mass in association with β cell proliferation and islet neogenesis (19Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar, 25Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1074) Google Scholar), we hypothesized that GLP-1 might also enhance β 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 β-cell apoptosis (29O'Brien B.A. Harmon B.V. Cameron D.P. Allan D.J. J. Pathol. 1996; 178: 176-181Crossref PubMed Scopus (168) Google Scholar), 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 (33Boushey R.P. Yusta B. Drucker D.J. Cancer Res. 2001; 61: 687-693PubMed Google Scholar, 41Boushey R.P. Yusta B. Drucker D.J. Am. J. Physiol. 1999; 277: E937-E947Crossref PubMed Google Scholar). 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 versusSTZ+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). 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. 2 a;p < 0.05 for day 15–52 glucose, STZ versusSTZ+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. 2 b; 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. 2 a). 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 β cells in separate groups of mice treated with STZ, with or without Ex-4. Only a rare apoptotic β cell was detectable in histological sections from pancreases of control or Ex-4-treated mice in the absence of STZ (Fig3 a). 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 β 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 β cell area (Fig. 3, b and d; p < 0.001, STZversus STZ+Ex-4). Because GLP-1 agonists have been shown to induce β-cell proliferation (19Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Egan J.M. Bonner-Weir S. Habener J.F. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (514) Google Scholar), we assessed the extent of β-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+ β cells, whether expressed as BrdUrd+ cells per islet, or normalized to β 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 micein vivo. To ascertain whether basal levels of endogenous GLP-1 receptor signaling protected β cells from external injury, we administered STZ to mice with a targeted disruption of theGlp-1R gene (GLP-1R−/− mice (32Scrocchi L.A. Brown T.J. MacLusky N. Brubaker P.L. Auerbach A.B. Joyner A.L. Drucker D.J. Nature Med. 1996; 2: 1254-1258Crossref PubMed Scopus (653) Google Scholar)). 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 β 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. Figure 1, Figure 2, Figure 3, Figure 4 demonstrate that activation or abrogation of GLP-1 receptor signaling regulates the extent of murine β cell apoptosis in vivo. To determine whether GLP-1 agonists exert direct anti-apoptotic effects on islet β cells in vitro using a different approach for generation of cytotoxic injury, we induced apoptosis in purified populations of s" @default.
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