THE J BIOLOGICAL C © 2003 by The American …

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 1, Issue of January 3, pp. 471 478, 2003. 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in Glucagon-like Peptide-1 Receptor signaling modulates Cell Apoptosis*. Received for publication, September 13, 2002, and in revised form, October 22, 2002. Published, JBC Papers in Press, October 29, 2002, DOI Yazhou Li , Tanya Hansotia , Bernardo Yusta , Frederic Ris**, Philippe A. Halban**, and Daniel J. Drucker . From the 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 Glucagon-like peptide-1 (GLP-1) stimulates insulin and is secreted from the distal gut after nutrient ingestion (2).

2 Secretion and augments cell mass via activation of The termination of GLP-1 action by the enzyme dipeptidyl cell proliferation and islet neogenesis. We examined peptidase IV occurs within minutes following GLP-1 secretion whether GLP-1 receptor signaling modifies the cellular (3 5), yet GLP-1 exerts several rapid metabolic actions includ- susceptibility to apoptosis. Mice administered strepto- ing stimulation and inhibition of insulin and glucagon secre- zotocin (STZ), an agent known to induce cell apoptosis, tion, respectively (6 10). GLP-1 action is essential for glucose exhibit sustained improvement in glycemic control and homeostasis, because GLP-1 receptor blockade with the antag- increased levels of plasma insulin with concomitant ad- onist exendin (9 39) increases blood glucose and decreases ministration of the GLP-1 agonist exendin-4 (Ex-4).

3 Levels of circulating insulin in human and rodent studies Blood glucose remained significantly lower for weeks (11 14). after cessation of exendin-4. STZ induced cell apopto- Activation of GLP-1 receptor signaling leads to enhanced sis, which was significantly reduced by co-administra- expression of mRNA transcripts for glucokinase, GLUT-2, tion of Ex-4. Conversely, mice with a targeted disruption Pdx-1, and insulin in cell lines (15 17) and in both normal of the GLP-1 receptor gene exhibited increased cell apoptosis after STZ administration. Exendin-4 directly and diabetic rodents (18 20). Furthermore, GLP-1 and ex- reduced cytokine-induced apoptosis in purified rat endin-4 promote differentiation of exocrine cell lines toward a.

4 cells exposed to interleukin 1 , tumor necrosis fator , cell phenotype (21), a process that appears to depend on the and interferon in vitro. Furthermore, Ex-4-treated expression of Pdx-1 (22, 23). BHK-GLP-1R cells exhibited significantly increased cell GLP-1 receptor signaling is also coupled to formation of new viability, reduced caspase activity, and decreased cleav- cells through enhanced proliferation of existing cells (24). age of -catenin after treatment with cycloheximide in and via induction of islet neogenesis (25). The mitogenic actions vitro. These findings demonstrate that GLP-1 receptor of GLP-1 are detectable in normal rodents (20, 24) and in the signaling directly modifies the susceptibility to apo- setting of experimental diabetes (19, 25).

5 Administration of ptotic injury, and provides a new potential mechanism GLP-1 or exendin-4 to newborn rats treated with the cell linking GLP-1 receptor activation to preservation or en- toxin streptozotocin (STZ) leads to increased cell mass at hancement of cell mass in vivo. postnatal day 7, which persists and remains increased at 2. months of age. The increased cell mass in the GLP-1/ex- endin-4 treated rats was attributed to both enhanced cell Glucagon-like peptide-1 (GLP-1)1 is derived from posttrans- proliferation and increased numbers of small budding islets lational processing of proglucagon in enteroendocrine L cells (1) (26). Because STZ is known to induce cell destruction in part through activation of apoptotic pathways (27 29), we examined whether GLP-1 receptor activation influences cell mass via * This work was partially supported by grants from the Juvenile Diabetes Research Foundation (JDRF 2000-559 to D.)

6 J. D. and JDRF regulation of cellular susceptibility to apoptotic cell death. 4-1999-844 to P. A. H.), the Canadian Diabetes Association and Ontario MATERIALS AND METHODS. Research and Development Challenge Fund (to D. J. D.), and by the Swiss National Science Foundation (Grant to P. A. H.). Materials Tissue culture medium, serum, flasks, plates, and anti- The costs of publication of this article were defrayed in part by the biotics, including G418, were from Invitrogen. Cycloheximide, forskolin, payment of page charges. This article must therefore be hereby marked and protease inhibitor mixture were purchased from Sigma. Exendin-4. advertisement in accordance with 18 Section 1734 solely to was from California Peptide Research (Napa, CA).

7 Indicate this fact. Animal Experiments Male C57BL/6 mice, 8 weeks of age, were used These authors contributed equally to this work. for experiments shown in Figs. 1 3. Age- and sex-matched CD-1 GLP- Supported by a postdoctoral fellowship Award from the Canadian 1R / control mice housed in the same animal facility were used for Diabetes Association. studies of GLP-1R / mice in the CD1 background (8-week-old male Supported by a Novo Nordisk-Banting and Best Diabetes Centre mice). All animals were maintained on standard laboratory chow under studentship. a 12 h:12 h light-dark schedule, and experiments were conducted ac- Supported by a Senior Scientist Award (Canadian Institutes of cording to protocols and guidelines approved by the Toronto General Health Research).

8 To whom correspondence should be addressed: Toronto General Hospital, 200 Elizabeth St. MBRW4R-402, Toronto, Hospital Animal Care Committee. STZ (Sigma) (50 mg/kg body weight, Ontario M5G 2C4, Canada. Tel.:416-340-4125; Fax: 416-978-4108; intraperitoneal injection once daily for 5 days) was administered as a E-mail: freshly prepared solution in mM sodium citrate pH Exendin-4. 1. The abbreviations used are: GLP-1, glucagon-like peptide-1; STZ, (Ex-4; 24 nmol/kg body weight, a dose selected based on therapeutic streptozotocin; Ex-4, exendin-4; TUNEL, terminal deoxynucleotide efficacy in previous mouse experiments (30)) was administered as a transferase-mediated dUTP nick end labeling; BrdUrd, 5 -bromo-2 - single daily intraperitoneal injection.

9 For studies depicted in Figs. 1 and deoxyuridine; ELISA, enzyme-linked immunosorbent assay; BHK, baby 2, morning blood glucose was measured periodically throughout the hamster kidney; CHX, cycloheximide. experimental period and an oral glucose tolerance tests was done at day This paper is available on line at 471. 472 GLP-1 and Cell Apoptosis 30. For histological studies of islet apoptosis, Ex-4 administration was software. The vast majority of BrdUrd cells were immunopositive for commenced either 2 or 7 days before STZ in separate experiments and insulin, and hence represented cells . The relative cross-sectional cell continued until the last injection of STZ; C57BL/6 mice were sacrificed area, as a percentage of total pancreatic area, was assessed quantita- within 24 h after the last STZ injection.

10 For studies of apoptosis in tively as previously described (34 36). The number of BrdUrd-positive GLP-1R / mice, wild-type GLP-1R / and GLP-1R / mice were cells are expressed both per islet and per 105 m2 -cell area. divided into separate groups (n 4 6) and administered a slightly Rat Islets and Sorted cells Islets were isolated from the pancreas lower dose of STZ (40 mg/kg body weight) because of the different of adult male Wistar rats (180 220 g) by collagenase digestion and sensitivities of CD-1 versus C57BL/6 mice to STZ as delineated in purified on a gradient of Ficoll (37). The islets were further dissociated preliminary dose-response studies caused by the known species-specific into single cells by trypsinization, and cells were sorted on the basis sensitivity to streptozotocin-induced apoptosis (31).