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Glucagon-like Peptide (GLP)-2 Action in the Murine Central Nervous System Is Enhanced by Elimination of GLP-1 Receptor Signaling

2001; Elsevier BV; Volume: 276; Issue: 24 Linguagem: Inglês

10.1074/jbc.m009382200

ISSN

1083-351X

Autores

Julie A. Lovshin, Jennifer L. Estall, Bernardo Yusta, Theodore J. Brown, Daniel J. Drucker,

Tópico(s)

Diabetes Treatment and Management

Resumo

Glucagon-like peptide-2 (GLP-2) regulates energy homeostasis via effects on nutrient absorption and maintenance of gut mucosal epithelial integrity. The biological actions of GLP-2 in the central nervous system (CNS) remain poorly understood. We studied the sites of endogenous GLP-2 receptor (GLP-2R) expression, the localization of transgenic LacZ expression under the control of the mouse GLP-2R promoter, and the actions of GLP-2 in the murine CNS. GLP-2R expression was detected in multiple extrahypothalamic regions of the mouse and rat CNS, including cell groups in the cerebellum, medulla, amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, and pituitary. A 1.5-kilobase fragment of the mouse GLP-2R promoter directed LacZ expression to the gastrointestinal tract and CNS regions in the mouse that exhibited endogenous GLP-2R expression, including the cerebellum, amygdala, hippocampus, and dentate gyrus. Intracerebroventricular injection of GLP-2 significantly inhibited food intake during dark-phase feeding in wild-type mice. Disruption of glucagon-like peptide-1 receptor (GLP-1R) signaling with the antagonist exendin-(9–39) in wild-type mice or genetically in GLP-1R−/− mice significantly potentiated the anorectic actions of GLP-2. These findings illustrate that CNS GLP-2R expression is not restricted to hypothalamic nuclei and demonstrate that the anorectic effects of GLP-2 are transient and modulated by the presence or absence of GLP-1R signaling in vivo.AF338223AF338224 Glucagon-like peptide-2 (GLP-2) regulates energy homeostasis via effects on nutrient absorption and maintenance of gut mucosal epithelial integrity. The biological actions of GLP-2 in the central nervous system (CNS) remain poorly understood. We studied the sites of endogenous GLP-2 receptor (GLP-2R) expression, the localization of transgenic LacZ expression under the control of the mouse GLP-2R promoter, and the actions of GLP-2 in the murine CNS. GLP-2R expression was detected in multiple extrahypothalamic regions of the mouse and rat CNS, including cell groups in the cerebellum, medulla, amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, and pituitary. A 1.5-kilobase fragment of the mouse GLP-2R promoter directed LacZ expression to the gastrointestinal tract and CNS regions in the mouse that exhibited endogenous GLP-2R expression, including the cerebellum, amygdala, hippocampus, and dentate gyrus. Intracerebroventricular injection of GLP-2 significantly inhibited food intake during dark-phase feeding in wild-type mice. Disruption of glucagon-like peptide-1 receptor (GLP-1R) signaling with the antagonist exendin-(9–39) in wild-type mice or genetically in GLP-1R−/− mice significantly potentiated the anorectic actions of GLP-2. These findings illustrate that CNS GLP-2R expression is not restricted to hypothalamic nuclei and demonstrate that the anorectic effects of GLP-2 are transient and modulated by the presence or absence of GLP-1R signaling in vivo.AF338223AF338224 glucagon-like peptide-1 and -2, respectively human GLP-2 GLP-1 and GLP-2 receptors, respectively central nervous system reverse transcription-polymerase chain reaction rapid amplification of cDNA ends base pair(s) kilobase(s) phosphate-buffered saline 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside Baby hamster kidney The glucagon-like peptides are liberated in the gut and central nervous system via tissue-specific post-translational processing of a common proglucagon precursor (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). Glucagon-like peptide-1 (GLP-1) 1 and GLP-2 are secreted from the gut following nutrient ingestion and regulate nutrient absorption and energy homeostasis (2Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar, 3Drucker D.J. Endocrinology. 2001; 142: 521-527Crossref PubMed Scopus (310) Google Scholar). The actions of GLP-1 include regulation of gastric emptying, gastric acid secretion, inhibition of food intake and glucagon secretion, and stimulation of glucose-dependent insulin secretion and insulin biosynthesis (2Drucker D.J. Diabetes. 1998; 47: 159-169Crossref PubMed Google Scholar, 3Drucker D.J. Endocrinology. 2001; 142: 521-527Crossref PubMed Scopus (310) Google Scholar, 4Holst J.J. Trends Endocrinol. Metab. 1999; 10: 229-235Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 5Kieffer T.J. Habener J.F. Endocr. Rev. 1999; 20: 876-913Crossref PubMed Google Scholar). GLP-1 also promotes expansion of islet mass via stimulation of β-cell proliferation and induction of islet neogenesis via increased ductal Pdx-1 expression (6Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1091) Google Scholar, 7Stoffers 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 (516) Google Scholar). Taken together, these actions maintain euglycemia, hence enhancing GLP-1 action represents a potential strategy for the treatment of diabetes mellitus. GLP-2 exhibits trophic properties in the small and large bowel characterized by expansion of the mucosal epithelium via stimulation of crypt cell proliferation and inhibition of apoptosis (8Drucker D.J. Ehrlich P. Asa S.L. Brubaker P.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7911-7916Crossref PubMed Scopus (715) Google Scholar, 9Tsai C.-H. 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The intestinotrophic and cytoprotective properties of GLP-2 have been evaluated in the setting of acute intestinal injury, where GLP-2 administration inhibits apoptosis and reduces the severity of mucosal damage in both the small and large intestine (15Drucker D.J. Yusta B. Boushey R.P. Deforest L. Brubaker P.L. Am. J. Physiol. 1999; 276: G79-G91PubMed Google Scholar, 16Boushey R.P. Yusta B. Drucker D.J. Am. J. Physiol. 1999; 277: E937-E947Crossref PubMed Google Scholar, 17Alavi K. Schwartz M.Z. Palazzo J.P. Prasad R. J. Pediatr. Surg. 2000; 35: 847-851Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 18Prasad R. Alavi K. Schwartz M.Z. J. Pediatr. Surg. 2000; 35: 357-359Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the central nervous system (CNS), the glucagon-like peptides are synthesized predominantly in the caudal brainstem and, to a lesser extent, in the hypothalamus (19Drucker D.J. Asa S. J. Biol. 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Nature. 1996; 379: 69-72Crossref PubMed Scopus (1582) Google Scholar, 25Beak S.A. Heath M.M. Small C.J. Morgan D.G.A. Ghatei M.A. Taylor A.D. Buckingham J.C. Bloom S.R. Smith D.M. J. Clin. Invest. 1998; 101: 1334-1341Crossref PubMed Scopus (98) Google Scholar, 26Beak S.A. Small C.J. Ilovaiskaia I. Hurley J.D. Ghatei M.A. Bloom S.R. Smith D.M. Endocrinology. 1996; 137: 4130-4138Crossref PubMed Scopus (61) Google Scholar, 27Seeley R.J. Blake K. Rushing P.A. Benoit S. Eng J. Woods S.C. D'Alessio D. J. Neurosci. 2000; 20: 1616-1621Crossref PubMed Google Scholar, 28Seeley R.J. Woods S.C. D'Alessio D. Endocrinology. 2000; 141: 473-475Crossref PubMed Scopus (7) Google Scholar, 29Rinaman L. Am. J. Physiol. 1999; 277: R582-R590PubMed Google Scholar). Peripheral administration of GLP-1 or the lizard GLP-1 analog exendin-4 also reduces food intake and body weight (30Toft-Nielsen M.B. Madsbad S. Holst J.J. Diabetes Care. 1999; 22: 1137-1143Crossref PubMed Scopus (305) Google Scholar, 31Szayna M. Doyle M.E. Betkey J.A. Holloway H.W. Spencer R.G. Greig N.H. Egan J.M. Endocrinology. 2000; 141: 1936-1941Crossref PubMed Scopus (283) Google Scholar), suggesting that gut-derived GLP-1 provides signals that influence feeding behavior either directly to the brain or indirectly, likely via vagal afferents. In contrast to the increasing number of studies describing CNS actions of GLP-1, much less is known about the potential function(s) of GLP-2 in the brain. Experiments using rat hypothalamic and pituitary membranes demonstrated GLP-2-mediated activation of adenylate cyclase (32Hoosein N.M. Gurd R.S. FEBS Lett. 1984; 178: 83-86Crossref PubMed Scopus (85) Google Scholar). Consistent with these findings, the actions of GLP-2 were subsequently shown to be transduced in a cAMP-dependent manner via a recently cloned GLP-2 receptor (GLP-2R) isolated from hypothalamic and intestinal cDNA libraries (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar). The GLP-2R is expressed in a highly tissue-specific manner predominantly in gut endocrine cells and in the brain (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar, 34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In comparison with GLP-1, little is known about either the expression or function of the GLP-2R in different regions of the CNS. Both GLP-1- and GLP-2-immunoreactive fiber tracts project from the brainstem to multiple CNS regions, including the hypothalamus, thalamus, cortex, and pituitary (21Larsen P.J. Tang-Christensen M. Holst J.J. Orskov C. Neuroscience. 1997; 77: 257-270Crossref PubMed Scopus (494) Google Scholar, 35Tang-Christensen M. Larsen P.J. Thulesen J. Romer J. Vrang N. Nat. Med. 2000; 6: 802-807Crossref PubMed Scopus (229) Google Scholar). Intracerebroventricular infusion of GLP-2 in rats inhibits food intake (35Tang-Christensen M. Larsen P.J. Thulesen J. Romer J. Vrang N. Nat. Med. 2000; 6: 802-807Crossref PubMed Scopus (229) Google Scholar), similar to results obtained following intracerebroventricular infusion of GLP-1 (24Turton M.D. O'Shea D. Gunn I. Beak S.A. Edwards C.M.B. Meeran K. Choi S.J. Taylor G.M. Heath M.M. Lambert P.D. Wilding J.P.H. Smith D.M. Ghatei M.A. Herbert J. Bloom S.R. Nature. 1996; 379: 69-72Crossref PubMed Scopus (1582) Google Scholar,36Tang-Christensen M. Larsen P.J. Goke R. Fink-Jensen A. Jessop D.S. Moller M. Sheikh S.P. Am. J. Physiol. 1996; 271: R848-R856PubMed Google Scholar). Unexpectedly, the anorectic effects of GLP-2 in rats are completely inhibited by the GLP-1R antagonist exendin-(9–39) (35Tang-Christensen M. Larsen P.J. Thulesen J. Romer J. Vrang N. Nat. Med. 2000; 6: 802-807Crossref PubMed Scopus (229) Google Scholar). These findings imply that CNS GLP-2 may exert its effects via the GLP-1R to inhibit food intake, or alternatively, exendin-(9–39) may also function as a CNS GLP-2R antagonist. Furthermore, although expression of the rat GLP-2R was reported to be restricted to the dorsomedial nucleus of the hypothalamus by in situhybridization (35Tang-Christensen M. Larsen P.J. Thulesen J. Romer J. Vrang N. Nat. Med. 2000; 6: 802-807Crossref PubMed Scopus (229) Google Scholar), other studies have reported a more widespread distribution of GLP-2R mRNA transcripts in various regions of the rat CNS (37White, R. B., Broqua, P., Meyer, J., Junien, J.-L., and Aubert, M. L. (2000) 82nd Annual Meeting of the Endocrine Society, June 21–24, Toronto, Ontario, Canada, p. 271, Abstr. 1115, The Endocrine Society Press, Bethesda, MDGoogle Scholar). To understand the biological function and mechanisms regulating control of GLP-2R expression in the brain, we have now studied GLP-2R expression and GLP-2 action in the rodent CNS using a combination of immunohistochemical, reverse transcription-polymerase chain reaction (RT-PCR), transgenic, and cell-based analyses. All animal experiments were approved and carried out strictly in accordance with the Canadian Council on Animal Care guidelines and the Animal Care Committee at the Toronto General Hospital, University Health Network (Toronto, Ontario, Canada). Animals were allowed to acclimatize to the animal care facilities for at least 1 week prior to any experimental procedure. A genomic clone containing the 5′-flanking, 5′-untranslated, and coding regions of the murine GLP-2R gene was isolated from a 129SVJ mouse genomic library. To identify additional GLP-2R nucleotide sequences 5′ to the translation start site (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar), the 5′-end of the rat GLP-2R cDNA was generated and characterized using adaptor-modified complementary DNA from rat brain (CLONTECH, Palo Alto, CA) in 5′-rapid amplification of cDNA ends (RACE) experiments. Separately, a 1516-base pair (bp) fragment of the mouse GLP-2R gene was subcloned from the mouse genomic library (Incyte Genomics, St. Louis, MO), sequenced, and ligated immediately 5′ to a cDNA encoding LacZ with a nuclear localization signal (a gift from A. Nagy). The GLP-2R promoter-lacZ transgene was gel-purified and used for generation of transgenic mice. In total, eight founder animals were identified by Southern blotting and PCR analysis and mated with non-transgenic mice to determine germ-line transmission of the transgene. Three transgenic founder mice (designated lines 2–4) exhibited germ-line transmission and were used to generate lines for further analysis of transgene expression. Male Harlan Sprague-Dawley rats (300–500 g) or GLP-2R promoter-lacZ transgenic mice were killed by CO2 inhalation and quickly decapitated. The brains were rapidly removed and placed ventral side up on a chilled glass plate. The pituitary glands were also removed and frozen in liquid nitrogen. The amygdala, cerebral cortex, cerebellum, pons/midbrain, and medulla were dissected and frozen in liquid nitrogen. The amygdala was dissected by first producing a 3-mm thick coronal section by making a coronal cut at the optic chiasm and at the posterior edge of the mamillary bodies. A cut connecting the rhinal fissures formed the dorsal boundary of the amygdaloid block, and cuts made continuous with the lateral ventricles to the lateral hypothalamic sulci formed the medial boundaries of the amygdaloid blocks. The cerebral cortex was also taken from this coronal section and consisted primarily of parietal and frontal cortices. The cerebellum was removed, and a coronal cut was made at the posterior edge of the pons. The neural tissue posterior to this cut comprised the medulla, which also contained the anterior-most portion of the spinal cord. The midbrain block, which also included the pons, extended from the posterior edge of the mamillary bodies to the posterior edge of the pons, with the cerebellar and cerebral cortices, hippocampus, and amygdala removed. Total RNA was isolated from CNS tissues using TrizolTM reagent (Life Technologies, Inc., Toronto) and from peripheral tissues using a modified guanidinium thiocyanate procedure (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar) and dissolved in ribonuclease-free water. RNA integrity was assessed on a 1% (w/v) agarose gel containing formaldehyde and visualized on a UV transilluminator (Fisher, Montreal, Quebec, Canada) using ethidium bromide staining. For RT-PCR experiments, RNA samples were treated with DNase I (Life Technologies, Inc.), primed with random hexamers (Life Technologies, Inc.), and reverse-transcribed with SuperscriptTM II reverse transcriptase (Life Technologies, Inc.). To control for contamination, reactions were also carried out in the absence of SuperscriptTM. Following first-strand cDNA synthesis, samples were treated with ribonuclease H (MBI Fermentas, Vilnius, Lithuania) to remove RNA. For subsequent PCR amplification, first-strand cDNA was used as template. Oligonucleotide primer pairs, annealing temperature, and cycle number for PCR amplification were as follows. For the rat GLP-2R, 5′-TTGTGAACGGGCGCCAGGAGA-3′ and 5′-GATCTCACTCTCTTCCAGAATCTC-3′ were annealed at 65 °C for 40 cycles; for the mouse GLP-2R, 5′-CTGCTGGTTTCCATCAAGCAA-3′ and 5′-TAGATCTCACTCTCTTCCAGA-3′ were annealed at 65 °C for 30 cycles; for rat glyceraldehyde-3-phosphate dehydrogenase, 5′-TCCACCACCCTGTTGCTGTAG-3′ and 5′-GACCACAGTCCATGACATCACT-3′ were annealed at 60 °C for 30 cycles; and for the GLP-2R-lacZ transgene, 5′-CGCTGATTTGTGTAGTCGGTT-3′ and 5′-CTTATTCGCCTTGCAGCACAT-3′ were annealed at 63 °C for 40 cycles. The expected PCR product for the mouse and rat GLP-2R cDNAs is ∼1.6 kilobases (kb), corresponding to the full-length GLP-2R (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar, 34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). The predictedlacZ PCR product is ∼580 bp; and for rat glyceraldehyde-3-phosphate dehydrogenase, the expected PCR product is ∼450 bp. To control for nonspecific amplification, PCR reactions were also carried out in the absence of first-strand cDNA. Following amplification, PCR products were separated by gel electrophoresis; transferred onto a nylon membrane (GeneScreen, Life Technologies, Inc.); and hybridized with 1) a 32P-labeled internal cDNA probe for the rat GLP-2R (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar, 34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), 2) a32P-labeled internal lacZ oligonucleotide (5′-TCAGGAAGATCGCACTCCAGC-3′), or 3) a 32P-labeled internal cDNA probe for rat glyceraldehyde-3-phosphate dehydrogenase (39Piechaczyk M. Blanchard J.M. Marty L. Dani C. Panabieres F. El Sabouty S. Fort P. Jeanteur P. Nucleic Acids Res. 1984; 12: 6951-6963Crossref PubMed Scopus (406) Google Scholar). Following hybridization, membranes were washed stringently, and hybridization signals were quantified on a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) using ImageQuantTM software (Version 5.0, Molecular Dynamics, Inc.). Rats or mice were deeply anesthetized following intraperitoneal injections of sodium pentobarbital. Following transcardial perfusion with 0.9% sodium chloride, animals were perfused with 4% neutral buffered Formalin for ∼15 min. Brains were removed and post-fixed at room temperature for 4 h or overnight at 4 °C. Brains were then cryopreserved overnight in a 20% sucrose in phosphate-buffered saline (PBS) gradient at 4 °C, frozen slowly in dry ice vapor, and either stored at −80 °C or sectioned immediately at 10 μm on a cryostat. All sections were collected and thaw-mounted onto Superfrost Plus slides (Fisher). For detection of β-galactosidase-immunopositive cells, slides were incubated for 4 h at 37 °C in a 1:8000 dilution of polyclonal anti-β-galactosidase antiserum (ICN Pharmaceuticals Inc., Costa Mesa, CA). Polyclonal anti-GLP-2R antiserum (1:800) that recognizes the GLP-2R, but not the glucagon, glucose-dependent inhibitory polypeptide, and GLP-1 receptors (34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), was a gift from NPS Allelix Corp. (Mississauga, Canada). To control for nonspecific immunopositivity, anti-GLP-2R antiserum was also preabsorbed overnight at 4 °C in the presence of recombinant GLP-2R immunogen (34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), and immunocytochemistry studies were carried out with 1) anti-GLP-2R antiserum, 2) preimmune serum, 3) antibody diluent alone, or 4) preabsorbed anti-GLP-2R antiserum. All slides were counterstained in hematoxylin. Brains were isolated from mice and placed in 2% paraformaldehyde and 0.2% glutaraldehyde in PBS fixative for 1 h at room temperature. Sixty minutes later, brains were rinsed in PBS and transferred to 4 °C in 15% sucrose in PBS solution for 4 h to overnight and subsequently to 30% sucrose in PBS solution for 4 h to overnight for cryopreservation. Brains were then frozen in dry ice vapor and stored at −80 °C. Tissues were sectioned at 10 μm in a −25–30 °C cryostat and subsequently thaw-mounted and stored at −80 °C. Prior to staining with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal; Bioshop Canada, Burlington, Ontario), slides were slowly warmed to room temperature and rinsed in PBS. Slides were treated with X-gal solution overnight at 37 °C. Following treatment with X-gal solution, slides were rinsed in PBS, counterstained with eosin, and dehydrated in an ethanol series. All slides were visualized and captured using a JVC video camera with a ½-inch chip device adapted (0.63 × c-mount) to a light microscope (Leica Ltd., Cambridge, United Kingdom). Magnification is reported as the objective magnification multiplied by the c-mount magnification multiplied by the electronic magnification (electronic magnification was corrected for by dividing the diagonal of the image captured by the camera chip size). Recombinant h[Gly2]GLP-2, a dipeptidyl peptidase IV-resistant GLP-2 analog (40Drucker D.J. Shi Q. Crivici A. Sumner-Smith M. Tavares W. Hill M. Deforest L. Cooper S. Brubaker P.L. Nat. Biotechnol. 1997; 15: 673-677Crossref PubMed Scopus (224) Google Scholar, 41DaCambra M.P. Yusta B. Sumner-Smith M. Crivici A. Drucker D.J. Brubaker P.L. Biochemistry. 2000; 39: 8888-8894Crossref PubMed Scopus (55) Google Scholar), was a gift from NPS Allelix Corp. Human GLP-1-(7–36)-NH2, exendin-4, and exendin-(9–39) were purchased from California Peptide Research Inc. (Napa, CA). Forskolin and 3-isobutyl-1-methylxanthine were obtained from Sigma. BHK fibroblast cells stably transfected with either the rat GLP-1R or GLP-2R were propagated as previously described (42Yusta B. Somwar R. Wang F. Munroe D. Grinstein S. Klip A. Drucker D.J. J. Biol. Chem. 1999; 274: 30459-30467Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and the levels of intracellular cAMP were assayed following exposure to individual peptides in Dulbecco's modified Eagle's medium containing 100 μm 3-isobutyl-1-methylxanthine as reported (41DaCambra M.P. Yusta B. Sumner-Smith M. Crivici A. Drucker D.J. Brubaker P.L. Biochemistry. 2000; 39: 8888-8894Crossref PubMed Scopus (55) Google Scholar, 42Yusta B. Somwar R. Wang F. Munroe D. Grinstein S. Klip A. Drucker D.J. J. Biol. Chem. 1999; 274: 30459-30467Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Cells were incubated for 5 min with exendin-(9–39) or medium alone before addition of an agonist (GLP-1, h[Gly2]GLP-2, or exendin-4). The treated cells were then incubated at 37 °C for 10 min. Absolute ethanol (−20 °C) was added to terminate the reaction, and the plates were stored at −80 °C until the cell extracts were collected (2–4 h later). Forskolin was used as a positive control. cAMP radioimmunoassays (Biomedical Technologies, Inc., Stoughton, MA) were performed on dried aliquots of extract, and data were normalized to cAMP/well. All treatments were performed in triplicate or quadruplicate, and the data are expressed as means ± SD. EC50 values were calculated using Prism Version 3.00 (GraphPAD Software Inc., San Diego, CA). For intracerebroventricular injections, adult male CD1 mice randomized into multiple experimental groups were anesthetized by inhalation of methoxyflurane (Metophane, Janssen, Toronto) (43Scrocchi L.A. Brown T.J. MacLusky N. Brubaker P.L. Auerbach A.B. Joyner A.L. Drucker D.J. Nat. Med. 1996; 2: 1254-1258Crossref PubMed Scopus (663) Google Scholar). Following intracerebroventricular injections of equal volumes of saline or peptide dissolved in saline, animals were allowed to recover for ∼15 min until the observation of a righting response. Mice were then weighed and given a pre-measured quantity of rodent chow, and food intake was quantified at 1, 2, 4, and 22 h. The accuracy of intracerebroventricular injection was verified at autopsy analysis by detection of bromphenol dye in the lateral ventricles of selected animals. Animals were injected with peptide either at 7 p.m. (for dark-phase feeding studies) or at 10 a.m. following an overnight fast of 15 h (for fasting studies). The observation that GLP-2R RNA transcripts were restricted to the dorsomedial nucleus of the rat hypothalamus as demonstrated by in situ hybridization (35Tang-Christensen M. Larsen P.J. Thulesen J. Romer J. Vrang N. Nat. Med. 2000; 6: 802-807Crossref PubMed Scopus (229) Google Scholar) differed from recent reports of more widespread expression of the GLP-2R in multiple regions of the CNS (37White, R. B., Broqua, P., Meyer, J., Junien, J.-L., and Aubert, M. L. (2000) 82nd Annual Meeting of the Endocrine Society, June 21–24, Toronto, Ontario, Canada, p. 271, Abstr. 1115, The Endocrine Society Press, Bethesda, MDGoogle Scholar). We detected GLP-2R mRNA transcripts not only in the rat hypothalamus, but also in the brainstem by RT-PCR (34Yusta B. Huang L. Munroe D. Wolff G. Fantaske R. Sharma S. Demchyshyn L. Asa S.L. Drucker D.J. Gastroenterology. 2000; 119: 744-755Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Accordingly, we reexamined the localization of rodent CNS GLP-2R expression using a combination of RT-PCR and immunohistochemistry analyses. Furthermore, we compared the localization of endogenous GLP-2R mRNA transcripts and GLP-2R immunopositivity with the regions of LacZ expression in tissues isolated from GLP-2R promoter-lacZ transgenic mice. To identify DNA regulatory sequences important for control of CNS GLP-2R expression, we focused initially on characterization of the 5′-end of the GLP-2R mRNA transcript. As GLP-2R cDNA sequences upstream of the translation start site had not been previously reported (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar), we carried out 5′-RACE experiments using cDNA template from rat brain to identify 5′-untranslated sequences of the rat GLP-2R. Multiple RACE reaction products were consistently obtained that were ∼500 bp in size. These products were cloned, and sequence analysis demonstrated the presence of previously identified rat GLP-2R cDNA sequences (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Scholar) and an additional 104 nucleotides of rat GLP-2R 5′-untranslated sequences upstream of the previously reported ATG codon (Fig. 1 a). Using a 213-bp ApaI/SmaI rat cDNA fragment containing 5′-coding sequences as a probe, we isolated an ∼2-kb subclone from a bacterial artificial chromosome clone derived from a mouse genomic library. The DNA sequences of the mouse GLP-2R genomic subclone were aligned with the known rat GLP-2R cDNA sequence (33Munroe D.G. Gupta A.K. Kooshesh P. Rizkalla G. Wang H. Demchyshyn L. Yang Z.-J. Kamboj R.K. Chen H. McCallum K. Sumner-Smith M. Drucker D.J. Crivici A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1569-1573Crossref PubMed Scopus (281) Google Sch

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