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BACKGROUND: The resistance to epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKIs) therapy is currently the major clinical challenge in the treatment of lung cancer. This study aims to reveal the role of glucagon-like peptide (GLP) 2 and GLP-2 receptor (GLP2R) signaling on the EGFR-TKIs and cisplatin resistance of lung cancer cells. METHODS: The common differentially expressed genes in PC9 and HCC827 cells that were individually resistant to one of the three EGFR-TKIs (dacomitinib, osimertinib and afatinib) were screened. The data were from GSE168043 and GSE163913. The expression of GLP2R in drug-resistant cells was detected by western blot. The effect of GLP2R expression down- or up-regulation on resistance to dacomitinib, osimertinib, afatinib or cisplatin was measured by CCK-8 and flow cytometry assays. The long-acting analog of GLP-2, teduglutide, treated the parental cells. RESULTS: A total of 143 common differentially expressed genes were identified. Compared with the parent cells, the GLP2R expression in drug-resistant cell lines was significantly up-regulated. The exogenous expression of GLP2R in parental cells enhanced cell viability, while knockdown of GLP2R levels in drug-resistant cell lines inhibited cell viability. In addition, teduglutide treatment also enhanced the viability of lung cancer cells. CONCLUSION: GLP2-GLP2R signal may change the sensitivity of cells to EGFR-TKIs and cisplatin. The development of GLP-2 or GLP2R inhibitors may be beneficial to the clinical treatment of lung cancer.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Receptores ErbB/efectos de los fármacos , Receptores de Péptidos Similares al Glucagón/efectos de los fármacos , Péptidos Similares al Glucagón/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Línea Celular Tumoral , Receptores ErbB/genética , Receptores de Péptidos Similares al Glucagón/genética , Humanos , Neoplasias Pulmonares/genética , Inhibidores de Proteínas QuinasasRESUMEN
BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is secreted postprandially by enteroendocrine L-cells and stimulates growth of the gut and bone. One GLP-2 analogue is approved for short bowel syndrome (SBS). To improve therapeutic efficacy, we developed biased GLP-2 receptor (GLP-2R) agonists through N-terminal modifications. EXPERIMENTAL APPROACH: Variants with Ala and Trp substitutions of the first seven positions of GLP-2(1-33) were studied in vitro for affinity, G protein activation (cAMP accumulation), recruitment of ß-arrestin 1 and 2, and internalization of the human and mouse GLP-2R. The intestinotrophic actions of the most efficacious (cAMP) biased variant were examined in mice. KEY RESULTS: Ala substitutions had more profound effects than Trp substitutions. For both, alterations at positions 1, 3 and 6 most severely impaired activity. ß-arrestin recruitment was more affected than cAMP accumulation. Among Ala substitutions, [H1A], [D3A] and [F6A] impaired potency (EC50 ) for cAMP-accumulation >20-fold and efficacy (Emax ) to 48%-87%, and were unable to recruit arrestins. The Trp substitutions, [A2W], [D3W] and [G4W] were partial agonists (Emax of 46%-59%) with 1.7-12-fold decreased potencies in cAMP and diminished ß-arrestin recruitment. The biased variants, [F6A], [F6W] and [S7W] induced less GLP-2R internalization compared with GLP-2, which induced internalization in a partly arrestin-independent manner. In mice, [S7W] enhanced gut trophic actions with increased weight of the small intestine, increased villus height and crypt depth compared with GLP-2. CONCLUSION AND IMPLICATIONS: G protein-biased GLP-2R agonists with diminished receptor desensitization have superior intestinotrophic effects and may represent improved treatment of intestinal insufficiency including SBS.
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Arrestina , Péptido 2 Similar al Glucagón , Ratones , Humanos , Animales , Péptido 2 Similar al Glucagón/farmacología , Arrestina/metabolismo , Proteínas de Unión al GTP/metabolismo , beta-Arrestinas/metabolismo , Arrestinas , beta-Arrestina 1/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is secreted postprandially from enteroendocrine Lcells and has anabolic action on gut and bone. Short-acting teduglutide is the only approved GLP-2 analog for the treatment of short-bowel syndrome (SBS). To improve the therapeutic effect, we created a series of lipidated GLP-2R agonists. EXPERIMENTAL APPROACH: Six GLP-2 analogs were studied in vitro for cAMP accumulation, ß-arrestin 1 and 2 recruitment, affinity, and internalization. The trophic actions on intestine and bone were examined in vivo in rodents. KEY RESULTS: Lipidations at lysines introduced at position 12, 16, and 20 of hGLP-2(1-33) were well-tolerated with less than 2.2-fold impaired potency and full efficacy at the hGLP-2R in cAMP accumulation. In contrast, N- and C-terminal (His1 and Lys30) lipidations impaired potency by 4.2- and 45-fold and lowered efficacy to 77% and 85% of hGLP-2, respectively. All variants were similarly active on the rat and mouse GLP-2Rs and the three most active variants displayed increased selectivity for hGLP-2R over hGLP-1R activation, compared to native hGLP-2. Impact on arrestin recruitment and receptor internalization followed that of Gαs-coupling, except for lipidation in position 20, where internalization was more impaired, suggesting desensitization protection. A highly active variant (C16 at position 20) with low internalization and a half-life of 9.5 h in rats showed improved gut and bone tropism with increased weight of small intestine in mice and decreased CTX levels in rats. CONCLUSION AND IMPLICATION: We present novel hGLP-2 agonists suitable for in vivo studies of the GLP-2 system to uncover its pharmacological potential.
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Péptido 2 Similar al Glucagón , Roedores , Humanos , Ratas , Ratones , Animales , Péptido 2 Similar al Glucagón/farmacología , Receptor del Péptido 2 Similar al GlucagónRESUMEN
Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα q activation and ß-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected ß-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.
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Glucagon-like peptide-1 (GLP-1) shows robust protective effects on ß-cell survival and function and GLP-1 based therapies are successfully applied for type-2 diabetes (T2D) and obesity. Another cleavage product of pro-glucagon, Glucagon-like peptide-2 (GLP-2; both GLP-1 and GLP-2 are inactivated by DPP-4) has received little attention in its action inside pancreatic islets. In this study, we investigated GLP-2 production, GLP-2 receptor (GLP-2R) expression and the effect of GLP-2R activation in human islets. Isolated human islets from non-diabetic donors were exposed to diabetogenic conditions: high glucose, palmitate, cytokine mix (IL-1ß/IFN-γ) or Lipopolysaccharide (LPS) in the presence or absence of the DPP4-inhibitor linagliptin, the TLR4 inhibitor TAK-242, the GLP-2R agonist teduglutide and/or its antagonist GLP-2(3-33). Human islets under control conditions secreted active GLP-2 (full-length, non-cleaved by DPP4) into the culture media, which was increased by combined high glucose/palmitate, the cytokine mix and LPS and highly potentiated by linagliptin. Low but reproducible GLP-2R mRNA expression was found in all analyzed human islet isolations from 10 donors, which was reduced by pro-inflammatory stimuli: the cytokine mix and LPS. GLP-2R activation by teduglutide neither affected acute or glucose stimulated insulin secretion nor insulin content. Also, teduglutide had no effect on high glucose/palmitate- or LPS-induced dysfunction in cultured human islets but dampened LPS-induced macrophage-dependent IL1B and IL10 expression, while its antagonist GLP-2(3-33) abolished such reduction. In contrast, the expression of islet macrophage-independent cytokines IL6, IL8 and TNF was not affected by teduglutide. Medium conditioned by teduglutide-exposed human islets attenuated M1-like polarization of human monocyte-derived macrophages, evidenced by a lower mRNA expression of pro-inflammatory cytokines, compared to vehicle treated islets, and a reduced production of itaconate and succinate, marker metabolites of pro-inflammatory macrophages. Our results reveal intra-islet production of GLP-2 and GLP-2R expression in human islets. Despite no impact on ß-cell function, local GLP-2R activation reduced islet inflammation which might be mediated by a crosstalk between endocrine cells and macrophages.
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Péptido 2 Similar al Glucagón/metabolismo , Inflamación , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/metabolismo , Macrófagos/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Femenino , Receptor del Péptido 2 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/fisiología , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Islotes Pancreáticos/efectos de los fármacos , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Masculino , Pancreatitis/inmunología , Pancreatitis/metabolismo , Pancreatitis/patologíaRESUMEN
The mammalian proglucagon gene (Gcg) encodes three glucagon like sequences, glucagon, glucagon-like peptide-1 (GLP-1), and glucagon-like peptide-2 that are of similar length and share sequence similarity, with these hormones having cell surface receptors, glucagon receptor (Gcgr), GLP-1 receptor (Glp1r), and GLP-2 receptor (Glp2r), respectively. Gcgr, Glp1r, and Glp2r are all class B1 G protein-coupled receptors (GPCRs). Despite their sequence and structural similarity, analyses of sequences from rodents have found differences in patterns of sequence conservation and evolution. To determine whether these were rodent-specific traits or general features of these genes in mammals I analyzed coding and protein sequences for proglucagon and the receptors for proglucagon-derived peptides from the genomes of 168 mammalian species. Single copy genes for each gene were found in almost all genomes. In addition to glucagon sequences within Hystricognath rodents (e.g., guinea pig), glucagon sequences from a few other groups (e.g., pangolins and some bats) as well as changes in the proteolytic processing of GLP-1 in some bats are suggested to have functional effects. GLP-2 sequences display increased variability but accepted few substitutions that are predicted to have functional consequences. In parallel, Glp2r sequences display the most rapid protein sequence evolution, and show greater variability in amino acids at sites involved in ligand interaction, however most were not predicted to have a functional consequence. These observations suggest that a greater diversity in biological functions for proglucagon-derived peptides might exist in mammals.
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Evolución Molecular , Variación Genética , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/genética , Glucagón/genética , Proglucagón/genética , Receptores de Glucagón/genética , Secuencia de Aminoácidos , Animales , Genoma , Péptido 1 Similar al Glucagón , Péptido 2 Similar al Glucagón , Mamíferos , FilogeniaRESUMEN
There is an increasing interest in the putative role of glucagon-like peptide 1 receptor (GLP-1R) agonists as novel therapeutic agents for mental disorders. Herein, we investigated the expressions of GLP-1R and GLP-2R genes, and its relationship with body mass index (BMI), in the post-mortem brain tissue of patients with mood (MD) and psychotic disorders. Brain samples were localized to the dorsolateral prefrontal cortex (dlPFC) (nâ¯=â¯459) and hippocampus (nâ¯=â¯378). After adjustment for age, sex, ethnicity, post-mortem interval (PMI) and BMI, we observed significant differences, between healthy controls and MD subjects, in GLP-1R and GLP-2R gene expression in the dlPFC (ßâ¯=â¯1.504, pâ¯=â¯0.004; and ßâ¯=â¯1.305, pâ¯=â¯0.011, respectively); whereas in the hippocampus, only GLP-1R expression was significantly associated with MD (ßâ¯=â¯-1.28, pâ¯=â¯0.029). No significant differences were found in relation to schizophrenia. In addition, we observed a moderating effect of MD diagnosis on the associations between BMI, GLP-1R and GLP-2R expression values in the dlPFC (ßâ¯=â¯-0.05, pâ¯=â¯0.003; and ßâ¯=â¯-0.04, pâ¯=â¯0.004, respectively). There was a similar moderating effect for GLP-1R in the hippocampus (ßâ¯=â¯0.043, 95% CI 0.003; 0.08 pâ¯=â¯0.03), but in an opposite direction than observed in the dlPFC. This is the first evidence of abnormal gene expression of GLP-1R and GLP-2R in postmortem brain of individuals with MD, providing a rationale for further inquiry and proof of principle interventional studies.
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Índice de Masa Corporal , Encéfalo/metabolismo , Receptores de Péptidos Similares al Glucagón/biosíntesis , Receptores de Péptidos Similares al Glucagón/genética , Trastornos del Humor/metabolismo , Trastornos Psicóticos/metabolismo , Adolescente , Adulto , Anciano , Autopsia , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Expresión Génica , Receptor del Péptido 1 Similar al Glucagón/biosíntesis , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/biosíntesis , Receptor del Péptido 2 Similar al Glucagón/genética , Hipocampo/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Corteza Prefrontal/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice. METHODS: We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor (Glp1r), GLP-2 receptor (Glp2r), and preproglucagon (Gcg) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG). RESULTS: Comparison of Glp1r, Glp2r, and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r-/- mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology. CONCLUSION: The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.
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Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 2 Similar al Glucagón/metabolismo , Homeostasis , Intestinos/crecimiento & desarrollo , Proglucagón/metabolismo , Animales , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 2 Similar al Glucagón/genética , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Proglucagón/genéticaRESUMEN
OBJECTIVE: Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice. METHODS: Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2r+/+ and Glp2r-/- mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2r+/+ and Glp2r-/- mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively. RESULTS: Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo. GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo. Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r-/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein gavage, was significantly attenuated in Glp2r-/- mice. CONCLUSIONS: These findings reveal an important role for GLP-2R signaling in the physiological and pharmacological control of enteral amino acid sensing and assimilation, defining an enteroendocrine cell-enterocyte axis for optimal energy absorption.
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Sistemas de Transporte de Aminoácidos/metabolismo , Péptido 2 Similar al Glucagón/metabolismo , Absorción Intestinal/efectos de los fármacos , Aminoácidos/metabolismo , Animales , Células Enteroendocrinas/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/fisiología , Péptido 2 Similar al Glucagón/fisiología , Receptor del Péptido 2 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Yeyuno/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/metabolismo , Receptores de Glucagón/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed. RESULTS: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy. CONCLUSIONS: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.
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Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.
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Receptor del Péptido 2 Similar al Glucagón/metabolismo , Mucosa Intestinal/metabolismo , Animales , Expresión Génica , Receptor del Péptido 2 Similar al Glucagón/genética , Yeyuno/citología , Masculino , Ratones Endogámicos C57BL , Plexo Mientérico/citología , Neuronas/metabolismo , Especificidad de Órganos , Ratas WistarRESUMEN
This study characterized the glucagon-like peptide 2 receptor (GLP2R) gene of chickens because relatively little is known about the underlying mechanism of GLP2 actions in nonmammalian species. With the use of reverse transcription PCR, we first cloned the chicken GLP2R (cGLP2R) from adult intestine, which was predicted to encode a 529-amino acid receptor precursor. With the use of a pGL3-CRE luciferase reporter system, we demonstrated that cGLP2R expressed in Chinese hamster ovary cells could be potently activated by cGLP2 (half maximal effective concentration, 1.06 nM) but not by its structurally related peptides, including the newly identified glucagon-like peptide, indicating that cGLP2R is a functional receptor specific to cGLP2. Reverse transcription PCR assay revealed that cGLP2R mRNA was widely expressed in adult chicken tissues, including pancreas and various parts of the gastrointestinal tract. With the use of quantitative real-time reverse transcription PCR assays, we further investigated the mRNA expression of cGLP2R and its potential downstream mediators, epidermal growth factor receptor (EGFR) ligands (heparin-binding EGF-like growth factor, epiregulin, and amphiregulin), in the distal duodenum of developing embryos. The mRNA expression levels of GLP2R and EGFR ligands (heparin-binding EGF-like growth factor and amphiregulin) were shown to increase (P < 0.05 or 0.01) during the late embryonic stages (E16 and E20), implying a potential coordinated action of GLP2 and EGFR ligands on embryonic intestine development. Taken together, our findings not only establish a molecular basis to explore the physiological roles of GLP2 in birds, but they also provide comparative insights into the roles of GLP2R and its ligand in vertebrates, such as its roles in embryonic intestine development.