RESUMO
Glucagon-like peptide-1 receptor agonists (GLP-1RAs) exert anti-inflammatory effects relevant to the chronic complications of type 2 diabetes. Although GLP-1RAs attenuate T cell-mediated gut and systemic inflammation directly through the gut intraepithelial lymphocyte GLP-1R, how GLP-1RAs inhibit systemic inflammation in the absence of widespread immune expression of the GLP-1R remains uncertain. Here, we show that GLP-1R activation attenuates the induction of plasma tumor necrosis factor alpha (TNF-α) by multiple Toll-like receptor agonists. These actions are not mediated by hematopoietic or endothelial GLP-1Rs but require central neuronal GLP-1Rs. In a cecal slurry model of polymicrobial sepsis, GLP-1RAs similarly require neuronal GLP-1Rs to attenuate detrimental responses associated with sepsis, including sickness, hypothermia, systemic inflammation, and lung injury. Mechanistically, GLP-1R activation leads to reduced TNF-α via α1-adrenergic, δ-opioid, and κ-opioid receptor signaling. These data extend emerging concepts of brain-immune networks and posit a new gut-brain GLP-1R axis for suppression of peripheral inflammation.
Assuntos
Diabetes Mellitus Tipo 2 , Sepse , Humanos , Exenatida , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeos/farmacologia , Agonistas do Receptor Semelhante a Toll , Peçonhas/farmacologia , Fator de Necrose Tumoral alfa , Inflamação , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismoRESUMO
Gut intraepithelial lymphocytes (IELs) are thought to calibrate glucagon-like peptide 1 (GLP-1) bioavailability, thereby regulating systemic glucose and lipid metabolism. Here, we show that the gut IEL GLP-1 receptor (GLP-1R) is not required for enteroendocrine L cell GLP-1 secretion and glucose homeostasis nor for the metabolic benefits of GLP-1R agonists (GLP-1RAs). Instead, the gut IEL GLP-1R is essential for the full effects of GLP-1RAs on gut microbiota. Moreover, independent of glucose control or weight loss, the anti-inflammatory actions of GLP-1RAs require the gut IEL GLP-1R to selectively restrain local and systemic T cell-induced, but not lipopolysaccharide-induced, inflammation. Such effects are mediated by the suppression of gut IEL effector functions linked to the dampening of proximal T cell receptor signaling in a protein-kinase-A-dependent manner. These data reposition key roles of the L cell-gut IEL GLP-1R axis, revealing mechanisms linking GLP-1R activation in gut IELs to modulation of microbiota composition and control of intestinal and systemic inflammation.
Assuntos
Microbioma Gastrointestinal , Linfócitos Intraepiteliais , Glicemia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Humanos , Inflamação , Intestinos , Linfócitos Intraepiteliais/metabolismo , Receptores de Antígenos de Linfócitos TRESUMO
BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide hormone secreted by enteroendocrine cells in response to nutrient ingestion. GLP-2 stimulates crypt cell proliferation leading to expansion of the mucosal epithelium; however, the mechanisms transducing the trophic effects of GLP-2 are incompletely understood. METHODS: We examined the gene expression profiles and growth-promoting actions of GLP-2 in normal mice in the presence or absence of an inhibitor of ErbB receptor signaling, in Glp2r(-/-) mice and in Egfr(wa2) mice harboring a hypomorphic point mutation in the epidermal growth factor receptor. RESULTS: Exogenous GLP-2 administration rapidly induced the expression of a subset of ErbB ligands including amphiregulin, epiregulin, and heparin binding (HB)-epidermal growth factor, in association with induction of immediate early gene expression in the small and large bowel. These actions of GLP-2 required a functional GLP-2 receptor because they were eliminated in Glp2r(-/-) mice. In contrast, insulin-like growth factor-I and keratinocyte growth factor, previously identified mediators of GLP-2 action, had no effect on the expression of these ErbB ligands. The GLP-2-mediated induction of ErbB ligand expression was not metalloproteinase inhibitor sensitive but was significantly diminished in Egfr(wa2) mice and completed abrogated in wild-type mice treated with the pan-ErbB inhibitor CI-1033. Furthermore, the stimulatory actions of GLP-2 on crypt cell proliferation and bowel growth were eliminated in the presence of CI-1033. CONCLUSIONS: These findings identify the ErbB signaling network as a target for GLP-2 action leading to stimulation of growth factor-dependent signal transduction and bowel growth in vivo.
Assuntos
Proliferação de Células , Colo/citologia , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Jejuno/citologia , Proteínas Oncogênicas v-erbB/metabolismo , Transdução de Sinais , Anfirregulina , Animais , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Família de Proteínas EGF , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Receptores ErbB/genética , Feminino , Glicoproteínas/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Mutação PuntualRESUMO
OBJECTIVE: Glucagon-like peptide-1 is a nutrient-sensitive hormone secreted from enteroendocrine L cells within the small and large bowel. Although GLP-1 levels rise rapidly in response to food ingestion, the greatest density of L cells is localized to the distal small bowel and colon. Here, we assessed the importance of the distal gut in the acute L cell response to diverse secretagogues. METHODS: Circulating levels of glucose and plasma GLP-1 were measured in response to the administration of L cell secretagogues in wild-type mice and in mice with (1) genetic reduction of Gcg expression throughout the small bowel and large bowel (GcgGut-/-) and (2) selective reduction of Gcg expression in the distal gut (GcgDistalGut-/-). RESULTS: The acute GLP-1 response to olive oil or arginine administration was markedly diminished in GcgGut-/- but preserved in GcgDistalGut-/- mice. In contrast, the increase in plasma GLP-1 levels following the administration of the GPR119 agonist AR231453, or the melanocortin-4 receptor (MC4R) agonist LY2112688, was markedly diminished in the GcgDistalGut-/- mice. The GLP-1 response to LPS was also markedly attenuated in the GcgGut-/- mice and remained submaximal in the GcgDistalGut-/- mice. Doses of metformin sufficient to lower glucose and increase GLP-1 levels in the GcgGut+/+ mice retained their glucoregulatory activity, yet they failed to increase GLP-1 levels in the GcgGut-/- mice. Surprisingly, the actions of metformin to increase plasma GLP-1 levels were substantially attenuated in the GcgDistalGut-/- mice. CONCLUSION: These findings further establish the importance of the proximal gut for the acute response to nutrient-related GLP-1 secretagogues. In contrast, we identify essential contributions of the distal gut to (i) the rapid induction of circulating GLP-1 levels in response to pharmacological selective agonism of G-protein-coupled receptors, (ii) the increased GLP-1 levels following the activation of Toll-Like Receptors with LPS, and iii) the acute GLP-1 response to metformin. Collectively, these results reveal that distal gut Gcg + endocrine cells are rapid responders to structurally and functionally diverse GLP-1 secretagogues.
Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucagon/metabolismo , Animais , Glicemia/análise , Colo/metabolismo , Colo/fisiologia , Células Enteroendócrinas/patologia , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/genética , Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/fisiologia , Masculino , Metformina/farmacologia , Camundongos , Camundongos Knockout , Proglucagon/metabolismoRESUMO
OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) conveys information from ingested nutrients to peripheral tissues, signaling energy availability. The GIP Receptor (GIPR) is also expressed in the bone marrow, notably in cells of the myeloid lineage. However, the importance of gain and loss of GIPR signaling for diverse hematopoietic responses remains unclear. METHODS: We assessed the expression of the Gipr in bone marrow (BM) lineages and examined functional roles for the GIPR in control of hematopoiesis. Bone marrow responses were studied in (i) mice fed regular or energy-rich diets, (ii) mice treated with hematopoietic stressors including acute 5-fluorouracil (5-FU), pamsaccharide (LPS), and Pam3CysSerLys4 (Pam3CSK4), with or without pharmacological administration of a GIPR agonist, and (iii) mice with global (Gipr-/-) or selective deletion of the GIPR (GiprTie2-/-) with and without bone marrow transplantation (BMT). RESULTS: Gipr is expressed within T cells, myeloid cells, and myeloid precursors; however, these cell populations were not different in peripheral blood, spleen, or BM of Gipr-/- and GiprTie2-/- mice. Nevertheless, gain and loss of function studies revealed that GIPR signaling controls the expression of BM Toll-like receptor (TLR) and Notch-related genes regulating hematopoiesis. Loss of the BM GIPR attenuates the extent of adipose tissue inflammation and dysregulates the hematopoietic response to BMT. GIPR agonism modified BM gene expression profiles following 5-FU and Pam3CSK4 whereas loss of the Gipr altered the hematopoietic responses to energy excess, two TLR ligands, and 5-FU. However, the magnitude of the cellular changes in hematopoiesis in response to gain or loss of GIPR signaling was relatively modest. CONCLUSION: These studies identify a functional gut hormone-BM axis positioned for the transduction of signals linking nutrient availability to the control of TLR and Notch genes regulating hematopoiesis. Nevertheless, stimulation or loss of GIPR signaling has minimal impact on basal hematopoiesis or the physiological response to hematopoietic stress.
Assuntos
Metabolismo Energético/genética , Hematopoese/genética , Receptores dos Hormônios Gastrointestinais/genética , Tecido Adiposo/metabolismo , Animais , Biomarcadores , Composição Corporal , Células da Medula Óssea/metabolismo , Fluoruracila/farmacologia , Expressão Gênica , Regulação da Expressão Gênica , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Paniculite/etiologia , Paniculite/metabolismo , Paniculite/patologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Dipeptidyl peptidase-4 (DPP4) modulates inflammation by enzymatic cleavage of immunoregulatory peptides and through its soluble form (sDPP4) that directly engages immune cells. Here we examine whether reduction of DPP4 activity alters inflammation. Prolonged DPP4 inhibition increases plasma levels of sDPP4, and induces sDPP4 expression in lymphocyte-enriched organs in mice. Bone marrow transplantation experiments identify hematopoietic cells as the predominant source of plasma sDPP4 following catalytic DPP4 inhibition. Surprisingly, systemic DPP4 inhibition increases plasma levels of inflammatory markers in regular chow-fed but not in high fat-fed mice. Plasma levels of sDPP4 and biomarkers of inflammation are lower in metformin-treated subjects with type 2 diabetes (T2D) and cardiovascular disease, yet exhibit considerable inter-individual variation. Sitagliptin therapy for 12 months reduces DPP4 activity yet does not increase markers of inflammation or levels of sDPP4. Collectively our findings dissociate levels of DPP4 enzyme activity, sDPP4 and biomarkers of inflammation in mice and humans.
Assuntos
Doenças Cardiovasculares/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/sangue , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Inflamação/imunologia , Idoso , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/prevenção & controle , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/imunologia , Dieta Aterogênica/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Dipeptidil Peptidase 4/imunologia , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Modelos Animais de Doenças , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Humanos , Inflamação/sangue , Inflamação/diagnóstico , Inflamação/tratamento farmacológico , Mediadores da Inflamação/análise , Mediadores da Inflamação/metabolismo , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/sangue , Isoformas de Proteínas/metabolismo , Fosfato de Sitagliptina/administração & dosagem , Fosfato de Sitagliptina/efeitos adversosRESUMO
The importance of pancreatic versus intestinal-derived GLP-1 for glucose homeostasis is controversial. We detected active GLP-1 in the mouse and human pancreas, albeit at extremely low levels relative to glucagon. Accordingly, to elucidate the metabolic importance of intestinal proglucagon-derived peptides (PGDPs), we generated mice with reduction of Gcg expression within the distal (GcgDistalGut-/-) or entire (GcgGut-/-) gut. Substantial reduction of gut Gcg expression markedly reduced circulating levels of GLP-1, and impaired glucose homeostasis, associated with increased levels of GIP, and accelerated gastric emptying. GcgDistalGut-/- mice similarly exhibited lower circulating GLP-1 and impaired oral glucose tolerance. Nevertheless, plasma levels of insulin remained normal following glucose administration in the absence of gut-derived GLP-1. Collectively, our findings identify the essential importance of gut-derived PGDPs for maintaining levels of circulating GLP-1, control of gastric emptying, and glucose homeostasis.
Assuntos
Microbioma Gastrointestinal , Animais , Glucose , Homeostase , Humanos , Insulina , Camundongos , Peptídeos , ProglucagonRESUMO
Glucagon-like peptide 1 (GLP-1) receptors are widely distributed throughout the nervous system, enabling physiological and pharmacological control of glucose and energy homeostasis. Here we elucidated the importance of Glp1r expression within cellular domains targeted by expression of Wnt1-Cre2 or Phox2b-Cre. Widespread loss of neural Glp1r in Glp1rΔWnt1-/- mice had no effect on basal food intake, gastric emptying, and glucose homeostasis. However, the glucoregulatory actions of GLP-1R agonists, but not gut-selective DPP-4 inhibition, were preserved in Glp1rΔWnt1-/- mice. Unexpectedly, selective reduction of Glp1r expression within neurons targeted by Phox2b-Cre impaired glucose homeostasis and gastric emptying and attenuated the extent of weight loss achieved with sustained GLP-1R agonism. Collectively, these studies identify discrete neural domains of Glp1r expression mediating GLP-1-regulated control of metabolism and the gut-brain axis and reveal the unexpected importance of neuronal Phox2b+ cells expressing GLP-1R for physiological regulation of gastric emptying, islet hormone responses, and glucose homeostasis.
Assuntos
Encéfalo/metabolismo , Ingestão de Alimentos , Esvaziamento Gástrico , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/metabolismo , Incretinas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Peptídeos Semelhantes ao Glucagon/análogos & derivados , Peptídeos Semelhantes ao Glucagon/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Fragmentos Fc das Imunoglobulinas/farmacologia , Incretinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Glucagon-like peptide-2 (GLP-2), secreted from enteroendocrine cells, attenuates gut motility, enhances barrier function, and augments nutrient absorption, actions mediated by a single GLP-2 receptor (GLP-2R). Despite extensive analyses, the precise distribution and cellular localization of GLP-2R expression remains controversial, confounded by the lack of suitable GLP-2R antisera. Here, we reassessed murine Glp2r expression using regular and real-time quantitative PCR (qPCR), in situ hybridization (ISH), and a Glp2rLacZ reporter mouse. Glp2r mRNA expression was detected from the stomach to the rectum and most abundant in the jejunum. Glp2r transcripts were also detected in cerebral cortex, mesenteric lymph nodes, gallbladder, urinary bladder, and mesenteric fat. Surprisingly, Glp2r mRNA was found in testis by qPCR at levels similar to jejunum. However, the testis Glp2r transcripts, detected by different primer pairs and qPCR, lacked 5' mRNA coding sequences, and only a minute proportion of them corresponded to full-length Glp2r mRNA. Within the gut, Glp2r-driven LacZ expression was localized to enteric neurons and lamina propria stromal cells, findings confirmed by ISH analysis of the endogenous Glp2r mRNA. Unexpectedly, vascular Glp2rLacZ expression was localized to mesenteric veins and not arteries. Moreover, mesenteric fat Glp2rLacZ expression was detected within blood vessels and not adipocytes. Reporter LacZ expression was not detected in all tissues expressing an endogenous Glp2r transcript, such as gallbladder, urinary bladder, and mesenteric lymph nodes. Collectively, these findings extend our understanding of the cellular domains of Glp2r expression and highlight limitations inherent in application of commonly used technologies to infer analysis of gene expression.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 2/genética , Animais , Trato Gastrointestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/análiseRESUMO
Glucagon-like peptide (GLP)-1 promotes beta-cell proliferation and survival through stimulation of its specific G-protein-coupled receptor; however, the potential for GLP-1 receptor (GLP-1R) agonists to promote growth and proliferation of human pancreatic-derived cells remains poorly understood. We identified five human pancreatic cancer cell lines that express the GLP-1R and analyzed cell growth and survival in response to GLP-1R activation. Although cholera toxin (an activator of Galphas) and forskolin (an activator of adenylyl cyclase) increased levels of intracellular cAMP in all cell lines, the GLP-1R agonist exendin-4 (Ex-4) increased cAMP only in CFPAC-1 cells. Conversely, Ex-4 induced extracellular regulated kinase (ERK) 1/2 activation in PL 45 cells in a GLP-1R-and epidermal growth factor receptor-dependent manner, whereas Ex-4 inhibited ERK1/2 phosphorylation in Hs 766T and CAPAN-1 cells. Ex-4 did not modulate the proliferation of these cell lines in vitro and did not inhibit apoptosis after exposure of cells to cytotoxic agents such as cycloheximide, indomethacin, LY294002, or cyclopamine. Furthermore, daily Ex-4 treatment for 4 weeks had no effect on the propagation of CFPAC-1 or PL 45 tumor cells evaluated in nude mice in vivo. Thus, acute or chronic (4 weeks) GLP-1R stimulation does not modify the growth or survival of human pancreatic cancer cells.
Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Neoplasias Pancreáticas/fisiopatologia , Receptores de Glucagon/genética , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Cromonas/farmacologia , Cicloeximida/farmacologia , Cicloparafinas/farmacologia , Inibidores Enzimáticos/farmacologia , Genes Reporter , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Indometacina/farmacologia , Cinética , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
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.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Homeostase , Intestinos/crescimento & desenvolvimento , Proglucagon/metabolismo , Animais , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 2/genética , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proglucagon/genéticaRESUMO
Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L cells promotes nutrient disposal via the incretin effect. However, the majority of L cells are localized to the distal gut, suggesting additional biological roles for GLP-1. Here, we demonstrate that GLP-1 receptor (GLP-1R) signaling controls mucosal expansion of the small bowel (SB) and colon. These actions did not require the epidermal growth factor (EGF) or intestinal epithelial insulin-like growth factor (IGF1) receptors but were absent in Glp1r(-/-) mice. Polyp number and size were increased in SB of exendin-4-treated Apc(Min/+) mice, whereas polyp number was reduced in SB and colon of Glp1r(-/-):Apc(Min/+) mice. Exendin-4 increased fibroblast growth factor 7 (Fgf7) expression in colonic polyps of Apc(Min/+) mice and failed to increase intestinal growth in mice lacking Fgf7. Exogenous exendin-4 and Fgf7 regulated an overlapping set of genes important for intestinal growth. Thus, gain and loss of GLP-1R signaling regulates gut growth and intestinal tumorigenesis.
Assuntos
Colo/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Animais , Carcinogênese/metabolismo , Proliferação de Células/fisiologia , Colo/fisiologia , Colo/fisiopatologia , Fator de Crescimento Epidérmico/metabolismo , Exenatida , Feminino , Incretinas/metabolismo , Mucosa Intestinal/fisiologia , Mucosa Intestinal/fisiopatologia , Intestino Delgado/patologia , Intestino Delgado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Peçonhas/metabolismoRESUMO
Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or ß-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67(+) cells in the exocrine compartment; however, pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 phosphorylation, and mediated through the GLP-1R. Rapamycin abrogated the GLP-1R-dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3ß gene expression. Mass spectrometry analysis identified GLP-1R-dependent upregulation of Reg family members, as well as proteins important for translation and export, including Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis, leading to increased pancreatic mass, independent of changes in DNA content or cell proliferation in mice.
Assuntos
Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Receptores de Glucagon/agonistas , Receptores de Glucagon/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Exenatida , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Liraglutida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas a Pancreatite , Peptídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Peçonhas/farmacologiaRESUMO
Obesity, accompanying or independent of type 2 diabetes mellitus (T2DM), is associated with higher rates of malignancy. Hence, there is considerable interest in understanding whether therapies used to treat obese patients with T2DM impact cancer cell growth. Glucagon-like peptide-1 (GLP-1) is produced in enteroendocrine cells and secreted after meal ingestion. GLP-1 regulates blood glucose through multiple mechanisms, principally inhibition of glucagon and stimulation of insulin secretion. GLP-1 also exerts independent effects promoting cell growth and survival, and sustained activation of GLP-1 receptor (GLP-1R) signaling in rodent thyroid glands leads to C-cell hyperplasia and medullary thyroid cancer. Hence, whether therapies based on GLP-1R activation modify growth or survival of cancer cells is of ongoing interest. We studied the biological actions of GLP-1 in mouse CT26 colon cancer cells that express a functional GLP-1R. The GLP-1R agonist exendin (Ex)-4 (exenatide) increased intracellular cAMP levels and inhibited the activity of signaling kinases glycogen synthase kinase 3 and ERK1/2 in CT26 cells. The Ex-4-induced inactivation of glycogen synthase kinase 3, but not ERK1/2, was dependent on protein kinase A and blocked by the GLP-1R antagonist Ex(9-39). Furthermore, Ex-4 altered cell morphology, induced apoptosis, and inhibited proliferation of CT26 cells in vitro. Moreover Ex-4 decreased CT26 colony formation in soft agar and augmented apoptosis induced by irinotecan. Twice-daily treatment of CT26 tumor-bearing BALB/c mice with Ex-4 for 2 wk increased tumor apoptosis. Hence, GLP-1R activation reduces growth and survival in CT26 colon cancer cells that express the endogenous classical GLP-1R.
Assuntos
Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Colo/metabolismo , Receptores de Glucagon/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , AMP Cíclico/metabolismo , Exenatida , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Camundongos , Peptídeos/farmacologia , Receptores de Glucagon/agonistas , Transdução de Sinais/efeitos dos fármacos , Peçonhas/farmacologiaRESUMO
OBJECTIVE: Clinical reports link use of the glucagon-like peptide-1 receptor (GLP-1R) agonists exenatide and liraglutide to pancreatitis. However, whether these agents act on the exocrine pancreas is poorly understood. RESEARCH DESIGN AND METHODS: We assessed whether the antidiabetic agents exendin (Ex)-4, liraglutide, the dipeptidyl peptidase-4 inhibitor sitagliptin, or the biguanide metformin were associated with changes in expression of genes associated with the development of experimental pancreatitis. The effects of Ex-4 when administered before or after the initiation of caerulein-induced experimental pancreatitis were determined. The importance of endogenous GLP-1R signaling for gene expression in the exocrine pancreas and the severity of pancreatitis was assessed in Glp1r(-/-) mice. RESULTS: Acute administration of Ex-4 increased expression of egr-1 and c-fos in the exocrine pancreas. Administration of Ex-4 or liraglutide for 1 week increased pancreas weight and induced expression of mRNA transcripts encoding the anti-inflammatory proteins pancreatitis-associated protein (PAP) (RegIIIbeta) and RegIIIalpha. Chronic Ex-4 treatment of high-fat-fed mice increased expression of PAP and reduced pancreatic expression of mRNA transcripts encoding for the proinflammatory monocyte chemotactic protein-1, tumor necrosis factor-alpha, and signal transducer and activator of transcription-3. Sitagliptin and metformin did not significantly change pancreatic gene expression profiles. Ex-4 administered before or after caerulein did not modify the severity of experimental pancreatitis, and levels of pancreatic edema and serum amylase were comparable in caerulein-treated Glp1r(-/-) versus Glp1r(+/+) mice. CONCLUSIONS: These findings demonstrate that GLP-1 receptor activation increases pancreatic mass and selectively modulates the expression of genes associated with pancreatitis. However, activation or genetic elimination of GLP-1R signaling does not modify the severity of experimental pancreatitis in mice.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Pancreatite/metabolismo , Pancreatite/fisiopatologia , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Animais , Ceruletídeo/toxicidade , Gorduras na Dieta/farmacologia , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/genética , Exenatida , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Genes fos/fisiologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/toxicidade , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipoglicemiantes/toxicidade , Liraglutida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/fisiologia , Pancreatite/induzido quimicamente , Proteínas Associadas a Pancreatite , Peptídeos/toxicidade , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Peçonhas/toxicidadeRESUMO
Glucagon-like peptide-2 (GLP-2) secreted from enteroendocrine cells exerts proabsorptive, regenerative, and cytoprotective actions in the normal and injured gut epithelium. Hence, sustained GLP-2 receptor (GLP-2R) activation represents a strategy under investigation for the prevention and treatment of chemotherapy-induced mucositis. Nevertheless, the consequences of increased GLP-2R signaling for the growth and survival of intestinal tumor cells remain poorly understood. We studied the proliferative and cytoprotective actions of GLP-2 in human colon cancer cells stably transfected with the GLP-2R and in nude mice harboring GLP-2R(+) human colon cancer cells. The importance of the GLP-2R for tumor growth was also examined in Apc(Min/+) mice chronically treated with exogenous GLP-2 and in Apc(Min/+):Glp2r(-/-) mice. GLP-2 increased cyclic AMP accumulation and produced cell-specific activation of growth and survival pathways in DLD-1, SW480, and HT29 cells. However, GLP-2 did not stimulate cell growth or attenuate cycloheximide-, LY294002-, indomethacin-, or chemotherapy-induced cytotoxicity in vitro. Moreover, chronic GLP-2 administration had no effect on the growth of human colon cancer cell xenografts in nude mice in vivo. Daily GLP-2 treatment for 7 weeks increased growth of normal gut mucosa but did not increase the number or size of polyps in Apc(Min/+) mice, and genetic disruption of the Glp2r gene in Apc(Min/+) mice did not modify polyp size or number. Taken together, although GLP-2R activation engages signaling pathways promoting cell proliferation and cytoprotection in the normal gut epithelium, sustained direct or indirect modulation of GLP-2R signaling does not modify intestinal tumor cell growth or survival.
Assuntos
Proliferação de Células/efeitos dos fármacos , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Neoplasias Intestinais/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Genes APC , Receptor do Peptídeo Semelhante ao Glucagon 2 , Células HT29 , Humanos , Neoplasias Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Células Tumorais CultivadasRESUMO
Classic models of receptor desensitization and internalization have been largely based on the behavior of Family A G-protein-coupled receptors (GPCRs). The glucagon-like peptide-2 receptor (GLP-2R) is a member of the Family B glucagon-secretin GPCR family, which exhibit significant sequence and structural differences from the Family A receptors in their intracellular and extracellular domains. To identify structural motifs that regulate GLP-2R signaling and cell surface receptor expression, we analyzed the functional properties of a series of mutant GLP-2Rs. The majority of the C-terminal receptor tail was dispensable for GLP-2-induced cAMP accumulation, ERK1/2 activation, and endocytosis in transfected cells. However, progressive truncation of the C terminus reduced cell surface receptor expression, altered agonist-induced GLP-2R trafficking, and abrogated protein kinase A-mediated heterologous receptor desensitization. Elimination of the distal 21 amino acids of the receptor was sufficient to promote constitutive receptor internalization and prevent agonist-induced recruitment of beta-arrestin-2. Site-directed mutagenesis identified specific amino acid residues within the distal GLP-2R C terminus that mediate the stable association with beta-arrestin-2. Surprisingly, although the truncated mutant receptors failed to interact with beta-arrestin-2, they underwent homologous desensitization and subsequent resensitization with kinetics similar to that observed with the wild-type GLP-2R. Our data suggest that, although the GLP-2R C terminus is not required for coupling to cellular machinery regulating signaling or desensitization, it may serve as a sorting signal for intracellular trafficking. Taken together with the previously demonstrated clathrin and dynamin-independent, lipid-raft-dependent pathways for internalization, our data suggest that GLP-2 receptor signaling has evolved unique structural and functional mechanisms for control of receptor trafficking, desensitization, and resensitization.