RESUMO
Perturbation of endoplasmic reticulum (ER) homeostasis impairs insulin biosynthesis, beta cell survival, and glucose homeostasis. We show that a murine model of diabetes is associated with the development of ER stress in beta cells and that treatment with the GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in vivo. Exendin-4 attenuated translational downregulation of insulin and improved cell survival in purified rat beta cells and in INS-1 cells following induction of ER stress in vitro. GLP-1R agonists significantly potentiated the induction of ATF-4 by ER stress and accelerated recovery from ER stress-mediated translational repression in INS-1 beta cells in a PKA-dependent manner. The effects of exendin-4 on the induction of ATF-4 were mediated via enhancement of ER stress-stimulated ATF-4 translation. Moreover, exendin-4 reduced ER stress-associated beta cell death in a PKA-dependent manner. These findings demonstrate that GLP-1R signaling directly modulates the ER stress response leading to promotion of beta cell adaptation and survival.
Assuntos
Fator 4 Ativador da Transcrição/farmacologia , Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/fisiologia , Peptídeos/farmacologia , Receptores de Glucagon/metabolismo , Estresse Fisiológico , Peçonhas/farmacologia , eIF-2 Quinase/fisiologia , Animais , Sobrevivência Celular , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Exenatida , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Homeostase , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Ratos , Ratos Wistar , Receptores de Glucagon/fisiologia , Regulação para CimaRESUMO
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
Glucagon-like peptide 1 (GLP-1) exhibits considerable potential for the treatment of type 2 diabetes because of its effects on stimulation of insulin secretion and the inhibition of gastric emptying, appetite, and glucagon secretion. However, native GLP-1 undergoes rapid enzymatic inactivation, prompting development of long-acting degradation-resistant GLP-1 receptor agonists such as exendin-4 (Ex-4). To study the consequences of sustained exposure to Ex-4, we generated metallothionein promoter-exendin-4 (MT-Exendin) mice that continuously express a proexendin-4 transgene in multiple murine tissues. We now report that MT-Exendin mice develop extensive tissue lymphocytic infiltration with increased numbers of CD4(+) and CD8a(+) cells in the liver and/or kidney and increased numbers of B220(+) cells present in the pancreas and liver. MT-Exendin mice generate antibodies directed against Ex-4, exendin NH(2)-terminal peptide (ENTP), and proexendin-4 as well as antibodies that cross-react with native GLP-1. Furthermore, lymphocytes isolated from MT-Exendin mice proliferate in response to proexendin-4 but not after exposure to Ex-4 or ENTP. These findings demonstrate that expression of a proexendin-4 transgene may be associated with activation of humoral and cellular immune responses in mice.
Assuntos
Linfócitos/imunologia , Metalotioneína/genética , Peptídeos/genética , Regiões Promotoras Genéticas/genética , Peçonhas/genética , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Exenatida , Feminino , Peptídeo 1 Semelhante ao Glucagon/imunologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Imunização , Imuno-Histoquímica , Rim/citologia , Rim/imunologia , Antígenos Comuns de Leucócito/imunologia , Fígado/citologia , Fígado/imunologia , Linfócitos/citologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pâncreas/citologia , Pâncreas/imunologia , Peptídeos/imunologia , Peptídeos/fisiologia , Receptores de Glucagon/fisiologia , Peçonhas/imunologiaRESUMO
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
Obesity and diabetes are characterized by increased inflammation reflecting disordered control of innate immunity. We reveal a local intestinal intraepithelial lymphocyte (IEL)-GLP-1 receptor (GLP-1R) signaling network that controls mucosal immune responses. Glp1r expression was enriched in intestinal IEL preparations and copurified with markers of Tαß and Tγδ IELs, the two main subsets of intestinal IELs. Exendin-4 increased cAMP accumulation in purified IELs and reduced the production of cytokines from activated IELs but not from splenocytes ex vivo. These actions were mimicked by forskolin, absent in IELs from Glp1r(-/-) mice, and attenuated by the GLP-1R agonist exendin (9-39) consistent with a GLP-1R-dependent mechanism of action. Furthermore, Glp1r(-/-) mice exhibited dysregulated intestinal gene expression, an abnormal representation of microbial species in feces, and enhanced sensitivity to intestinal injury following administration of dextran sodium sulfate. Bone marrow transplantation using wild-type C57BL/6 donors normalized expression of multiple genes regulating immune function and epithelial integrity in Glp1r(-/-) recipient mice, whereas acute exendin-4 administration robustly induced the expression of genes encoding cytokines and chemokines in normal and injured intestine. Taken together, these findings define a local enteroendocrine-IEL axis linking energy availability, host microbial responses, and mucosal integrity to the control of innate immunity.
Assuntos
Mucosa Intestinal/imunologia , Linfócitos/imunologia , Receptores de Glucagon/agonistas , Receptores de Glucagon/fisiologia , Animais , Citocinas/análise , Exenatida , Feminino , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Transdução de Sinais , Peçonhas/farmacologiaRESUMO
Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce lipid accumulation in peripheral tissues, attenuating atherosclerosis and hepatic steatosis in preclinical studies. We examined whether GLP-1R activation decreases atherosclerosis progression in high-fat diet-fed male ApoE(-/-) mice after administration of streptozotocin and treatment with the long-acting GLP-1R agonist taspoglutide administered once monthly vs. metformin in the drinking water for 12 wk. Taspoglutide did not reduce plaque area or lipid content in the aortic arch or abdominal aorta, and no significant change in aortic macrophage accumulation was detected after taspoglutide or metformin. In contrast, hepatic triglyceride levels were significantly reduced in livers from taspoglutide-treated mice. Both peripheral and intracerebroventricular administration of exendin-4 rapidly decreased plasma triglyceride levels in fasted mice, and taspoglutide therapy in ApoE(-/-) mice modulated the expression of hepatic genes controlling fatty acid uptake and oxidation. We were unable to detect expression of the entire Glp1r coding sequence in macrophages isolated from ApoE(-/-), C57BL/6, and IL10(-/-) mice. Similarly, Glp1r mRNA transcripts were not detected in RNA from isolated murine hepatocytes. Using Western blotting and tissue extracts from Glp1r(+/+) and Glp1r(-/-) mice, and cells transfected with a tagged murine GLP-1R cDNA, we could not validate the sensitivity and specificity of three different GLP-1R antisera commonly used for the detection of GLP-1R protein. Taken together, these findings illustrate divergent actions of GLP-1R agonists on atherosclerosis progression and accumulation of ectopic lipid in ApoE(-/-) mice and highlight the importance of indirect GLP-1R actions for the control of hepatic lipid accumulation.
Assuntos
Aterosclerose/metabolismo , Fígado/metabolismo , Receptores de Glucagon/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Western Blotting , Composição Corporal/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Teste de Tolerância a Glucose , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Interleucina-10/deficiência , Interleucina-10/genética , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Receptores de Glucagon/agonistas , Receptores de Glucagon/genéticaRESUMO
The enteroendocrine and enteric nervous systems convey signals through an overlapping network of regulatory peptides that act either as circulating hormones or as localized neurotransmitters within the gastrointestinal tract. Because recent studies invoke an important role for vasoactive intestinal peptide (VIP) as a downstream mediator of glucagon-like peptide-2 (GLP-2) action in the gut, we examined the importance of the VIP-GLP-2 interaction through analysis of Vip(-/-) mice. Unexpectedly, we detected abnormal villous architecture, expansion of the crypt compartment, increased crypt cell proliferation, enhanced Igf1 and Kgf gene expression, and reduced expression of Paneth cell products in the Vip(-/-) small bowel. These abnormalities were not reproduced by antagonizing VIP action in wild-type mice, and VIP administration did not reverse the intestinal phenotype of Vip(-/-) mice. Exogenous administration of GLP-2 induced the expression of ErbB ligands and immediate-early genes to similar levels in Vip(+/+) vs. Vip(-/-) mice. Moreover, GLP-2 significantly increased crypt cell proliferation and small bowel growth to comparable levels in Vip(+/+) vs. Vip(-/-) mice. Unexpectedly, exogenous GLP-2 administration had no therapeutic effect in mice with dextran sulfate-induced colitis; the severity of colonic injury and weight loss was modestly reduced in female but not male Vip(-/-) mice. Taken together, these findings extend our understanding of the complex intestinal phenotype arising from loss of the Vip gene. Furthermore, although VIP action may be important for the antiinflammatory actions of GLP-2, the Vip gene is not required for induction of a gene expression program linked to small bowel growth after enhancement of GLP-2 receptor signaling.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Transdução de Sinais/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 2 , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Mucosa Intestinal/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestinos/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/metabolismo , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Peptídeo Intestinal Vasoativo/genéticaRESUMO
Exogenous glucagon-like peptide-2 receptor (GLP-2R) activation elicits proliferative and cytoprotective responses in the gastrointestinal mucosa and ameliorates experimental small and large bowel gut injury. Nevertheless, the essential physiological role(s) of the endogenous GLP-2R remain poorly understood. We studied the importance of the GLP-2R for gut growth, epithelial cell lineage allocation, the response to mucosal injury, and host-bacterial interactions in Glp2r(-/-) and littermate control Glp2r(+/+) mice. Glp2r(-/-) mice exhibit normal somatic growth and preserved small and large bowel responses to IGF-I and keratinocyte growth factor. However, Glp2r(-/-) mice failed to up-regulate intestinal epithelial c-fos expression in response to acute GLP-2 administration and do not exhibit changes in small bowel conductance or small or large bowel growth after administration of GLP-2R agonists. The crypt and villus compartment and the numbers and localization of Paneth, enteroendocrine, and goblet cells were comparable in Glp2r(+/+) vs. Glp2r(-/-) mice. Although the severity and extent of colonic mucosal injury in response to 3% oral dextran sulfate was similar across Glp2r genotypes, Glp2r(-/-) mice exhibited significantly increased morbidity and mortality and increased bacterial translocation after induction of enteritis with indomethacin and enhanced mucosal injury in response to irinotecan. Moreover, bacterial colonization of the small bowel was significantly increased, expression of Paneth cell antimicrobial gene products was reduced, and mucosal bactericidal activity was impaired in Glp2r(-/-) mice. Although the Glp2r is dispensable for gut development and the response to colonic injury, Glp2r(-/-) mice exhibit enhanced sensitivity to small bowel injury, and abnormal host-bacterial interactions in the small bowel.
Assuntos
Enterite/patologia , Predisposição Genética para Doença , Intestinos/patologia , Celulas de Paneth/citologia , Receptores de Glucagon/genética , Receptores de Glucagon/fisiologia , Animais , Peso Corporal , Éxons , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 2 , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Permeabilidade , RNA Ribossômico 16S/metabolismoRESUMO
G protein-coupled receptor 119 (GPR119) was originally identified as a ß-cell receptor. However, GPR119 activation also promotes incretin secretion and enhances peptide YY action. We examined whether GPR119-dependent control of glucose homeostasis requires preservation of peptidergic pathways in vivo. Insulin secretion was assessed directly in islets, and glucoregulation was examined in wild-type (WT), single incretin receptor (IR) and dual IR knockout (DIRKO) mice. Experimental endpoints included plasma glucose, insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and peptide YY. Gastric emptying was assessed in WT, Glp1r-/-, DIRKO, Glp2r-/-, and GPR119-/- mice treated with the GPR119 agonist AR231453. AR231453 stimulated insulin secretion from WT and DIRKO islets in a glucose-dependent manner, improved glucose homeostasis, and augmented plasma levels of GLP-1, GIP, and insulin in WT and Gipr-/- mice. In contrast, although AR231453 increased levels of GLP-1, GIP, and insulin, it failed to lower glucose in Glp1r-/- and DIRKO mice. Furthermore, AR231453 did not improve ip glucose tolerance and had no effect on insulin action in WT and DIRKO mice. Acute GPR119 activation with AR231453 inhibited gastric emptying in Glp1r-/-, DIRKO, Glp2r-/-, and in WT mice independent of the Y2 receptor (Y2R); however, AR231453 did not control gastric emptying in GPR119-/- mice. Our findings demonstrate that GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact IR signaling and enteral glucose exposure for optimal control of glucose tolerance in vivo. In contrast, AR231453 inhibits gastric emptying independent of incretin, Y2R, or Glp2 receptors through GPR119-dependent pathways. Hence, GPR119 engages multiple complementary pathways for control of glucose homeostasis.