RESUMO
OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Glucagon , Glucose , Homeostase , Secreção de Insulina , Células Secretoras de Insulina , Receptores de Glucagon , Transdução de Sinais , Animais , Glucagon/metabolismo , Camundongos , Células Secretoras de Insulina/metabolismo , Glucose/metabolismo , Receptores de Glucagon/metabolismo , Receptores de Glucagon/genética , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dieta Hiperlipídica , Glicemia/metabolismo , FemininoRESUMO
In response to oral glucose, glucagon-like peptide-1 receptor (Glp1r) knockout (Glp1r−/−) mice become hyperglycaemic due to impaired insulin secretion. Exercise also induces hyperglycaemia in Glp1r−/− mice. In contrast to oral glucose, exercise decreases insulin secretion. This implies that exercise-induced hyperglycaemia in Glp1r−/− mice results from the loss of a non-insulinotropic effect mediated by the Glp1r. Muscle glucose uptake (MGU) is normal in exercising Glp1r−/− mice. Thus, we hypothesize that exercise-induced hyperglycaemia in Glp1r−/− mice is due to excessive hepatic glucose production (HGP). Wild-type (Glp1r+/+) and Glp1r−/− mice implanted with venous and arterial catheters underwent treadmill exercise or remained sedentary for 30 min. [3-3H]glucose was used to estimate rates of glucose appearance (Ra), an index of HGP, and disappearance (Rd). 2[14C]deoxyglucose was used to assess MGU. Glp1r−/− mice displayed exercise-induced hyperglycaemia due to an excessive increase in Ra but normal Rd and MGU. Exercise-induced glucagon levels were â¼2-fold higher in Glp1r−/− mice, resulting in a â¼2-fold higher glucagon:insulin ratio. Since inhibition of the central Glp1r stimulates HGP, we tested whether intracerebroventricular (ICV) infusion of the Glp1r antagonist exendin(939) (Ex9) in Glp1r+/+ mice would result in exercise-induced hyperglycaemia. ICV Ex9 did not enhance glucose levels or HGP during exercise, suggesting that glucoregulatory effects of Glp1 during exercise are mediated via the pancreatic Glp1r. In conclusion, functional disruption of the Glp1r results in exercise-induced hyperglycaemia associated with an excessive increase in glucagon secretion and HGP. These results suggest an essential role for basal Glp1r signalling in the suppression of alpha cell secretion during exercise.
Assuntos
Glucose/fisiologia , Hiperglicemia/fisiopatologia , Condicionamento Físico Animal/fisiologia , Receptores de Glucagon/fisiologia , Animais , Corticosterona/sangue , Glucagon/sangue , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hiperglicemia/sangue , Hiperglicemia/etiologia , Insulina/sangue , Cinética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Type 2 diabetes mellitus is characterised by beta cell failure, which frequently develops in the setting of insulin resistance. Inflammation contributes to the pathophysiology of type 2 diabetes by impairing insulin action in peripheral tissues and via reduction of beta cell function. Inflammation may also play an important role in the development of complications that arise in patients with type 2 diabetes. Hence, the anti-inflammatory actions of commonly used glucose-lowering drugs may contribute, indirectly, to their mechanisms of action and therapeutic benefit. Herein we highlight the anti-inflammatory actions of glucagon-like peptide-1 (GLP-1), which exerts direct and indirect actions on immune function. The observations that GLP-1 receptor agonists exert anti-inflammatory actions in preclinical studies, taken together with case reports linking improvements in psoriasis with GLP-1 receptor agonist therapy, illustrates the emerging clinical implications of non-classical anti-inflammatory actions of incretin-based therapeutics.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células T Matadoras Naturais/metabolismo , Psoríase/complicações , Psoríase/tratamento farmacológico , Receptores de Glucagon/metabolismo , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , MasculinoRESUMO
AIMS/HYPOTHESIS: Metformin is widely used for the treatment of type 2 diabetes. Although it reduces hepatic glucose production, clinical studies show that metformin may reduce plasma dipeptidyl peptidase-4 activity and increase circulating levels of glucagon-like peptide 1 (GLP-1). We examined whether metformin exerts glucoregulatory actions via modulation of the incretin axis. METHODS: Metformin action was assessed in Glp1r(-/-), Gipr(-/-), Glp1r:Gipr(-/-), Pparα (also known as Ppara)(-/-) and hyperglycaemic obese wild-type mice with or without the GLP-1 receptor (GLP1R) antagonist exendin(9-39). Experimental endpoints included glucose tolerance, plasma insulin levels, gastric emptying and food intake. Incretin receptor expression was assessed in isolated islets from metformin-treated wild-type and Pparα(-/-) mice, and in INS-1 832/3 beta cells with or without peroxisome proliferator-activated receptor (PPAR)-α or AMP-activated protein kinase (AMPK) antagonists. RESULTS: In wild-type mice, metformin acutely increased plasma levels of GLP-1, but not those of gastric inhibitory polypeptide or peptide YY; it also improved oral glucose tolerance and reduced gastric emptying. Metformin significantly improved oral glucose tolerance despite loss of incretin action in Glp1r(-/-), Gipr(-/-) and Glp1r(-/-) :Gipr(-/-) mice, and in wild-type mice fed a high-fat diet and treated with exendin(9-39). Levels of mRNA transcripts for Glp1r, Gipr and Pparα were significantly increased in islets from metformin-treated mice. Metformin directly increased Glp1r expression in INS-1 beta cells via a PPAR-α-dependent, AMPK-independent mechanism. Metformin failed to induce incretin receptor gene expression in islets from Pparα(-/-) mice. CONCLUSIONS/INTERPRETATION: As metformin modulates multiple components of the incretin axis, and enhances expression of the Glp1r and related insulinotropic islet receptors through a mechanism requiring PPAR-α, metformin may be mechanistically well suited for combination with incretin-based therapies.
Assuntos
Hipoglicemiantes/farmacologia , Metformina/farmacologia , PPAR alfa/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/sangue , Ingestão de Alimentos/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/sangue , Peptídeo 1 Semelhante ao Glucagon/sangue , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipoglicemiantes/uso terapêutico , Masculino , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , PPAR alfa/genética , Fragmentos de Peptídeos/uso terapêutico , Receptores dos Hormônios Gastrointestinais/genética , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/sangue , Transdução de Sinais/efeitos dos fármacosRESUMO
Glucagon-like peptide 1 (GLP1) is postulated to regulate blood glucose and satiety, but the biological importance of GLP1 as an incretin and neuropeptide remains controversal. The regulation of nutrient-induced insulin secretion is dependent on the secretion of incretins, gut-derived peptides that potentiate insulin secretion from the pancreatic islets. To ascertain the relative physiological importance of GLP1 as a regulator of feeding behavior and insulin secretion, we have generated mice with a targeted disruption of the GLP1 receptor gene (GLP1R). These GLP1R-/- mice are viable, develop normally but exhibit increased levels of blood glucose following oral glucose challenge in association with diminished levels of circulating insulin. It is surprising that they also exhibit abnormal levels of blood glucose following intraperitoneal glucose challenge. Intracerebroventricular administration of GLP1 inhibited feeding in wild-type mice but not in GLP1R-/- mice; however, no evidence for abnormal body weight or feeding behavior was observed in GLP1R-/- mice. These observations demonstrate that GLP1 plays a central role in the regulation of glycemia; however, disruption of GLP1/GLP1R signaling in the central nervous system is not associated with perturbation of feeding behavior or obesity in vivo.
Assuntos
Glicemia/análise , Receptores de Glucagon/metabolismo , Animais , Feminino , Deleção de Genes , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Glucose/farmacologia , Intolerância à Glucose , Insulina/sangue , Masculino , Camundongos , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Ratos , Receptores de Glucagon/genéticaRESUMO
AIMS/HYPOTHESIS: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and nonobese diabetic (NOD) and Glp1r-/- mice. METHODS: Glp1r mRNA expression was examined in sorted immune subpopulations by RT-PCR. The effects of GLP-1R activation were assessed on cAMP production and proliferation, migration and survival of primary immune cells from WT and NOD mice. The ability of primary cells from Glp1r-/- mice to proliferate, migrate or survive apoptosis was determined. Immunophenotyping studies were performed to assess the frequency of immune subpopulations in Glp1r-/- mice. RESULTS: Ex vivo activation of the GLP-1R resulted in a modest but significant elevation of cAMP in primary thymocytes and splenocytes from both WT and NOD mice. GLP-1R activation did not increase proliferation of primary thymocytes, splenocytes or peripheral lymph node cells. In contrast, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. Activation or loss of GLP-1R signalling did not modify apoptosis or chemotaxis in primary lymphocytes. Male Glp1r-/- mice exhibited a significantly lower percentage of peripheral regulatory T cells, although no differences were observed in the numbers of CD4+ and CD8+ T cells and B cells in the spleen and lymph nodes of Glp1r-/- mice. CONCLUSIONS/INTERPRETATION: These studies establish that GLP-1R signalling may regulate lymphocyte proliferation and maintenance of peripheral regulatory T cells.
Assuntos
Ativação Linfocitária/imunologia , Camundongos Endogâmicos NOD/imunologia , Receptores de Glucagon/imunologia , Linfócitos T Reguladores/imunologia , Animais , Divisão Celular , Movimento Celular , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Citometria de Fluxo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Receptores de Glucagon/deficiência , Receptores de Glucagon/genética , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/citologiaRESUMO
AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. METHODS: Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. RESULTS: Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. CONCLUSIONS/INTERPRETATION: Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.
Assuntos
Receptores de Glucagon/metabolismo , Animais , Colesterol/química , Colesterol/metabolismo , Cricetinae , Enterócitos/metabolismo , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1 , Incretinas/metabolismo , Lipídeos/química , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Obesidade/metabolismo , Receptores de Glucagon/metabolismoRESUMO
The proglucagon gene is expressed in a highly restricted tissue-specific manner in the A cells of the pancreatic islet and the L cells of the small and large intestines. The results of previous experiments indicate that cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element. We show here that the G1 element contains several AT-rich subdomains that bind proteins present in islet and enteroendocrine cell extracts. Electrophoretic mobility shift assay experiments using specific antisera identified the homeobox protein cdx-2/3 (which designates the same homeobox protein called cdx-2 for mice and cdx-3 for hamsters) as a major component of the G1-Gc2 complex in islet and intestinal cells. Mutations of the Gc element that decreased cdx-2/3 binding also resulted in decreased proglucagon promoter activity in islet and intestinal cell lines. The finding that cdx-2/3 mediates activation of the proglucagon promoter in both islet and enteroendocrine cells is consistent with the common endodermal lineage of these tissues and provides new insight into the coordinate regulation of genes expressed in both pancreatic and intestinal endocrine cell types.
Assuntos
Regulação da Expressão Gênica , Glucagon/genética , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Animais , Sequência de Bases , Sítios de Ligação , Fator de Transcrição CDX2 , Linhagem Celular , Cricetinae , DNA/genética , DNA/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Proglucagon , Ratos , Homologia de Sequência do Ácido Nucleico , TransativadoresRESUMO
The state of differentiation of various neoplastic cell lines is inversely correlated with the rate of cellular growth. To delineate the changes in hormone gene expression associated with an induced decrease in the growth rate of rat insulinoma cells, we studied the effects of sodium butyrate on the expression of the genes encoding insulin, glucagon, and angiotensinogen. Sodium butyrate inhibited cellular proliferation and decreased levels of c-myc mRNA. Concomitantly, steady-state levels of mRNAs encoding insulin and glucagon increased by 10- and 8.5-fold, respectively, as a result of a specific increase in the transcription of both genes. Sodium butyrate also inhibited angiotensinogen gene expression, which was ectopic in the insulinoma cells. These observations suggest that sodium butyrate induces a pattern of events leading to the differentiation of the rat insulinoma cells.
Assuntos
Angiotensinogênio/genética , Butiratos/farmacologia , Regulação da Expressão Gênica , Genes/efeitos dos fármacos , Glucagon/genética , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Animais , Ácido Butírico , Linhagem Celular , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , RatosRESUMO
The glucagon gene is expressed specifically in the alpha cells of the pancreatic islets. We show here that 300 base pairs of the 5'-flanking region of the rat glucagon gene, linked to a chloramphenicol acetyltransferase reporter plasmid transfected into islet cell lines of different hormone-producing phenotypes, directs transcription only in glucagon-producing islet cells. Deletional and linker-scanning mutations and DNase I footprinting assays identify three transcriptional control elements within these 300 base pairs. Two of these elements (G2 and G3) independently display enhancerlike functions on both homologous and heterologous promoters in glucagon (alpha) cells, but only on heterologous promoters in insulin- (beta) and somatostatin- (delta) expressing cells, and not in non-islet cells. The proximal promoter element (G1), characterized by low intrinsic transcriptional activity, is critical for specific expression of the glucagon gene in alpha cells. However, nuclear extracts prepared from all three islet cell phenotypes give similar protection to the three control elements of the glucagon 5'-flanking sequence. We conclude that these phenotypically distinct islet cell lines all contain regulatory DNA-binding proteins interacting with the three control elements of the glucagon gene, but that factors interacting with the glucagon promoter result in transcriptional activation only in alpha cells, to restrict glucagon gene expression to these cells. These observations suggest that interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glucagon/genética , Ilhotas Pancreáticas/metabolismo , Animais , Sequência de Bases , DNA/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Transcrição GênicaRESUMO
Species-specific differences in the enzymatic inactivation of peptides is an important consideration in the evaluation of therapeutic efficacy. We demonstrate that glucagon-like peptide 2 (GLP-2), shown to be highly intestinotrophic in mice, promotes an increase in intestinal villus height but has no trophic effect on small bowel weight in rats. The reduced intestinotrophic activity of GLP-2 in rats is attributable to inactivation by the enzyme dipeptidyl peptidase IV (DPP-IV). GLP-2(1-33) was degraded to GLP-2(3-33) following incubation with human placental DPP-IV or rat serum but not by serum from DPP-IV-deficient rats. Administration of rat GLP-2 to DPP-IV-deficient rats was associated with markedly increased bioactivity of rat GLP-2 resulting in a significant increase in small bowel weight. A synthetic GLP-2 analog, r[Gly2]GLP-2, with an alanine to glycine substitution at position 2, was resistant to cleavage by both DPP-IV and rat serum in vitro. Treatment of wild-type rats with r[Gly2]GLP-2 produced a statistically significant increase in small bowel mass. DPP-IV-mediated inactivation of GLP-2 is a critical determinant of the growth factor-like properties of GLP-2.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Glucagon/farmacologia , Peptídeos/farmacologia , Animais , Biotecnologia , Glucagon/antagonistas & inibidores , Glucagon/metabolismo , Peptídeo 2 Semelhante ao Glucagon , Peptídeos Semelhantes ao Glucagon , Humanos , Técnicas In Vitro , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/crescimento & desenvolvimento , Camundongos , Peptídeos/antagonistas & inibidores , Peptídeos/metabolismo , Engenharia de Proteínas , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Glucagon and the glucagon-like peptides are derived from a common proglucagon precursor, and regulate energy homeostasis through interaction with a family of distinct G protein coupled receptors. Three proglucagon-derived peptides, glucagon, GLP-1, and GLP-2, play important roles in energy intake, absorption, and disposal, as elucidated through studies utilizing peptide antagonists and receptor knockout mice. The essential role of glucagon in the control of hepatic glucose production, taken together with data from studies employing glucagon antagonists, glucagon receptor antisense oligonucleotides, and glucagon receptor knockout mice, suggest that reducing glucagon action may be a useful strategy for the treatment of type 2 diabetes. GLP-1 secreted from gut endocrine cells controls glucose homeostasis through glucose-dependent enhancement of beta-cell function and reduction of glucagon secretion and gastric emptying. GLP-1 administration is also associated with reduction of food intake, prevention of weight gain, and expansion of beta-cell mass through stimulation of beta-cell proliferation, and prevention of apoptosis. GLP-1R agonists, as well as enzyme inhibitors that prevent GLP-1 degradation, are in late stage clinical trials for the treatment of type 2 diabetes. Exenatide (Exendin-4) has been approved for the treatment of type 2 diabetes in the United States in April 2005. GLP-2 promotes energy absorption, inhibits gastric acid secretion and gut motility, and preserves mucosal epithelial integrity through enhancement of crypt cell proliferation and reduction of epithelial apoptosis. A GLP-2R agonist is being evaluated in clinical trials for the treatment of inflammatory bowel disease and short bowel syndrome. Taken together, the separate receptors for glucagon, GLP-1, and GLP-2 represent important targets for developing novel therapeutic agents for the treatment of disorders of energy homeostasis.
Assuntos
Glucagon/metabolismo , Receptores de Glucagon/metabolismo , Sequência de Aminoácidos , Animais , Diabetes Mellitus/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptídeos Semelhantes ao Glucagon/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Dados de Sequência Molecular , OxintomodulinaRESUMO
Glucagon-like peptide-2 (GLP-2) regulates proliferative and cytoprotective pathways in the intestine; however GLP-2 receptor (GLP-2R) signal transduction remains poorly understood, and cell lines that express the endogenous GLP-2R have not yet been isolated. We have now identified several expressed sequence tags from human cervical carcinoma cDNA libraries that correspond to GLP-2R nucleotide sequences. GLP-2R mRNA transcripts were detected by RT-PCR in two human cervical carcinoma cell lines, including HeLa cells. GLP-2 increased cAMP accumulation and activated ERK1/2 in HeLa cells transiently expressing the cloned human HeLa cell GLP-2R cDNA. However, the GLP-2R-induced activation of ERK1/2 was not mediated through Galphas, adenylyl cyclase, or transactivation of the epidermal growth factor receptor, but was pertussis toxin sensitive, inhibited by dominant negative Ras, and dependent on betagamma-subunits. GLP-2 also induced a significant increase in bromodeoxyuridine incorporation that was blocked by dominant negative Ras. Furthermore, GLP-2 inhibited HeLa cell apoptosis induced by LY294002 in a protein kinase A-dependent, but ERK-independent, manner. These findings demonstrate that the HeLa cell GLP-2R differentially signals through both Galphas/cAMP- and Gi/Go-dependent pathways, illustrating for the first time that the GLP-2R is capable of coupling to multiple heterotrimeric G proteins defining distinct GLP-2R-dependent biological actions.
Assuntos
Apoptose , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Glucagon/química , Adenilil Ciclases/metabolismo , Southern Blotting , Western Blotting , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Colo do Útero/metabolismo , Cromonas/farmacologia , Clonagem Molecular , AMP Cíclico/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , Feminino , Biblioteca Gênica , Genes Dominantes , Receptor do Peptídeo Semelhante ao Glucagon 1 , Células HeLa , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , Modelos Biológicos , Morfolinas/farmacologia , Toxina Pertussis/farmacologia , Plasmídeos/metabolismo , Receptores de Glucagon/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Ativação Transcricional , TransfecçãoRESUMO
Parathyroid hormone-related peptide (PTHrP) was originally isolated from tumors associated with the development of hypercalcemia in vivo. Analyses of PTHrP gene expression have demonstrated that PTHrP is also produced in a wide variety of normal fetal and adult nonneoplastic tissues. The results of recent experiments have demonstrated that PTHrP is a growth factor-regulated gene, and different molecular forms of synthetic PTHrP display variable activities in assays for growth factor-like properties in vitro. We have studied the growth factor-like activity of PTHrP in cells transfected with a human PTHrP (hPTHrP) expression vector. Transfected cell lines contained increased amounts of PTHrP mRNA transcripts as assessed by Northern blot analysis. The PTHrP mRNA transcripts were translated into immunoreactive hPTHrP as measured by radioimmunoassay, and conditioned medium from transfected cell lines stimulated cyclic AMP (cAMP) formation in ROS 17/2.8 osteosarcoma cells. The transforming growth factor-beta-like properties of hPTHrP-producing NRK 49F clones were examined using the large-colony transformation assay in soft agar. PTHrP-producing NRK 49F clones did not form large colonies in the presence of epidermal growth factor. In contrast, PTHrP-producing and wild-type NRK 49F cells formed large colonies in the presence of epidermal growth factor and transforming growth factor-beta. No effect on cell growth was observed in PTHrP-producing NRK 49F rat kidney fibroblasts or mouse NIH 3T3 fibroblasts. In contrast, RCB 2.2 osteoblast cells expressing the hPTHrP cDNA were growth inhibited. Incubation of wild-type RCB 2.2 cells with synthetic hPTHrP[1-34] (at concentrations of 1.0-10.0 nM) also produced growth inhibition. PTHrP increased cAMP formation in RCB 2.2 cells but not in NIH 3T3 or NRK 49F cells. Incubation of RCB 2.2 cells with dibutyryl cAMP was also associated with inhibition of cell growth. The results of these studies demonstrate that PTHrP may function as an autocrine growth inhibitor of specific cell types, possibly through a cAMP-dependent pathway.
Assuntos
Proteínas/fisiologia , Fatores de Crescimento Transformadores/fisiologia , Células 3T3/fisiologia , Animais , Linhagem Celular , DNA/genética , Expressão Gênica/genética , Humanos , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Biossíntese de Proteínas/genética , Proteínas/genética , Proteínas/imunologia , RNA Mensageiro/genética , Ratos , Transcrição Gênica/genética , Transfecção , Fatores de Crescimento Transformadores/genéticaRESUMO
The parathyroid hormone-like peptide (PLP) gene is widely expressed in normal and neoplastic tissues. Previous studies have demonstrated that PLP gene expression is regulated by serum and cycloheximide, features common to the regulation of a number of different early response genes. We now report that PLP mRNA transcripts are induced within 5 min of exposure of rat keratinocytes to serum, return to control values at 20 min, and then increase and remain elevated for at least 4 h, following which they return to baseline levels. The PLP mRNA t1/2 was approximately 90 min in both serum-deprived and serum-stimulated cells. The serum induction was blocked by actinomycin D. Cycloheximide alone induced PLP gene expression; however, PLP mRNA transcripts were not superinduced in the presence of both serum and cycloheximide. Dexamethasone and 1,25-dihydroxyvitamin D3 inhibited the basal levels of PLP mRNA transcripts but did not eliminate the serum induction of PLP gene expression. Epidermal growth factor or transforming growth factor-beta alone induced PLP mRNA transcripts, but no induction was observed following exposure of cells to epidermal growth factor and transforming growth factor-beta together. Treatment with 12-O-tetradecanoylphorbol-13-acetate for 90 min did not induce PLP mRNA transcripts, but 12-O-tetradecanoylphorbol-13-acetate blocked the rapid serum induction of PLP gene expression. These features of PLP gene expression suggest that PLP is a member of the growth factor-regulated early response gene family. The rapid serum stimulation of PLP gene expression raises the possibility that PLP may contribute in an autocrine fashion to the early cellular response to growth factor stimulation.
Assuntos
Cicloeximida/farmacologia , Substâncias de Crescimento/farmacologia , Queratinócitos/fisiologia , Proteínas/genética , Transcrição Gênica , Animais , Animais Recém-Nascidos , Calcitriol/farmacologia , Linhagem Celular , Meios de Cultura , Dactinomicina/farmacologia , Dexametasona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Família Multigênica , Proteína Relacionada ao Hormônio Paratireóideo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Tubulina (Proteína)/genéticaRESUMO
UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.
Assuntos
Queratinócitos/efeitos da radiação , Ornitina Descarboxilase/biossíntese , Raios Ultravioleta/efeitos adversos , Animais , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Queratinócitos/enzimologia , Ornitina Descarboxilase/genética , RNA Mensageiro/análise , RatosRESUMO
Chemotherapeutic agents produce cytotoxicity via induction of apoptosis and cell cycle arrest. Rapidly proliferating cells in the bone marrow and intestinal crypts are highly susceptible to chemotherapy, and damage to these cellular compartments may preclude maximally effective chemotherapy administration. Glucagon-like peptide (GLP)-2 is an enteroendocrine-derived regulatory peptide that inhibits crypt cell apoptosis after administration of agents that damage the intestinal epithelium. We report here that a human degradation-resistant GLP-2 analogue, h[Gly2]-GLP-2 significantly improves survival, reduces bacteremia, attenuates epithelial injury, and inhibits crypt apoptosis in the murine gastrointestinal tract after administration of topoisomerase I inhibitor irinotecan hydrochloride or the antimetabolite 5-fluorouracil. h[Gly2]-GLP-2 significantly improved survival and reduced weight loss but did not impair chemotherapy effectiveness in tumor-bearing mice treated with cyclical irinotecan. Furthermore, h[Gly2]-GLP-2 reduced chemotherapy-induced apoptosis, decreased activation of caspase-8 and -3, and inhibited poly(ADP-ribose) polymerase cleavage in heterologous cells transfected with the GLP-2 receptor. These observations demonstrate that the antiapoptotic effects of GLP-2 on intestinal crypt cells may be useful for the attenuation of chemotherapy-induced intestinal mucositis.
Assuntos
Antineoplásicos/efeitos adversos , Camptotecina/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Receptores de Glucagon/fisiologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Camptotecina/efeitos adversos , Camptotecina/uso terapêutico , Linhagem Celular , Feminino , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Peptídeo 2 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptídeos Semelhantes ao Glucagon , Humanos , Intestinos/efeitos dos fármacos , Intestinos/patologia , Irinotecano , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias/mortalidade , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/mortalidade , Ratos , Receptores de Glucagon/genética , Taxa de Sobrevida , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Studies of parathyroid hormone-like peptide (PLP) have demonstrated that PLP gene expression is inducible by serum, growth factors, and cycloheximide. Rapid induction of PLP gene expression has also been observed following the induction of cell differentiation. These features of PLP gene expression are consistent with a role for PLP in the regulation of cell growth and differentiation. To understand the biology of PLP in developing cells and tissues, we have studied the distribution of PLP gene expression in the fetal and neonatal rat. PLP was localized by immunocytochemistry to skin, vascular smooth muscle, skeletal muscle, heart, liver, kidney, lung, and gastrointestinal tract in Day 14 fetal rat. By Day 18 PLP immunopositivity was also detected in both fetal pituitary and adrenal medulla, as well as in endocrine pancreas. Whereas few PLP-immunopositive cells were detected in Day 14 brain, scattered areas of PLP immunopositivity were evident in Day 18 brain, in regions such as the choroid plexus. Immunostaining for PLP was also detected in Day 18 tissues that were positive on Day 14. The pattern of staining in fetal testis, where PLP was strongly localized to seminiferous tubules, differed from adult testis, where PLP was localized predominantly in Leydig cells. PLP was localized to the hepatocytes but not to the hematopoietic elements in fetal liver. Neonatal hepatocytes were weakly PLP immunopositive, and PLP was not detected in adult rat liver. Northern blot analysis demonstrated the presence of a single 1.4-kilobase PLP mRNA transcript in fetal brain, liver, heart, lung, and intestine. The results of these studies demonstrate that the PLP gene is widely expressed in a diverse number of fetal rat tissues. The cellular and tissue localization of PLP immunopositivity remains fairly constant in the transition from fetal to neonatal and adult tissues except in the testis, where a cellular switch in PLP-producing cells occurs, and the liver, where PLP gene expression is progressively extinguished postnatally.
Assuntos
Feto/química , Proteínas/análise , Adolescente , Animais , Feminino , Idade Gestacional , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/análise , Proteína Relacionada ao Hormônio Paratireóideo , Gravidez , RatosRESUMO
Glucocorticoid regulation of peptide hormone gene expression was studied in two cell lines derived from rodent islet cell tumors. In rat RIN1056A cells, dexamethasone reduced the levels of glucagon mRNA transcripts while markedly inducing the expression of the angiotensinogen gene. In contrast, dexamethasone had no effect on the regulation of glucagon gene expression in hamster InR1-G9 cells. Wild type InR1-G9 cells did not support the induction of the murine mammary tumor virus promoter by glucocorticoids, suggesting that these cells lacked the necessary cellular factor(s) for glucocorticoid responsiveness. Introduction of the glucocorticoid receptor into wild type InR1-G9 cells restored glucocorticoid induction of the murine mammary tumor virus promoter, but not glucocorticoid regulation of glucagon gene expression. Dexamethasone treatment of Sprague-Dawley rats had no effect on the levels of pancreatic glucagon mRNA transcripts. The results of these studies demonstrate that glucocorticoid regulation of glucagon gene expression is restricted to the immortalized RIN1056A cell line, providing additional evidence for cell-specific diversity in the regulation of peptide hormone gene expression in neuroendocrine tumors.