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1.
Cytotherapy ; 15(1): 122-31, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23260092

RESUMO

BACKGROUND: Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established. METHODS: D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon(+) cells. RESULTS: Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon(+) cells co-expressed neurofilament middle and some glucagon-like peptide-1(+) cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1(+) cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon(+) cells did not display Ca(2+) oscillations typical of pancreatic α-cells. DISCUSSION: These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins.


Assuntos
Células-Tronco Embrionárias/citologia , Animais , Diferenciação Celular/fisiologia , Glucagon , Camundongos , Hormônios Pancreáticos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
2.
PLoS One ; 7(4): e34650, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22506040

RESUMO

The non-steroidal compound STX modulates the hypothalamic control of core body temperature and energy homeostasis. The aim of this work was to study the potential effects of STX on pancreatic ß-cell function. 1-10 nM STX produced an increase in glucose-induced insulin secretion in isolated islets from male mice, whereas it had no effect in islets from female mice. This insulinotropic effect of STX was abolished by the anti-estrogen ICI 182,780. STX increased intracellular calcium entry in both whole islets and isolated ß-cells, and closed the K(ATP) channel, suggesting a direct effect on ß-cells. When intraperitoneal glucose tolerance test was performed, a single dose of 100 µg/kg body weight STX improved glucose sensitivity in males, yet it had a slight effect on females. In agreement with the effect on isolated islets, 100 µg/kg dose of STX enhanced the plasma insulin increase in response to a glucose load, while it did not in females. Long-term treatment (100 µg/kg, 6 days) of male mice with STX did not alter body weight, fasting glucose, glucose sensitivity or islet insulin content. Ovariectomized females were insensitive to STX (100 µg/kg), after either an acute administration or a 6-day treatment. This long-term treatment was also ineffective in a mouse model of mild diabetes. Therefore, STX appears to have a gender-specific effect on blood glucose homeostasis, which is only manifested after an acute administration. The insulinotropic effect of STX in pancreatic ß-cells is mediated by the closure of the K(ATP) channel and the increase in intracellular calcium concentration. The in vivo improvement in glucose tolerance appears to be mostly due to the enhancement of insulin secretion from ß-cells.


Assuntos
Acrilamidas/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Cálcio/metabolismo , Diabetes Mellitus/metabolismo , Estrogênios/farmacologia , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose/métodos , Homeostase/efeitos dos fármacos , Insulina/sangue , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores Sexuais
3.
Endocrinology ; 151(8): 3665-74, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20555029

RESUMO

Atrial natriuretic peptide (ANP) and its guanylyl cyclase-A (GC-A) receptor are being involved in metabolism, although their role in the endocrine pancreas is still greatly unknown. The aim of this work is to study a possible role for the ANP/GC-A system in modulating pancreatic beta-cell function. The results presented here show a direct effect of the GC-A receptor in regulating glucose-stimulated insulin secretion (GSIS) and beta-cell mass. GC-A activation by its natural ligand, ANP, rapidly blocked ATP-dependent potassium (K(ATP)) channel activity, increased glucose-elicited Ca(2+) signals, and enhanced GSIS in islets of Langerhans. The effect in GSIS was inhibited in islets from GC-A knockout (KO) mice. Pancreatic islets from GC-A KO mice responded to increasing glucose concentrations with enhanced insulin secretion compared with wild type (WT). Remarkably, islets from GC-A KO mice were smaller, presented lower beta-cell mass and decreased insulin content. However, glucose-induced Ca(2+) response was more vigorous in GC-A KO islets, and basal K(ATP) channel activity in GC-A KO beta-cells was greatly diminished compared with WT. When protein levels of the two K(ATP) channel constitutive subunits sulfonylurea receptor 1 and Inward rectifier potassium channel 6.2 were measured, both were diminished in GC-A KO islets. These alterations on beta-cell function were not associated with disruption of glucose tolerance or insulin sensitivity in vivo. Glucose and insulin tolerance tests were similar in WT and GC-A KO mice. Our data suggest that the ANP/GC-A system may have a modulating effect on beta-cell function.


Assuntos
Fator Natriurético Atrial/fisiologia , Células Secretoras de Insulina/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Animais , Fator Natriurético Atrial/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Glucose/metabolismo , Glucose/farmacologia , Insulina/análise , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Canais KATP/efeitos dos fármacos , Canais KATP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo
4.
J Endocrinol ; 200(2): 127-38, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19017711

RESUMO

PPAR alpha is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR alpha is involved in the regulation of in vivo triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. Some nuclear receptors have been involved in rapid effects mediated by non-genomic mechanisms. In this paper, we report the rapid non-genomic effects of PPAR alpha ligands on the intracellular calcium concentration ([Ca2+]i), mitochondrial function, reactive oxygen species (ROS) generation, and secretion of insulin in freshly isolated mouse islets of Langerhans. The hypolipidemic fibrate PPAR alpha agonist WY-14 643 decreased the glucose-induced calcium oscillations in intact islets. This effect was mimicked by the synthetic agonist GW7647 and the endogenous agonist oleylethanolamide. The WY-14 643 action was rapid in onset (5 min) and was still produced in the presence of protein and mRNA synthesis inhibitors, cycloheximide, and actinomycin-d. This suggests that it is independent of gene transcription. In addition, WY-14 623 impaired mitochondrial function, increased ROS formation and decreased insulin release. PPAR alpha is present in beta-cells, mainly in the cytosol and nucleus, with a small subpopulation localized in the plasma membrane. However, the presence of the PPAR alpha ligand effects in mice bearing a disrupted Ppar alpha gene raises the possibility that the rapid effects of the agonists in pancreatic beta-cells are independent of the receptor. We conclude that PPAR alpha agonists produce a decrease in glucose-induced [Ca2+]i signals and insulin secretion in beta-cells through a rapid, non-genomic mechanism.


Assuntos
Anticolesterolemiantes/farmacologia , Cálcio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Butiratos/farmacologia , Secreção de Insulina , Masculino , Subunidade 1 do Complexo Mediador , Camundongos , Mitocôndrias/efeitos dos fármacos , Ácidos Oleicos/metabolismo , Compostos de Fenilureia/farmacologia , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo
5.
Eur J Pharmacol ; 565(1-3): 207-11, 2007 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-17499236

RESUMO

Here we show that the activation of cannabinoid CB2 receptors improved glucose tolerance after a glucose load. Blockade of cannabinoid CB2 receptors counteracted this effect, leading to glucose intolerance. Since blockade of cannabinoid CB1 receptors mimics the actions of cannabinoid CB2 receptor agonists, we propose that the endocannabinoid system modulates glucose homeostasis through the coordinated actions of cannabinoid CB1 and CB2 receptors. We also describe the presence of both cannabinoid CB1 and CB2 receptor immunoreactivity in rat pancreatic beta- and non-beta-cells, adding the endocrine pancreas to adipose tissue and the liver as potential sites for endocannabinoid regulation of glucose homeostasis.


Assuntos
Glucose/metabolismo , Homeostase , Receptor CB2 de Canabinoide/fisiologia , Animais , Moduladores de Receptores de Canabinoides/fisiologia , Imuno-Histoquímica , Ilhotas Pancreáticas/química , Masculino , Piperidinas/farmacologia , Pirazóis/farmacologia , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/análise , Receptor CB2 de Canabinoide/análise
6.
Eur J Pharmacol ; 531(1-3): 282-4, 2006 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-16423347

RESUMO

Recent reports have described the presence of cannabinoid CB1 receptors in pancreatic islets. Here we show that administration of the endogenous cannabinoid anandamide or the selective cannabinoid CB1 receptor agonist Arachidonyl-2'-chloroethylamide (ACEA) results in glucose intolerance after a glucose load. This effect is reversed by the selective cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251). These results suggest that targeting cannabinoid CB1 receptors may serve as new therapeutic alternatives for metabolic disorders such as diabetes.


Assuntos
Intolerância à Glucose/fisiopatologia , Receptor CB1 de Canabinoide/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Glicemia/metabolismo , Moduladores de Receptores de Canabinoides/farmacologia , Endocanabinoides , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/prevenção & controle , Masculino , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas , Pirazóis/farmacologia , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores
7.
Cell Calcium ; 39(2): 155-62, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16321437

RESUMO

Insulin is the main hormone involved in the regulation of glycaemia, its impaired secretion is a hallmark of type I and type II diabetic individuals. Additionally, insulin is involved in lipogenesis and weight gain, provoking an anorexigenic action. The endocannabinoid system contributes to the physiological regulation of energy balance, food intake and lipid and glucose metabolisms. Despite that, an experimental link between the endocannabinoid system and the endocrine pancreas has not yet been described. Using quantitative real-time PCR and immunocytochemistry, we have demonstrated the existence of both CB1 and CB2 receptors in the endocrine pancreas. While the CB1 receptor is mainly expressed in non-beta-cells, the CB2 type exists in beta- and non-beta-cells within the islet. The endocannabinoid 2-arachidonylglycerol (2-AG) through CB2 receptors regulates [Ca(2+)](i) signals in beta-cells and as a consequence, it decreases insulin secretion. This effect may be a new component involved in the orexigenic effect of endocannabinoids and constitutes a potential target for pharmacologic manipulation of the energy balance.


Assuntos
Sinalização do Cálcio/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Sequência de Bases , Sinalização do Cálcio/efeitos dos fármacos , Endocanabinoides , Expressão Gênica , Glicerídeos/farmacologia , Técnicas In Vitro , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Alcamidas Poli-Insaturadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo
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