RESUMO
PURPOSE: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. METHODS: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). RESULTS: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. CONCLUSIONS: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.
Assuntos
Envelhecimento/metabolismo , Expressão Gênica , Aparelho Lacrimal/metabolismo , Envelhecimento/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Córnea/citologia , Córnea/metabolismo , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Aparelho Lacrimal/citologia , Peroxidação de Lipídeos , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Lágrimas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Vitamina E/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Pancreatic beta cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in beta cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic beta cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.
Assuntos
Antioxidantes/metabolismo , Catalase/metabolismo , Dexametasona/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Catalase/biossíntese , Catalase/genética , Morte Celular , Células Cultivadas , Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Prolactina/farmacologia , Proteínas SNARE/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Cinesinas/genética , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Fatores de TempoRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic beta-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Proteínas SNARE/genética , Sinaptotagminas/genética , Animais , Animais Recém-Nascidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imunoquímica , Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/embriologia , Microscopia Confocal , Gravidez , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic â-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.
Assuntos
Animais , Feminino , Gravidez , Ratos , Regulação da Expressão Gênica no Desenvolvimento/genética , Insulina , Ilhotas Pancreáticas , Prolactina/farmacologia , Proteínas SNARE/genética , Sinaptotagminas/genética , Animais Recém-Nascidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imunoquímica , Insulina/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/embriologia , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , Proteínas SNARE/metabolismo , /genética , /metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , /genética , /metabolismoRESUMO
Prolactin (PRL) exerts its biological effects mainly by activating the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling pathway. We have recently demonstrated that PRL also stimulates the insulin receptor substrates/phosphatidylinositol 3-kinase (IRSs/PI3K) and SH2-plekstrin homology domain (SHC)/ERK pathways in islets of neonatal rats. In the present study, we investigated the involvement of the PI3K and MAP kinase (MAPK) cascades in islet development and growth in pregnant rats. The protein expression of AKT1, p70S6K and SHC was higher in islets from pregnant compared with control rats. Higher basal levels of tyrosine phosphorylation were found in classic transducers of insulin cell signaling (IRS1, IRS2 and SHC). Increased levels of threonine/tyrosine phosphorylation of ERK1/2 and serine phosphorylation of AKT and p70S6K were also detected. To assess the participation of PRL in these phenomena, pregnant and control rats were treated with an antisense oligonucleotide to reduce the expression of the PRL receptor (PRLR). Phosphorylation of AKT was reduced in islets from pregnant and control rats, whereas p70S6K protein levels were reduced only in islets from treated pregnant rats. Finally, glucose-induced insulin secretion was reduced in islets from pregnant but not from control rats treated with the PRLR antisense oligonucleotide. In conclusion, downstream proteins of the PI3K (AKT and p70S6K) and MAPK (SHC and ERK1/2) cascades are regulated by PRL signaling in islets from pregnant rats. These findings indicate that these pathways participate in the increase in islet mass and the sensitivity to glucose during pregnancy.
Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Receptores da Prolactina/metabolismo , Animais , Células Cultivadas , Feminino , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Receptores da Prolactina/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
The effects of prolactin (PRL) on transcript profile expression in 24h cultured pancreatic adult rat islets were investigated by cDNA expression array analysis to identify possible candidate mRNA species that encode proteins involved in the maturation and growth of the endocrine pancreas. The expression of 54 out of 588 genes was altered by treatment with PRL. The differentially expressed transcripts identified were distributed in six main categories involved in cell proliferation and differentiation, namely, cell cycle regulation, signal transduction, transcription factors and coactivators, translational machinery, Ca(2+)-mediated exocytosis, and immuno-response. Treatment with PRL also reduced the expression of genes related to apoptosis. Several genes, whose expression was previously not known to be modulated by PRL were also identified including macrophage migration inhibitory factor and Ca(2+)/calmodulin-dependent protein kinase IV. These genes have recently been shown to play a crucial role in insulin secretion and insulin gene expression, respectively. Treatment with PRL also modified the expression of AKT2 and bone morphogenetic protein receptor 1A that control glucose homeostasis and directly affect the behavior of endocrine pancreas and/or the sensitivity of target tissues to insulin. In conclusion, PRL induces several patterns of gene expression in pancreatic islet cells. The analysis of these different patterns will be useful for understanding the complex mechanism of action of PRL in the maturation and differentiation of pancreatic islets.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Animais , Western Blotting , Células Cultivadas , Feminino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Insulin produces pleiotropic effects on sensitive tissues, including the ocular surface, through the tyrosine kinase insulin receptor. Cerebrospinal fluid and secreted fluids, such as milk and saliva, have been reported to contain insulin. In the present study, the presence of insulin was examined in tear film, and the expression of insulin and insulin-like growth factor (IGF)-1 receptor was examined in the human cornea and conjunctiva. METHODS: Stimulated tear samples collected from 33 volunteers (17 men, 16 women), aged 23 to 51 years, who were fed or fasted for 12 hours, were assayed for total protein and insulin content by the biuret dye test and a radioimmunoassay, respectively. Frozen sections of human cornea (n = 4) and conjunctiva (n = 3) were incubated with anti-insulin receptor and anti-IGF-1 receptor antibodies and developed with a secondary antibody-peroxidase conjugate. RESULTS: Insulin was detected in all tear samples analyzed, the mean concentration being 0.404 +/- 0.129 ng/mL. There were no gender-related differences. In fed subjects, tears tended toward a higher insulin content than those in fasted individuals. There was no linear correlation between insulin and total protein content (mean, 4.61 +/- 0.79 mg/mL) in the tear film. Insulin and IGF-1 receptors were detected in the plasma membrane and cytoplasm of corneal and conjunctival epithelial cells. CONCLUSIONS: To the best of the authors' knowledge, this study represents the first demonstration of insulin in human tear film and the presence of insulin and IGF-1 receptor on the human ocular surface. These results suggest that the pancreatic hormone may play a metabolic and/or mitogenic role on the ocular surface.