O efeito da hiperglicemia materna sobre a geração de espécies reativas de oxigênio e o estresse do retículo endoplasmático em ilhotas pancreáticas isoladas da prole de ratas
Arquivos
Data
2017-12-20
Autores
Carvalho, Diego Soares 
Orientadores
Hirata, Aparecida Emiko
Tipo
Tese de doutorado
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ISSN da Revista
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Resumo
A hiperglicemia durante a gravidez pode resultar em defeitos no metabolismo de glicose e na função das células β pancreáticas na prole. As origens fetais da doença no adulto estão associadas a vários mecanismos causadores que são dependentes das condições de estresse intrauterino. O papel do estresse oxidativo na programação fetal é apoiado por evidências epidemiológicas, correlacionado a elevados índices oxidantes em indivíduos com baixo peso ao nascer com o diabetes melitus tipo 2 (DM2) e doença cardiovascular. Outro mecanismo que tem sido estudado nesse contexto da programação fetal é o estresse do retículo endoplasmático (RE), através de proteínas sinalizadoras da resposta à proteína mal dobrada (UPR: Unfolded Protein Response) situadas na membrana do RE (PERK, IRE1α e ATF6). Esta via é altamente preservada nos organismos eucariotos e visa corrigir os erros de enovelamento das proteínas e restabelecer a homeostasia, porém quando isto não ocorre, vias apoptóticas são ativadas. As proteínas JNK, p38 MAPK e ERK1/2 fazem parte da família das MAP cinases (MAPKs) e desempenham um papel fundamental em eventos de proliferação e diferenciação celular e apoptose, além de mediar respostas ao estresse oxidativo e ao estresse do retículo endoplasmático. É possível que o estresse oxidativo e o estresse do retículo endoplasmático sejam um elo de conexão entre o insulto intrauterino e as consequências da programação fetal na vida adulta da prole. Desta forma, o objetivo deste trabalho foi avaliar o efeito da hiperglicemia materna sobre a geração de espécies reativas de oxigênio e o estresse do retículo endoplasmático em ilhotas pancreáticas isoladas da prole de ratas. Foram utilizadas ratas da linhagem wistar com três meses de idade e a indução do diabetes foi realizada através da administração de estreptozotocina (STZ) – 60 mg/kg em dose única via intraperitoneal. A partir das proles das mães diabéticas e controles, foram selecionados apenas os machos, que foram estudados aos 3 meses de idade. Os animais filhos de mãe diabética (FMD) apresentaram uma redução do peso corporal ao nascimento quando comparados ao grupo filho de mãe controle (CLT) que se manteve por todo o período experimental. Encontramos redução do peso do tecido adiposo epididimal, aumento dos triglicérides, VLDL e colesterol total, além de resistência à insulina evidenciada por teste in vivo. Observamos aumento da secreção de insulina estimulada pela glicose, na condição 16,7 mM. A produção do ânion superóxido encontrou-se reduzida no grupo FMD enquanto que a atividade da enzima Catalase (CAT) encontrou-se aumentada. Evidenciamos um aumento da fosforilação de eIF2α e das proteínas JNK e p-38 MAPK no grupo FMD quando comparado ao grupo CTL. Estes resultados demonstram que a hiperglicemia materna durante a concepção, gestação e lactação é capaz de promover alterações permanentes no peso corporal do nascimento aos três meses de idade, dislipidemia e resistência à insulina. Nas ilhotas pancreáticas isoladas, promoveu aumento no processo secretório de insulina quando desafiado com alta glicose associado à redução da geração do ânion superóxido (O2•) e aumento da atividade da enzima catalase. Observamos indicativo de estresse celular mediado pelo aumento da fosforilação das MAP kinases JNK, p-38MAPK e do fator de iniciação eucariótico 2 (eIF2α).
Hyperglycaemia during pregnancy may result in defects in glucose metabolism and pancreatic β cell function in offspring. The fetal origins of the disease in the adult are associated with several causative mechanisms that are dependent on the conditions of intrauterine stress. The role of oxidative stress in fetal programming is supported by epidemiological evidence, correlated with high oxidant rates presented in subjects with low birth weight with type 2 diabetes mellitus (DM2) and cardiovascular disease. Another mechanism that has been evaluated in the context of fetal programming is the stress of the endoplasmic reticulum (ER), through proteins that signaling the response to poorly folded protein (UPR: Unfolded Protein Response) located on the membrane of the ER (PERK, IRE1α e ATF6). This pathway is highly conserved in eukaryotic organisms and aims to correct protein folding errors and reestablish homeostasis, but when this does not occur, apoptotic pathways are activated. The JNK, p38 MAPK, and ERK1/2 proteins are part of the family of MAP kinases (MAPKs) and play a key role in cell proliferation, differentiation, and apoptosis, as well as mediate responses to oxidative stress and endoplasmic reticulum stress. It is possible that oxidative stress and endoplasmic reticulum stress are a link between intrauterine insult and the consequences of fetal programming in adulthood. Thus, the objective of this work was to evaluate the effect of maternal hyperglycemia on the generation of reactive oxygen species and the endoplasmic reticulum stress in isolated pancreatic islets from the offspring. Wistar rats were used at three months of age and diabetes induction was performed by administration of streptozotocin (STZ) - 60 mg/kg in a single intraperitoneal dose. Male rats were selected from the offspring of diabetic mothers and controls, which were studied at 3 months of age. The offspring of diabetic mothers (OD) presented a reduction in body weight at birth when compared to the offspring of controls mothers (OC) that remained throughout the experimental period. We found a reduction of epididymal adipose tissue weight while increased triglycerides, VLDL, and total cholesterol, as well as insulin resistance evidenced by in vivo experiments. We observed increased insulin secretion stimulated by glucose in the presence of high glucose16.7 mM. The production of the superoxide anion was reduced in the OD group whereas the activity of the catalase enzyme (CAT) was found to be increased. We showed an increase in phosphorylation of eIF2α, JNK and p-38 MAPK in the OD group when compared to OC group. These results demonstrate that maternal hyperglycemia during conception, gestation, and lactation is capable of promoting permanent changes in body weight from birth to three months of age, dyslipidemia and insulin resistance. In isolated pancreatic islets, it promoted alteration in the insulin secretory process when challenged with high glucose. This effect was associated with the reduction of superoxide anion generation (O2•) and increased activity of the catalase enzyme. We observed indicative of cell stress mediated by increased phosphorylation of MAP kinases JNK, p-38MAPK and eukaryotic initiation factor 2 (eIF2α).
Hyperglycaemia during pregnancy may result in defects in glucose metabolism and pancreatic β cell function in offspring. The fetal origins of the disease in the adult are associated with several causative mechanisms that are dependent on the conditions of intrauterine stress. The role of oxidative stress in fetal programming is supported by epidemiological evidence, correlated with high oxidant rates presented in subjects with low birth weight with type 2 diabetes mellitus (DM2) and cardiovascular disease. Another mechanism that has been evaluated in the context of fetal programming is the stress of the endoplasmic reticulum (ER), through proteins that signaling the response to poorly folded protein (UPR: Unfolded Protein Response) located on the membrane of the ER (PERK, IRE1α e ATF6). This pathway is highly conserved in eukaryotic organisms and aims to correct protein folding errors and reestablish homeostasis, but when this does not occur, apoptotic pathways are activated. The JNK, p38 MAPK, and ERK1/2 proteins are part of the family of MAP kinases (MAPKs) and play a key role in cell proliferation, differentiation, and apoptosis, as well as mediate responses to oxidative stress and endoplasmic reticulum stress. It is possible that oxidative stress and endoplasmic reticulum stress are a link between intrauterine insult and the consequences of fetal programming in adulthood. Thus, the objective of this work was to evaluate the effect of maternal hyperglycemia on the generation of reactive oxygen species and the endoplasmic reticulum stress in isolated pancreatic islets from the offspring. Wistar rats were used at three months of age and diabetes induction was performed by administration of streptozotocin (STZ) - 60 mg/kg in a single intraperitoneal dose. Male rats were selected from the offspring of diabetic mothers and controls, which were studied at 3 months of age. The offspring of diabetic mothers (OD) presented a reduction in body weight at birth when compared to the offspring of controls mothers (OC) that remained throughout the experimental period. We found a reduction of epididymal adipose tissue weight while increased triglycerides, VLDL, and total cholesterol, as well as insulin resistance evidenced by in vivo experiments. We observed increased insulin secretion stimulated by glucose in the presence of high glucose16.7 mM. The production of the superoxide anion was reduced in the OD group whereas the activity of the catalase enzyme (CAT) was found to be increased. We showed an increase in phosphorylation of eIF2α, JNK and p-38 MAPK in the OD group when compared to OC group. These results demonstrate that maternal hyperglycemia during conception, gestation, and lactation is capable of promoting permanent changes in body weight from birth to three months of age, dyslipidemia and insulin resistance. In isolated pancreatic islets, it promoted alteration in the insulin secretory process when challenged with high glucose. This effect was associated with the reduction of superoxide anion generation (O2•) and increased activity of the catalase enzyme. We observed indicative of cell stress mediated by increased phosphorylation of MAP kinases JNK, p-38MAPK and eukaryotic initiation factor 2 (eIF2α).
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Citação
CARVALHO, Diego Soares. O efeito da hiperglicemia materna sobre a geração de espécies reativas de oxigênio e o estresse do retículo endoplasmático em ilhotas pancreáticas isoladas da prole de ratas. 2017. São Paulo, [134] p. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2017.