ABSTRACT
Pdx-1, an important transcription factor highlighting in the early pancreatic development, islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research. Our aim was to explore the role of pdx-1 in pancreatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse transcriptase-PCR (RT-PCR), in situ hybridization and immunofluorescent staining were used in this research, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 (siPDX-1) or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hybridization and immunofluorescent staining results showed that siPDX-1 disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.
Subject(s)
Embryo, Nonmammalian , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/embryology , Islets of Langerhans/metabolism , RNA Interference , RNA, Small Interfering/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , ZebrafishABSTRACT
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.
Subject(s)
Animals , Female , Pregnancy , Rats , Gene Expression Regulation, Developmental/genetics , Insulin , Islets of Langerhans , Prolactin/pharmacology , SNARE Proteins/genetics , Synaptotagmins/genetics , Animals, Newborn , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Developmental/drug effects , Immunoblotting , Immunochemistry , Insulin/genetics , Islets of Langerhans/drug effects , Islets of Langerhans/embryology , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/analysis , SNARE Proteins/metabolism , /genetics , /metabolism , Synaptotagmins/metabolism , Syntaxin 1/genetics , Syntaxin 1/metabolism , /genetics , /metabolismABSTRACT
A gravidez é modelo natural de análise da reserva funcional do pâncreas materno. O pâncreas normal responde a este estímulo fisiológico com hiperatividade. Na falta de reserva funcional adequada, manifesta-se o diabete gestacional ou exacerba-se o diabete clínico. A glicose, elevada no meio intrauterino, é responsável por efeitos deletéricos no feto e no recém-nascido. As características morfológicas do pâncreas endócrino dos recém-nascidos indicam hiperatividade e säo semelhantes no diabete moderado e grave. O diabete moderado é modelo de macrossomia e o grave determina retardo de crescimento intra-uterino. É necessário esclarecer as causas destes desvios do crescimento fetal. Objetivos- Analisar a atividade endócrina-pancreática materna e fetal na associaçäo diabete e prenhez. Relacionar a funçäo do pâncreas endócrino fetal com os desvios do crescimento intra-uterino. Animais- Ratas Wistar jovens, näo-diabéticas e com diabete moderado e grave, näo-prenhez de 18 e 21 dias com seus respectivos recém-nascidos. Material de Estudo- Pâncreas endócrino, amostras de sangue de ratas e recém-nascidos e peso corpóreo fetal no 18§e 21§ dias da prenhez. Determinaçöes- Avaliaçöes morfológicas, morfométricas, imunohistoquímicas e bioquímicas do pâncreas endócrino materno e fetal. Avaliaçöes do peso fetal.