Increased expression of SNARE proteins and synaptotagmin IV in islets from pregnant rats and in vitro prolactin-treated neonatal islets.
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.
Cunha
Daniel A
DA
Department of Physiology, State University of Campinas (UNICAMP), Brazil.
Amaral
Maria E C
ME
Carvalho
Carolina P F
CP
Collares-Buzato
Carla B
CB
Carneiro
Everardo M
EM
Boschero
Antonio C
AC
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics*; D015151:Immunoblotting; D007120:Immunochemistry; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D018613:Microscopy, Confocal; D011247:Pregnancy; D011388:Prolactin / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050600:SNARE Proteins / Q000235:genetics* / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050857:Synaptotagmins / Q000235:genetics* / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D012333:RNA, Messenger; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; D050857:Synaptotagmins; D011388:Prolactin