title: New insights concerning insulin synthesis and its secretion in rat hippocampus and cerebral cortex: amyloid-β1-42-induced reduction of proinsulin level via glycogen synthase kinase-3β.
abstract: The reduction of insulin levels in hippocampal areas is associated with Alzheimer's disease. The present study using rat brain explores the mechanisms of insulin synthesis and secretion, as well as amyloid-β1-42 (Aβ(1-42))-induced reduction of proinsulin expression. After confirming the expression of insulin mRNA and proinsulin in rat brain, we visualized and analyzed the motion of insulin secretion in rat hippocampal neurons using pH-sensitive green fluorescent protein (pHluorin) fused to the insulin. In the rat hippocampal neurons expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy revealed the appearance of fluorescent spots induced by depolarization after stimulation with 50 mM KCl. In these fluorescent spots, Ca(2+)-dependent activator protein for secretion 2 (CAPS2), which is the regulator of the dense-core vesicle involving neuronal peptides, was co-localized with insulin-pHluorin. However, Aβ(1-42)-induced reduction of proinsulin in rat hippocampal neurons was inhibited by treatment with lithium and transfection with glycogen synthase kinase-3β (GSK-3β) siRNA. These results demonstrate that synthesized insulin is secreted from rat hippocampal and cortical neuron's dense-core vesicles, and that activation of GSK-3β in Aβ(1-42)-induced Alzheimer's model hippocampal neurons decreases the insulin synthesis.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan. Electronic address: tnemoto@fc.miyazaki-u.ac.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nemoto
authors.affiliation: Department of Anatomy, Ultrastructural Cell Biology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Fumiyo
authors.initials: F
authors.lastname: Toyoshima-Aoyama
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Yanagita
authors.affiliation: Department of Anesthesiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Toyoaki
authors.initials: T
authors.lastname: Maruta
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujita
authors.affiliation: Department of Anesthesiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Koshida
authors.affiliation: Department of Anesthesiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Tetsu
authors.initials: T
authors.lastname: Yonaha
authors.affiliation: Department of Sports Health and Welfare, Faculty of Social Welfare, Kyushu University of Health and Welfare, Nobeoka, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Wada
authors.affiliation: Department of Anatomy, Ultrastructural Cell Biology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Sawaguchi
authors.affiliation: Department of Pharmacology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Murakami
mesh_terms: D016229:Amyloid beta-Peptides / Q000494:pharmacology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D018021:Lithium Chloride / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; C538900:CAPS2 protein, rat; D002135:Calcium-Binding Proteins; D007328:Insulin; D009419:Nerve Tissue Proteins; C485187:PHluorin; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42); D049452:Green Fluorescent Proteins; D011384:Proinsulin; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride
keywords: Alzheimer's disease; Aβ(1–42); CAPS2; CSF; Ca(2+)-dependent activator protein for secretion 2; DMSO; GSK-3α; GSK-3β; Hippocampal neurons; Proinsulin; RT-PCR; amyloid-β(1–42); cerebrospinal fluid; dimethyl sulfoxide; glycogen synthase kinase-3α; glycogen synthase kinase-3β; pH-sensitive green fluorescent protein; pHluorin; reverse transcriptase polymerase chain reaction