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      Compartmentalization of GABA Synthesis by GAD67 Differs between Pancreatic Beta Cells and Neurons

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          Abstract

          The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.

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          Most cited references 51

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          Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

          We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.
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            Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines.

            New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.
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              Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase.

              The pancreatic islet beta-cell autoantigen of relative molecular mass 64,000 (64K), which is a major target of autoantibodies associated with the development of insulin-dependent diabetes mellitus (IDDM) has been identified as glutamic acid decarboxylase, the biosynthesizing enzyme of the inhibitory neurotransmitter GABA (gamma-aminobutyric acid). Pancreatic beta cells and a subpopulation of central nervous system neurons express high levels of this enzyme. Autoantibodies against glutamic acid decarboxylase with a higher titre and increased epitope recognition compared with those usually associated with IDDM are found in stiff-man syndrome, a rare neurological disorder characterized by a high coincidence with IDDM.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 February 2015
                2015
                : 10
                : 2
                Affiliations
                [1 ]Departments of Medicine and Microbiology/Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America
                [2 ]Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
                [3 ]Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
                [4 ]Institute of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
                University of Lille Nord de France, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SB JK CC EAP. Performed the experiments: JK CC EAP MP. Analyzed the data: JK CC EAP MP SB. Contributed reagents/materials/analysis tools: JK CC EAP MP EB NB SB. Wrote the paper: SB JK CC EAP. Developed a method to generate monolayer cultures of islets of Langerhans: NB JK EAP CC. Developed a method to transfect islets of Langerhans: JK EAP CC. Isolated human islets of Langerhans: EB.

                PONE-D-14-45542
                10.1371/journal.pone.0117130
                4315522
                25647668

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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                Figures: 8, Tables: 0, Pages: 24
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                Funding
                This study was supported by the Nora Eccles Treadwell Foundation (S. B., J. K.), by an International Network Program from the Danish Ministry of Science, Innovation and Higher Education (INP-2010-0102) (N. B., S. B.), by a National Institute of Health Diabetes Education and Research Center grant (P30 DK063720) Microscopy Core and Islet Isolation Core, a JDRF award (31-2012-783) (E. B.) to the ECIT Islets for Basic Research Program, and by a JFRF Advanced Postdoctoral Fellowship (3-APF-2014-208-A-N) (E. A. P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
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                All relevant data are within the paper and its Supporting Information files as well as in laboratory notebooks, files, and servers.

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