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      AAV GCG-EGFP, a new tool to identify glucagon-secreting α-cells

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          Abstract

          The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca 2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

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          Most cited references 49

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          The unique cytoarchitecture of human pancreatic islets has implications for islet cell function.

          The cytoarchitecture of human islets has been examined, focusing on cellular associations that provide the anatomical framework for paracrine interactions. By using confocal microscopy and multiple immunofluorescence, we found that, contrary to descriptions of prototypical islets in textbooks and in the literature, human islets did not show anatomical subdivisions. Insulin-immunoreactive beta cells, glucagon-immunoreactive alpha cells, and somatostatin-containing delta cells were found scattered throughout the human islet. Human beta cells were not clustered, and most (71%) showed associations with other endocrine cells, suggesting unique paracrine interactions in human islets. Human islets contained proportionally fewer beta cells and more alpha cells than did mouse islets. In human islets, most beta, alpha, and delta cells were aligned along blood vessels with no particular order or arrangement, indicating that islet microcirculation likely does not determine the order of paracrine interactions. We further investigated whether the unique human islet cytoarchitecture had functional implications. Applying imaging of cytoplasmic free Ca2+ concentration, [Ca2+]i, we found that beta cell oscillatory activity was not coordinated throughout the human islet as it was in mouse islets. Furthermore, human islets responded with an increase in [Ca2+]i when lowering the glucose concentration to 1 mM, which can be attributed to the large contribution of alpha cells to the islet composition. We conclude that the unique cellular arrangement of human islets has functional implications for islet cell function.
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            Glucose Sensing in L Cells: A Primary Cell Study

            Summary Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and α-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K+ channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.
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              Gene therapy using adeno-associated virus vectors.

               S Daya,  K Berns (2008)
              The unique life cycle of adeno-associated virus (AAV) and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector. An additional attractive feature of the wild-type virus is the lack of apparent pathogenicity. Gene transfer studies using AAV have shown significant progress at the level of animal models; clinical trials have been noteworthy with respect to the safety of AAV vectors. No proven efficacy has been observed, although in some instances, there have been promising observations. In this review, topics in AAV biology are supplemented with a section on AAV clinical trials with emphasis on the need for a deeper understanding of AAV biology and the development of efficient AAV vectors. In addition, several novel approaches and recent findings that promise to expand AAV's utility are discussed, especially in the context of combining gene therapy ex vivo with new advances in stem or progenitor cell biology.
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                Author and article information

                Contributors
                tim.kieffer@ubc.ca
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                25 July 2019
                25 July 2019
                2019
                : 9
                Affiliations
                [1 ]ISNI 0000 0001 2288 9830, GRID grid.17091.3e, Department of Cellular and Physiological Sciences, Life Sciences Institute, , University of British Columbia, ; Vancouver, British Columbia Canada
                [2 ]ISNI 0000 0000 9314 1427, GRID grid.413448.e, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ; Madrid, Spain
                [3 ]Instituto de Investigación, Desarrollo e innovación en Biotecnología Sanitaria de Elche (IDiBE), Elche, Spain
                [4 ]ISNI 0000 0001 2288 9830, GRID grid.17091.3e, Department of Surgery, , University of British Columbia, ; Vancouver, British Columbia Canada
                [5 ]ISNI 0000 0001 0684 7788, GRID grid.414137.4, Department of Pathology and Laboratory Medicine, , BC Children’s Hospital Research Institute, ; Vancouver, British Columbia Canada
                [6 ]ISNI 0000 0001 0722 3678, GRID grid.186587.5, Department of Biological Sciences, , San Jose State University, ; San Jose, CA USA
                Article
                46735
                10.1038/s41598-019-46735-2
                6658537
                31346189
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef https://doi.org/10.13039/501100000024, Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada);
                Funded by: FundRef https://doi.org/10.13039/100007476, Canadian Diabetes Association (Association Canadienne du Diabète);
                Categories
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                © The Author(s) 2019

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                diabetes, genetic vectors

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