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      Long-Term in vitro Growth of Human Insulin-Secreting Insulinoma Cells

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          Abstract

          Objective: Long-term in vitro maintenance of human insulin-secreting insulinoma cells. Methods: (1) Cell culture of ex vivo-derived insulinoma cell suspensions from 8 individual human donors, using various cell culture medium supplementations; (2) determination of insulin synthesis and secretion using immunocytochemistry and insulin and pro-insulin radioimmunoassays; (3) nestin-immunostaining of long-term in vitro grown insulinoma cell suspensions, and (4) phase-contrast light microscopy for analyzing the in vitro growth characteristics of the insulinoma cells. Results: (1) Parallel persistence of in vitro insulinoma cell proliferation as well as insulin-synthesizing and -secreting capacity depended on both the co-culture of insulinoma cells with human fibroblasts and the supplementation of the cell culture medium with tissue culture supernatant derived from the rodent pituitary adenoma cell line GH-3; (2) immunostaining for insulin and secretagogin confirmed the neuroendocrine origin of the insulinoma cells grown in vitro; (3) insulin secretion capability persisted up to an observation period of 25 weeks; (4) insulin secretion rates after 6 weeks of in vitro growth ranged from 3.5 to 83.3 µU/ml/h/60,000 cells plated, and (5) after long-term in vitro growth of insulinoma-derived cell suspensions with persistent insulin-secreting capacity, nestin staining was observed predominantly in co-cultured fibroblasts. Conclusion: Our data describe for the first time the long-term in vitro culture of insulin-secreting human insulinomas and highlight the importance of β-cell trophic factors for insulinoma cell growth.

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          Most cited references 41

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          Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system.

          Gut peptides exert diverse effects regulating satiety, gastrointestinal motility and acid secretion, epithelial integrity, and both nutrient absorption and disposal. These actions are initiated by activation of specific G protein-coupled receptors and may be mediated by direct or indirect effects on target cells. More recent evidence demonstrates that gut peptides, exemplified by glucagon-like peptides-1 and 2 (GLP-1 and GLP-2), directly regulate signaling pathways coupled to cell proliferation and apoptosis. GLP-1 receptor activation enhances beta-cell proliferation and promotes islet neogenesis via activation of pdx-1 expression. The proliferative effects of GLP-1 appear to involve multiple intracellular pathways, including stimulation of Akt, activation of protein kinase Czeta, and transactivation of the epidermal growth factor receptor through the c-src kinase. GLP-1 receptor activation also promotes cell survival in beta-cells and neurons via increased levels of cAMP leading to cAMP response element binding protein activation, enhanced insulin receptor substrate-2 activity and, ultimately, activation of Akt. These actions of GLP-1 are reflected by expansion of beta-cell mass and enhanced resistance to beta-cell injury in experimental models of diabetes in vivo. GLP-2 also promotes intestinal cell proliferation and confers resistance to cellular injury in a variety of cell types. Administration of GLP-2 to animals with experimental intestinal injury promotes regeneration of the gastrointestinal epithelial mucosa and confers resistance to apoptosis in an indirect manner via yet-to-be identified GLP-2 receptor-dependent regulators of mucosal growth and cell survival. These proliferative and antiapoptotic actions of GLP-1 and GLP-2 may contribute to protective and regenerative actions of these peptides in human subjects with diabetes and intestinal disorders, respectively.
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            Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes.

            The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell-specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (approximately 8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as alpha-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.
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              Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells.

              Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.
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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                2006
                August 2006
                25 August 2006
                : 83
                : 2
                : 123-130
                Affiliations
                aDepartment of Medicine III, Medical University Vienna, Vienna, Austria; bAcademy of Science, Sofia, Bulgaria; cDepartment of Endocrine Surgery, and dDivision of Endocrinology and Metabolism, Medical University Vienna, Vienna, Austria
                Article
                94875 Neuroendocrinology 2006;83:123–130
                10.1159/000094875
                16888402
                © 2006 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 3, Tables: 1, References: 51, Pages: 8
                Categories
                Original Paper

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