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      The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

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          Abstract

          Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon + cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

          Author Summary

          Type 1 diabetes is a condition that results from the loss of insulin-producing β-cells. Despite current therapies, diabetic patients are prone to vascular complications. Using the mouse as a model, we previously found that pancreatic glucagon-expressing cells can be regenerated and converted into β-like cells by the forced expression of a single gene, Pax4. Here, we generated transgenic mice allowing both the permanent labeling of α-cells and the inactivation of Arx solely in this cell subtype. Our results indicate that, upon Arx inactivation, α-cells can be continuously regenerated from duct-lining precursors and converted into β-like cells. Importantly, the additional loss of Pax4 does not impact these processes, suggesting that Arx is the main trigger of α-cell-mediated β-like cell neogenesis. Most interestingly, upon chemical induction of diabetes/β-cell loss, while control animals die or remain severely hyperglycemic, a normalization of the glycemia, a clear regeneration of the β-like cell mass, and an extended lifespan are noted in animals with the conditional inactivation of Arx. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

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          Most cited references34

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          Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.

          Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.
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            PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.

            It has been proposed that the Xenopus homeobox gene, XlHbox8, is involved in endodermal differentiation during pancreatic and duodenal development (Wright, C.V.E., Schnegelsberg, P. and De Robertis, E.M. (1988). Development 105, 787-794). To test this hypothesis directly, gene targeting was used to make two different null mutations in the mouse XlHbox8 homolog, pdx-1. In the first, the second pdx-1 exon, including the homeobox, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N terminus of PDX-1, replacing most of the homeodomain. Neonatal pdx-1 -/- mice are apancreatic, in confirmation of previous reports (Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Nature 371, 606-609). However, the pancreatic buds do form in homozygous mutants, and the dorsal bud undergoes limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. This outgrowth does not contain insulin or amylase-positive cells, but glucagon-expressing cells are found. The rostral duodenum shows a local absence of the normal columnar epithelial lining, villi, and Brunner's glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. The PDX-1/beta-galactosidase fusion allele is expressed in pancreatic and duodenal cells in the absence of functional PDX-1, with expression continuing into perinatal stages with similar boundaries and expression levels. These results offer additional insight into the role of pdx-1 in the determination and differentiation of the posterior foregut, particularly regarding the proliferation and differentiation of the pancreatic progenitors.
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              neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas.

              In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the alpha-, beta-, delta-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix-loop-helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                October 2013
                October 2013
                31 October 2013
                : 9
                : 10
                : e1003934
                Affiliations
                [1 ]Université de Nice Sophia Antipolis, iBV, UMR 7277, Nice, France
                [2 ]Inserm, iBV, U1091, Nice, France
                [3 ]CNRS, iBV, UMR 7277, Nice, France
                [4 ]Diabetes Research Center, Vrije Universiteit Brussel, Brussel, Belgium
                [5 ]Centre Commun de Microscopie, Université de Nice Sophia Antipolis, Nice, France
                [6 ]Laboratoire central d'Anatomie Pathologique, CHU de Nice, Nice, France
                [7 ]Hagedorn Research Institute, Department of Developmental Biology, Gentofte, Denmark
                [8 ]Biotechnology and Biotherapy Laboratory, Centre de Recherche de l'Institut du Cerveau et de la Moelle, CNRS UMR 7225; INSERM UMRS 975; University Pierre et Marie Curie, Hôpital Pitié Salpétrière, Paris, France
                [9 ]Max-Planck Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Göttingen, Germany
                [10 ]Department of Clinical Neurophysiology, University of Göttingen, Göttingen, Germany
                [11 ]Genome and Stem Cell Center, GENKOK, Erciyes University, Kayseri, Turkey
                Harvard Medical School, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MC AM PC. Performed the experiments: MC EG ND CR AV NBO AP FA GL SLG FBV DA JHS PR. Analyzed the data: MC HH AM PC. Contributed reagents/materials/analysis tools: FBV DA JHS PR HH AM. Wrote the paper: MC AM PC.

                Article
                PGENETICS-D-13-00829
                10.1371/journal.pgen.1003934
                3814322
                24204325
                1870beb5-0a77-492d-8e96-e30998c51f9c
                Copyright @ 2013

                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.

                History
                : 26 March 2013
                : 17 September 2013
                Page count
                Pages: 18
                Funding
                This work was supported by the Juvenile Diabetes Research foundation (17-2011-16, 2-2010-567, 26-2008-639, 17-2013-426), the INSERM AVENIR program, the INSERM, the European Research Council (StG-2011-281265) the FMR (DRC20091217179), the ANR/BMBF (2009 GENO 105 01/01KU0906), the “Investments for the Future” LABEX SIGNALIFE (ANR-11-LABX-0028-01), the Max-Planck Society, Club Isatis, Mr and Mrs Dorato, the Fondation Générale de Santé, the Foundation Schlumberger pour l'Education et la Recherche, the DON Foundation ( www.sdon.nl), and the Fund for Scientific Research-Flanders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Genetics
                Genetics

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