ORIGINAL ARTICLE Heterozygous Inactivation of the Na/Ca Exchanger Increases Glucose-Induced Insulin Release, b-Cell Proliferation, and Mass Evrard Nguidjoe,1 Sophie Sokolow,1,2 Serge Bigabwa,1 Nathalie Pachera,1 Florent Allagnat,3 Jean-Marie Vanderwinden,4 Abdullah Sener,5 Mario Manto,6 Marianne Depreter,7 Jan Mast,7 Geraldine Joanny,8 Eduard Montanya,8 Jacques Rahier,9 Alessandra K. Cardozo,3 Décio L. Eizirik,3 Stéphane Schurmans,2 and André Herchuelz1 +/+ OBJECTIVE—We have previously shown that overexpression of diabetes cure than Ncx1 islets when transplanted into di- of the Na-Ca exchanger (NCX1), a protein responsible for Ca2+ abetic animals. extrusion from cells, increases b-cell programmed cell death (ap- CONCLUSIONS—Downregulation of the Na/Ca exchanger optosis) and reduces b-cell proliferation. To further characterize leads to an increase in b-cell function, proliferation, mass, and the role of NCX1 in b-cells under in vivo conditions, we devel- resistance to physiologic stress, namely to various changes in oped and characterized mice deficient for NCX1. b-cell function that are opposite to the major abnormalities seen RESEARCH DESIGN AND METHODS—Biologic and morpho- in type 2 diabetes. This provides a unique model for the preven- logic methods (Ca2+ imaging, Ca2+ uptake, glucose metabolism, tion and treatment of b-cell dysfunction in type 2 diabetes and insulin release, and point counting morphometry) were used to after islet transplantation. assess b-cell function in vitro. Blood glucose and insulin levels were measured to assess glucose metabolism and insulin sensi- tivity in vivo. Islets were transplanted under the kidney capsule to assess their performance to revert diabetes in alloxan-diabetic he prevalence of type 2 diabetes is progressing in mice. an alarming way in most regions of the world — (1,2). Type 2 diabetes is a complex disease RESULTS Heterozygous inactivation of Ncx1 in mice induced characterized by insulin resistance and b-cell an increase in glucose-induced insulin release, with a major en- T hancement of its first and second phase. This was paralleled by dysfunction. One of the earliest abnormalities occurring in an increase in b-cell proliferation and mass. The mutation also this disease is the alteration in pulsatile insulin release increased b-cell insulin content, proinsulin immunostaining, glu- with the suppression of the first phase of insulin response cose-induced Ca2+ uptake, and b-cell resistance to hypoxia. In to glucose (3). The second phase of insulin release is also addition, Ncx1+/2 islets showed a two- to four-times higher rate diminished and a number of abnormalities of continuous insulin release have been observed (4,5). In addition to a defect in b-cell function, a reduction in islet and b-cell mass has been observed (6,7). This reduction could be From the 1Laboratory of Pharmacology, Experimental Neurology, Université related to increased programmed cell death (apoptosis), to Libre de Bruxelles (ULB), Faculté de Médecine, Brussels, Belgium; the decreased b-cell replication, or both (8). 2 Institute of Interdisciplinary Research, Institute of Molecular Biology and In a previous work, we observed that overexpression of Medicine (IRIBHM-IBMM), Experimental Neurology, Université Libre de Bruxelles (ULB), Faculté de Médecine, Brussels, Belgium; the 3Experimen- the Na/Ca exchanger (isoform 1: Na-Ca exchanger [NCX1]), 2+ tal Medicine, Experimental Neurology, Université Libre de Bruxelles (ULB), a protein responsible for Ca extrusion from cells (9,10), Faculté de Médecine, Brussels, Belgium; the 4Neurophysiology, Experimen- increased b-cell apoptosis and reduced b-cell proliferation tal Neurology, Université Libre de Bruxelles (ULB), Faculté de Médecine, 5 (11). The increase in apoptosis resulted from endoplasmic Brussels, Belgium; the Experimental Hormonology, Experimental Neurol- 2+ ogy, Université Libre de Bruxelles (ULB), Faculté de Médecine, Brussels, reticulum (ER) Ca depletion with resulting ER stress (11). Belgium; the 6Experimental Neurology, Université Libre de Bruxelles (ULB), If it is possible to increase apoptosis and to decrease Faculté de Médecine, Brussels, Belgium; the 7Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Brussels, Belgium; the 8Laboratory of b-cell proliferation by increasing the activity of NCX1, it Diabetes and Experimental Endocrinology, Institut d’Investigació Biomèd- may be possible to obtain the opposite effects by down- ica de Bellvitge (IDIBELL)–University of Barcelona, Centro de Investigación regulating such a mechanism. To test this hypothesis, we Biomédica en Red (CIBER) de Diabetes y Enfermedades Metabolicas Aso- +/2 9 generated Ncx1 heterozygous deficient mice (Ncx1 ). ciadas (CIBERDERM), Barcelona, Spain; and the Department of Pathology, +/2 Faculty of Medicine, Université Catholique de Louvain, Brussels, Belgium. Our data show that Ncx1 heterozygous inactivation Corresponding author: André Herchuelz, [email protected]. induces several b-cell modifications, including an increase Received 1 July 2010 and accepted 19 April 2011. in glucose-induced insulin release and in b-cell pro- DOI: 10.2337/db10-0924 +/2 This article contains Supplementary Data online at http://diabetes. liferation and mass. Ncx1 islets also displayed an in- diabetesjournals.org/lookup/suppl/doi:10.2337/db10-0924/-/DC1. creased resistance to hypoxia, and when transplanted in E.N. and S.S. contributed equally to this work. diabetic animals, showed a two- to four-times higher rate S.S. is currently affiliated with the University of California–Los Angeles School +/+ of Nursing, Los Angeles, California. of diabetes cure than Ncx1 islets. S.Sc is currently affiliated with Unité de Biochimie, Département des Sciences Fonctionnelles, Faculté de Médecine Vétérinaire, Groupe Interdisciplinaire de Génoprotéomique Appliquée Research, Université de Liège, Liège, RESEARCH DESIGN AND METHODS Belgium. 2/2 Ó 2011 by the American Diabetes Association. Readers may use this article as Generation of Ncx1 mice. Exon 11 of the murine Ncx1 gene (GenBank, long as the work is properly cited, the use is educational and not for profit, accession number AF409089) was cloned from a 129/Sv genomic phage library. and the work is not altered. See http://creativecommons.org/licenses/by The first 206-bp were amplified by PCR and a BamHI site was introduced for -nc-nd/3.0/ for details. cloning. A targeting vector was constructed by inserting the neomycin diabetes.diabetesjournals.org DIABETES 1 Diabetes Publish Ahead of Print, published online June 9, 2011 NCX1 HETEROZYGOUS INACTIVATION IN b-CELLS resistance cassette (neo) into the BamHI restriction site of that amplified Statistics. The results are expressed as means 6 SEM. The statistical sig- exon. Standard procedures were used to generate Ncx1+/2 mice (12). Except nificance of differences between data was assessed by using ANOVA, followed as otherwise stated, experimental mice were 2 to 6 months old, of both sexes, by the Tukey post-test. and had F2 genetic backgrounds from 129/Sv and CD1 mice. Ncx1+/+ mice consisted of age-matched littermates with two wild-type (WT) alleles at the Ncx1 locus (Ncx1+/+). RESULTS Intracellular Ca2+ concentration measurements. The measurement of Generation of Ncx12/2 mice. A mutant allele was con- intracellular Ca2+ concentration using fura-2 was done, as previously de- structed as described in RESEARCH DESIGN AND METHODS (Fig. scribed (11,13), using a camera-based image analysis system (MetaFluor, 1A). After electroporation with the targeting vector, the Universal Imaging, Ypsilanti, MI). fi Time-frequency analysis of spectral densities of the Ca2+ oscillations was recombinant embryonic stem cell clones were identi ed computed using Ackqnowledge Software, (Biopac Systems, Goleta, CA), with by DNA hybridization and used to produce chimeric mice a Hamming window. Data were segmented in successive epochs of 1 min. We (Fig. 1B). Transmission of the mutant allele produced computed the power spectral density (PSD), the integral below the power Ncx1 heterozygous mice that were mated to generate spectrum for the frequency band 0–0.17 Hz, and the crest factor for the same Ncx1 null mutants (Ncx12/2). The Ncx12/2 mice were not frequency band (ratio amplitude of the power spectra/integral 0–0.17 Hz). 45Ca uptake, insulin content, and insulin release from isolated islets. viable and died during embryogenesis as described (28). The methods used to measure 45Ca uptake in intact islets and insulin release Heterozygous and WT mice genotyping from tail biopsy from perifused islets are described in detail elsewhere (11,13–15). Insulin was specimen DNA revealed an Ncx1 WT amplicon of ;250 bp assayed using an ELISA kit (Mercodia, Uppsala, Sweden). For the measure- and an Ncx1 recombinant allele of ;100 bp, which were ment of insulin content, the islets were homogenized in water in the absence simultaneously amplified by PCR (Fig. 1C). NCX1 mRNA of glucose. levels in batches of islets from Ncx1+/+ and Ncx1+/2 mice, Measurement of glucose metabolism and morphology. The methods used +/2 3 14 measured using RT-PCR, showed a clear decrease in Ncx1 to measure D-[5- H]glucose metabolism and D-[U- C]glucose oxidation in in- D +/2 tact islets, have been previously described (16,17). mice (Fig. 1 ). Ncx1 mice were viable, fertile, had Golgi proinsulin/b-cell area was evaluated on slides stained for proinsulin a normal body weight, and showed no macro- or micro- with immunoperoxidase (18) digitized through a JVC KY-F58 color digital scopic abnormalities at the level of the brain, heart, lung, camera (Japan Victor, Brussels, Belgium). Tissues sections (5-mm thick) were liver, spleen, stomach, intestine, and thymus (Supplemen- prepared according to Jacoby et al. (19) and processed for hematoxylin-eosin tary Fig. 1). Likewise, serum levels of glucagon, growth staining. fi hormone (GH) in the fasting state and GLP-1 in the fed For electron microscopy, groups of 30 islets were xed, epoxide-embedded, +/+ +/2 and analyzed as described previously (20). state were comparable in Ncx1 and Ncx1 mice at 12 Measurement of b-cell proliferation, apoptosis, mass, and size.