Loss of Connexin36 Channels Alters -Cell Coupling, Islet Synchronization of Glucose-Induced Ca 2 and Insulin Oscillations, and Basal Insulin Release Magalie A. Ravier, 1 Martin Gu ¨ ldenagel, 2 Anne Charollais, 3 Asllan Gjinovci, 3 Dorothe ´ e Caille, 3 Goran So ¨ hl, 2 Claes B. Wollheim, 3 Klaus Willecke, 2 Jean-Claude Henquin, 1 and Paolo Meda 3 Normal insulin secretion requires the coordinated func- tioning of -cells within pancreatic islets. This coordi- nation depends on a communications network that involves the interaction of -cells with extracellular signals and neighboring cells. In particular, adjacent -cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozy- gous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36 / ) featured -cells devoid of gap junc- tions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36 / mice did not display the regular oscillations of intracel- lular calcium concentrations ([Ca 2 ] i ) seen in controls due to the loss of cell-to-cell synchronization of [Ca 2 ] i changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lack- ing Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is es- sential for the proper functioning of -cells, particularly for the pulsatility of [Ca 2 ] i and insulin secretion during glucose stimulation. Diabetes 54:1798 –1807, 2005 N ormal insulin secretion requires the coordi- nated functioning of -cells within pancreatic islets. This coordination is achieved through extracellular signals and ligands of the connec- tive matrix as well as through the interaction of -cells and their neighboring islet cells. Thus, cell interactions medi- ated by surface receptors and cell adhesion and junctional molecules have been implicated in the regulation of -cell functions (1–3). Specifically, -cells are interconnected by gap junctions, in which connexin channels linking adja- cent cells are concentrated (3). The interaction of these channels across the extracellular space allows direct exchanges of low-molecular weight cytoplasmic mole- cules and ensures electrical coupling. Previous studies have implicated connexins and/or the cell-to-cell ex- changes (3) and electrical coupling (4,5) that these pro- teins permit in the control of -cell function. However, because of the uncertainty about the connexin species that functionally connects native -cells (6,7), the in vivo relevance of the connexin-dependent signaling and the nature of the mechanism linking connexin expression to -cell function has not been resolved. We recently showed that connexin36 (Cx36) is ex- pressed by primary -cells (6,7) and insulin-producing cell lines that retain some ability to increase insulin secretion during glucose stimulation (6,8). We further documented that, at variance with most other cell types that coexpress several connexin species (3), -cells could not be shown to be linked by connexins other than Cx36 (7). These observations have opened the way to a direct experimen- tal test of the function of Cx36. Thus, alterations in the Cx36 content of insulin-producing cells affect their secre- tion, with both decreases and increases in the amount of Cx36 resulting in an inhibited secretory response to glu- cose (8 –11). However, in view of the limitations of these in vitro approaches, the many differences among cell lines and primary -cells and the multiple signaling pathways that converge to regulate the function of native pancreatic islets (1–3), the contribution of Cx36 to the in vivo function of the endocrine pancreas remains to be demon- strated. Here we have addressed this issue by studying mutant mice that do not express Cx36 (after targeted deletion of the cognate gene) (12) and feature deficits in the neural and retinal networks of cells that natively express this connexin isoform (12–15). RESEARCH DESIGN AND METHODS Transgenic mice. Cx36-deficient (12) and C57BL/6 mice were crossed to obtain heterozygous offspring. Progenies were intercrossed to obtain Cx36 -/- , Cx36 +/- , and Cx36 +/+ littermates. Genotyping was done by PCR analysis and Southern blot hybridization (12). Male and female mice age 3– 6 months and with an 87.5 (F2) to 97% (F4) C57BL/6 background were used for most experiments. For the intracellular calcium concentration ([Ca 2+ ] i ) and insulin- From the 1 Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, Brussels, Belgium; the 2 Institute of Genetics, University of Bonn, Bonn, Germany; and the 3 Department of Cell Physiology and Metabolism, Geneva University Medical Center, Geneva, Switzerland. Address correspondence and reprint requests to Paolo Meda, MD, Depart- ment of Cell Physiology and Metabolism, University of Geneva, C.M.U., 1 rue Michel Servet, 1211 Geneva 4, Switzerland. E-mail: paolo.meda@medecine. unige.ch. Jean-Claude Henquin, Unit of Endocrinology and Metabolism, Univer- sity of Louvain Faculty of Medicine, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium. E-mail: henquin@endo.ucl.ac.be. Received for publication 8 December 2004 and accepted in revised form 11 March 2005. M.A.R., M.G., and A.C. contributed equally to this work. [Ca 2+ ] i , intracellular calcium concentration; Cx36, connexin36; LY, Lucifer yellow. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1798 DIABETES, VOL. 54, JUNE 2005