Prolonged culture in low glucose induces apoptosis of rat pancreatic b-cells through induction of c-myc Mark Van de Casteele, a, * Benjamin Ate Kefas, a Ying Cai, a Harry Heimberg, a Donald K. Scott, b Jean-Claude Henquin, c Danie ¨l Pipeleers, a and Jean-Christophe Jonas c a Diabetes Research Center, Brussels Free University VUB, Brussels, Belgium, Partner of the Juvenile Diabetes Research Center for Beta Cell Therapy in Europe, Laarbeeklaan 103, B-1090 Brussels, Belgium b Department of Biochemistry, LSU Health Sciences Center, New Orleans, LA 70112, USA c Unit of Endocrinology and Metabolism, University of Louvain, UCL 55.30, Avenue Hippocrate, 55, B-1200 Brussels, Belgium Received 9 October 2003 Abstract Prolonged culture in low-glucose concentrations ( 65 mM) induces apoptosis in pancreatic b-cells by a poorly defined mechanism. We now show that, in both purified rat b-cells and isolated rat islets, culture in the presence of 3 or 5 mM (G3–G5) instead of 10 mM glucose (G10) induces a large increase in c-myc expression before onset of a caspase-dependent apoptosis. These effects were pre- vented by addition of leucine and glutamine to G3 and G5, and were mimicked by addition of the mitochondrial poison azide to G10. In contrast, inhibition of Ca 2þ influx and insulin secretion with diazoxide under control conditions did not stimulate islet c-myc expression nor b-cell apoptosis. In rat b-cells, adenovirus-mediated c-myc overexpression increased their rate of apoptosis, whereas antisense-c-myc expression reduced low-glucose-induced apoptosis by 50%. In the insulin producing MIN6 cell line, apoptosis induction by either low glucose or an activator of AMP-activated protein kinase (AMPK) was associated with c-myc mRNA and protein upregulation. In conclusion, stimulation of b-cell apoptosis by prolonged culture at low glucose partly results from early and sustained induction of b-cell c-myc expression. These effects may be due to sustained restriction in nutrient-derived metabolic signals. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Pancreatic islet; Gene expression; MIN6; Metabolism; Cell death; c-Myc antisense Stimulation of pancreatic b-cells with glucose induces an initial rise in b-cell energetic metabolism, which leads to the closure of ATP-sensitive K þ channels, plasma membrane depolarization, Ca 2þ influx, and a rise in the cytosolic Ca 2þ concentration that triggers insulin secre- tion [1]. Besides these acute effects on b-cell function, glucose also chronically exerts pleiotropic effects in pancreatic b-cells. Previous studies have indicated that the viability of cultured b-cells depends on the prevailing glucose concentration, with an optimal glucose con- centration for rodent b-cell survival of 10 mM [2,3]. Culture of b-cells at suboptimal glucose concentrations (<10 mM) triggers their apoptosis, suggesting that glu- cose suppresses an apoptosis program in these cells [4]. However, the mechanism leading to apoptosis induction under glucose limitation remains undefined. Glucose deprivation has been associated with increased c-myc expression in other cell systems [5,6]. c-Myc is a transcription factor of the basic helix-loop- helix-leucine zipper (bHLH-LZ) family that can induce both proliferation and apoptosis in a variety of cell types [7]. In pancreatic b-cells, chronic hyperglycemia or cul- ture in the presence of 20–30 mM glucose induces c-myc expression in a Ca 2þ -dependent manner [8,9]. Further- more, b-cell-targeted c-myc overexpression decreases insulin gene expression and stimulates b-cell apoptosis more than proliferation, leading to net b-cell loss and development of diabetes [10–12]. The aim of this study was to investigate whether deregulated expression of c-myc could contribute to b-cell apoptosis induced by culture at low glucose concentrations. * Corresponding author. Fax: +32-2-477-45-45. E-mail address: mvdcaste@vub.ac.be (M. Van de Casteele). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.013 Biochemical and Biophysical Research Communications 312 (2003) 937–944 BBRC www.elsevier.com/locate/ybbrc