with INS-1E cells transduced with REST-expressing adenoviral vectors led to the identification of several new REST target genes, with previous identified role in cell survival, including Irs2, Ptprn, and CDK5 activator subunit 2 (Cdk5r2, also called p39). siRNA- specific down-regulation of Cdk5r2 increased the susceptibility of INS-1E cells to major beta cell death effectors cytokines and palmitate, indicating that this activator of CDK5 has an anti- apoptotic activity in beta cells. Results Diabetic RIP-REST mice feature hyperglycemia and altered insulin secretion We previously reported the characterization of a glucose- intolerant mouse line (referred to as RIP-REST) featuring defects in insulin secretion as well as decreased insulin production, without detectable transgene expression in hypothalamus [13]. Mice from a novel line (referred to as diabetic RIP-REST) showed frank diabetes independently of the gender. These mice featured a glycemia of 23.662.6 mmol/l at 4 weeks of age, which increased at 3 month-old up to 33 mmol/l, resulting in lethality after few months. Wild type littermates had a basal glucose level of 9.261 mmol/l (Fig. 1A). To assess insulin secretion, the pancreas of 4–5 month-old transgenic and wild type animals were perfused in situ. Infusion of 8.0 and 16.0 mmol/l glucose led to a typical biphasic response of insulin secretion in control mice. In contrast, the secretion of the hormone in response to glucose was barely detectable in the diabetic animals (Fig. 1B). The addition of 1 nmol/l GLIP-1 to 8.0 mmol/l glucose largely potentiated insulin release from wild type mice, and to a much lesser extent also in diabetic RIP-REST mice (Fig. 1B). Altered insulin secretion of diabetic mice results from massive loss of beta cells Immunofluorescent labeling of glucagon and insulin in pancreas of 6 month-old mice showed that wild type islets featured, as expected, numerous central beta cells and lower number of alpha cells at the periphery (Fig. 2A left panel). In contrast, those of diabetic RIP-REST mice had much fewer beta cells, and displayed glucagon-positive cells scattered throughout the islets (Fig. 2A middle panel). Strikingly, only a small fraction of the surviving beta cells expressed the REST transgene (Fig. 2A right panel). When the same staining was performed on pancreas of 7 day-old mice (P7), a significant number of insulin positive cells was observed in the diabetic mice (Fig. 2B middle panel), of which most expressed the REST transgene (Fig. 2B right panel). However, and in contrast to controls (Fig. 2B left panel), alpha cells were already distributed throughout the islets of diabetic mice (Fig. 2B middle panel). To further characterize the time course of the beta cell loss occurring in diabetic mice, we quantified beta- and alpha-cell mass in P2, P7 and adult animals. Compared to wild type mice, we observed a gradual decrease in the beta cell fraction, which was 30 (p,0.05), 45 (p,0.05), and 90% (p,0.001) in P2, P7 and adult diabetic mice, respectively. In contrast, the alpha-cell mass was statistically similar to that of controls at all ages (Fig. 2C). Confocal microscopy also revealed the infrequent occurrence of double insulin- and glucagon-positive cells in adult diabetic RIP-REST mice, which was not observed in wild type mice (Fig. 2D). REST expression is associated with beta cell loss due to apoptosis To investigate the postnatal beta cell loss in diabetic RIP-REST mice, we first examined beta cell proliferation by PCNA REST Target Genes and Beta Cell Survival immunostaining in P2 pancreas. The same proportion of beta cells was labeled for this marker of cell proliferation in wild type (4.9%61.1; n = 1598), and transgenic mice (6.2%60.26; n = 833) (Fig. 3A). We next assessed beta cell apoptosis using TUNEL experiments. Whereas no apoptotic beta cell was observed in wild type mice, we detected several in islets of diabetic RIP-REST mice at P2 (Fig. 3B). We did not quantify the proportion of apoptosis, in vivo, because apoptotic cells are rapidly cleared by the immune system [17,18]. However, we assessed apoptosis in INS-1E cells transduced with REST- and GFP-expressing adenoviruses and observed that REST expression per se led to an increase in cell death under normal conditions and in a dose-dependent manner (Fig. 3C). The susceptibility of these cells to undergo apoptosis was also increased when cell death was triggered by cytokines (Fig. 3C). Together with the quantifications made in Fig. 2C, these observations suggest that diabetes of transgenic mice was accounted for by postnatal apoptosis of beta cells. The levels of REST expression inversely correlate with pancreatic insulin content in RIP-REST transgenic mice We analyzed the mice of a third transgenic line (referred to as RIP-REST 5), which was obtained after the pronuclear injection of the RIP-REST transgene. As the other RIP-REST mice, these mice also featured glucose intolerance, as observed by IPGTT (Fig. 4A). Measurement of pancreatic insulin content in each three transgenic line indicated that RIP-REST 5 mice featured a mild 30% decrease in insulin content, whereas RIP-REST mice had a 50% decrease, and diabetic RIP-REST mice a drastic 85% reduction, as compared to wild type mice (Fig. 4B). qPCR analysis of islet mRNA indicated that the expression of the REST transgene was six-fold higher in RIP-REST than in RIP-REST 5 mice (Fig. 4C). Diabetic RIP-REST mice could not be investigated by qPCR because of the low number of residual islets. Therefore, REST protein abundance in the three mouse lines was evaluated by semi-quantitative peroxydase REST immunostaining (Fig. 4D upper panel). After image processing, quantification of the average pixel intensity per nuclei revealed that REST abundance was significantly higher in beta cells from diabetic RIP-REST than from RIP-REST mice, itself being higher than in beta cells from RIP-REST 5 mice (Fig. 4D lower panel). These data demonstrate an inverse relationship between the levels of REST expression and insulin production, and suggest that high levels of REST in beta cells were sufficient to induce diabetes in transgenic mice. RE-1 database screening identifies several RE-1- containing genes involved in beta cell survival We screened a RE-1 database (http://www.bioinformatics. leeds.ac.uk/cgi-bin/RE1db/nrse.cgi) [19] to search for RE-1- containing genes thought to be involved in neuroendocrine-cell protection. We searched genes with anti-apoptotic properties and a conserved RE-1 sequence. We identified 14 RE-1-containing genes that may be involved in growth signals transduction pathways (including members of the insulin-like growth factors pathway, AKT1, and catenin isoforms), in transduction of apoptosis/proliferation (including mitogen-activated protein ki- nases) and in downstream mechanisms of mitogenesis (including cyclins, cyclin-dependent kinase activators) (Fig. 5A). We then used qPCR analysis to evaluate the expression of the corresponding mRNAs upon REST expression in INS-1E cells. These experiments showed that the majority of the selected candidates were not transcriptionally regulated by REST (data not shown). However, the expression of the genes coding for IRS2, PTPRN, CDK5R2, neuregulin1 (NRG1), delta2 catenin PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45844