REST Target Genes and Beta Cell Survival Figure 4. Increasing REST levels in beta cells lead to worsened glucose homeostasis. A. Blood glucose levels in 6 month-old wild type (open triangles; n = 8), RIP-REST 5 (gray triangles; n = 8) and RIP-REST males (dark triangles; n = 10) during an IPGTT. After a 14 h fasting, blood samples were taken before (t = 0) and after (t = 15, 30, 60 and 120 min) intraperitoneal injection of glucose (2 g/kg). Results are mean 6 SD. *P,0.05, **P,0.01. B. Insulin content in pancreas of 5 month-old RIP-REST 5 (light gray bar; n = 8), RIP-REST (dark gray bar; n = 6) and diabetic RIP-REST (black bar; n = 6) transgenic mice reveal a 30, 50 and 85% reduction, respectively, when compared with wild type mice (white bars, n = 6). Results are mean 6 SD. **P,0.01, ***P,0.001 versus values of wild type animals. C. Quantitative RT-PCR analysis of human REST mRNA levels in islets of 5 month-old WT (white bar; n = 5), RIP-REST 5 (light gray bar; n = 6) and RIP-REST (dark gray bar; n = 6) mice. REST mRNA levels are six-fold higher in RIP-REST than in RIP-REST 5 animals. Results are mean 6 SD. **P,0.01 versus values of wild type animals. D. REST transgene abundance in islets from P2 RIP-REST 5, RIP-REST and diabetic RIP-REST mice. Upper panel shows representative images of REST protein levels revealed using specific antibodies against REST and AEC staining of peroxydase activity (nuclear black dots). Parallel immunostaining of insulin (red) and the merge shows colocalization of the two signals. Scale bar, 25 mm. Lower panel shows the quantification of the corresponding average pixel intensity per nuclei for each group. *P,0.05 versus the two other groups. doi:10.1371/journal.pone.0045844.g004 expression of several genes coding for known effectors of beta cell survival/proliferation. REST expression does not impact beta cell proliferation at P2, and our observations suggest that the major consequence of high levels of REST expression is a gradual postnatal loss of beta cell mass. In a separate study, using an inducible REST transgene under the control of the Pdx1 promoter, we also observed postnatal beta cell loss leading to hyperglycemia (unpublished data). While the effect of REST expression on INS-1E cells survival was modest in normal conditions, we suggest that the expression of REST target genes is crucial during the postnatal remodeling of primary islet cells [27]. Searching for RE-1-containing genes bearing pro-survival activity, we identified MAPK8IP1 [9,28], GJD2 [29], IRS2 [4] and PTPRN [10]. We further discovered that Cdk5r2 [30] is an additional, hitherto disregarded RE-1-containing gene that is essential for beta cell survival. CDK5R2, like CDK5R1, is a neuron and beta cell-specific activator of the atypical kinase CDK5. This kinase phosphorylates a large number of substrates, involved in a variety of neuronal and non-neuronal functions (reviewed in [31]). Conflicting reports have described the role of PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45844