Survivin Regulation of Beta-Cell Mass Expansion Figure 4. Absence of survivin in beta cells leads to glucose intolerance after PDL. (a) At PDL day 7, female RIPCre+Survivinfl/flmice exhibit markedly higher glucose excursions during an IPGTT compared with other groups (n = 10 per genotype). (b) In vivo GSIS after i.p. glucose injection is markedly compromised in the mutant mice compared with that in other groups (n = 10 per genotype). &, RIPCre+Survivin+/+ sham day 7; %, RIPCre+Survivinfl/fl sham day 7; m, RIPCre+Survivin+/+ PDL day 7; g, RIPCre+Survivinfl/fl PDL day 7. The data represent as means 6 SE. *, p,0.05 vs sham operation. doi:10.1371/journal.pone.0041976.g004 that of control wild-type mice (P,0.05, Fig. 5a). Similarly, the increase in beta-cell number was markedly attenuated in the mutant mice (P,0.05, Fig. 5b). The average beta cell size was significantly reduced in the ligated pancreas tail of the mutant mice after PDL compared with sham group, likely due to beta cell division (P,0.05, Fig. 5c). The beta cell nucleus size remained increased in the pancreas tail of the mutant mice compared with that in controls after PDL (P,0.05, Fig. 5d). Although the increment of total islet number was significantly attenuated in the mutant mice, it was attributed mainly by the lack of increase in the number of larger islets while the increment of smaller islets were similar to that of wild-type mice (P,0.05, Fig. 5e), suggesting that survivin deletion impairs islet growth after PDL, rather than new islet formation. Our previous data have shown that the decreased beta-cell mass in the mutant mice was not accompanied by abnormal islet development [20]. However, at day 7 post PDL, survivin-deficient islets had an abnormal distribution of alpha and beta cells likely due to impaired beta-cell expansion. The proportion of alpha cells to beta cells was markedly increased in the mutant mice (P,0.01, Fig. 6a). However, alpha cell mass was similar in the two groups. Furthermore, there were no differences in genes expression related to islet neogenesis such as Ngn3, NeuroD1, Nkx2.2, Nkx6.1, Pdx1 and MafA between the two genotypes (Fig. 6b), suggesting that survivin deletion in beta cells did not affect islet neogenesis from endocrine progenitor cells after PDL. Cell Proliferation is Defective in Survivin-deficient Betacells after PDL To elucidate which specific cellular processes are affected in beta-cell mass expansion in survivin mutant mice after PDL, we first assessed beta-cell proliferation at PDL day 7. In control wildtype mice, PDL dramatically increased beta-cell proliferation by 9-fold in the ligated tail of pancreas compared with the sham- operated group. However, the induction in beta-cell proliferation in RIPcre+survivinfl/fl mice was significantly blunted, by 4.4-fold (P,0.05,Fig. 7a). This blunting in the increase of beta cell proliferation in response to PDL appears to be mainly accounted by a defect in proliferation of larger islets. In contrast, proliferation of small islet beta cells was better preserved following PDL compared to sham operation. These data further suggest that replication of preexisting beta cells is largely survivin-dependent. To attempt to gain insight into potential cell cycle proteins abnormalities, we first performed quantitative PCR on RNA from day 7 PDL or sham-operated pancreas for genes involved in cell cycle progression. Compared with sham-operated groups, there was a marked increase in the expression of Rb1, Ccne1, Cdk2, Ccna2, Ccnb1 and forkhead box M1 (FoxM1) in wildtype mice after PDL. However, compared with wildtype controls, there was an attenuation in the rise of Rb1, Ccne1, Cdk2, and Ccna2 in response to PDL in the survivin mutant mice. On the other hand, there was a significant further increase in expression of Ccnb1 and FoxM1 in survivin mutant mice (Fig. 7b). The attenuation in the rise of Rb1 gene expression could reflect a compensatory down- regulation in an attempt to enhance G1/S transition in cell cycle [25]. The upregulation of FoxM1 and Ccnb1 gene expression could also be adaptive changes in an attempt to promote cell cycle progression [26]. We further explored the expression of mitosis- related proteins in the islets of ligated pancreas tail. While p21 is mostly known as a cell cycle inhibitor, it can also promote mitosis by forming a complex with Caspase-3 and survivin [27]. Aurora B kinase binds to survivin to form the chromosomal passenger complex. In the absence of survivin, a decrease in p21, Caspase-3 and Aurora B kinase expression islets were observed after PDL, which may lead to defective mitosis (Fig. 7c). Our findings support the hypothesis that survivin is required for beta-cell proliferation in adults after PDL through cell cycle regulation. Moreover, p-Akt was significantly decreased in islets of mutant mice (Fig. 7c), indicating that survivin may also regulate the phosphatidylinositol 3-kinase-Akt signaling pathway which plays an important role in beta-cell proliferation. We also examined beta-cell apoptosis in the ligated tail of pancreas at PDL day 7. The rate of beta-cell apoptosis was low and not significantly different between the mutant mice and control wild-type mice after PDL or sham operation (Fig.7d). Furthermore, we did not detect any significant changes in cleaved Caspase-3 in the islets of mutant mice compared with controls (Fig. 7d), indicating that apoptosis did not significantly contribute to the defect in islet expansion in survivin-deficient beta cells after PDL. Discussion Survivin is highly expressed within pancreatic islets during embryogenesis and neonatal period, however eventually becomes undetectable by 2-month of age [20,21]. We previously showed that mice with survivin deleted throughout the pancreatic epithelium exhibit impaired beta-cell mass establishment and PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e41976