Role of Wdr13 Gene in Beta Cell Proliferation Figure 4. Histology of pancreatic islet, pancreatic beta cell proliferation, in-vivo and in-vitro insulin secretion in Wdr13 knockout mice. A) Islet morphology showing increased islet mass by H&E staining. B) Islet mass in mg at 6 months on high fat diet showing increased total islet mass in Wdr13 knockout mice (n = 4). C) Percent of dividing cells in islets by BrdU labeling (n = 309–517). D) Analysis of pancreatic beta cells proliferation analysis by BrdU labeling showing more proliferating beta cells in Wdr13 knockout islets. E) In-vivo insulin secretion in response to glucose at 6 months (n = 4 to 6). F) In-vitro insulin secretion from isolated pancreatic islet of the wild type and Wdr13 knockout mice at 2.8 mM or 16 mM glucose concentration (+/0 wild type male; 2/0 knockout male). doi:10.1371/journal.pone.0038685.g004 rule out the possibility that the origin of obesity in our mutant mice reflected the deficiency of this protein in any one or all of these tissues. In this context, it may be noted that the food intake in Wdr13 knockout mice was marginally higher than that in wild type littermates (Figure S1); the difference was statistically non- significant. Although our in-vitro data in MIN6 cells as well as hyperinsulinemia and better glucose clearance in Wdr13 knockout mice show functional significance of WDR13 protein in pancreatic beta cells, a tissue-specific knockout of Wdr13 gene will be necessary to confirm that the gross changes observed by us in the mutant mice are direct consequences of alterations in beta cell function in these mice. We have earlier reported relatively higher level of expression of Wdr13 in pancreas [3], and the expression of this gene in the pancreatic islets is much more as compared to extremely low levels seen in the acinar cells (Figure 1D). Wdr13 knockout mice had more pancreatic islet mass than their littermates when fed on a high fat diet for five months after weaning at one month. To understand the phenomenon of the increased islet mass, we assayed in vivo beta cell proliferation after one-month old mice were fed on a high fat diet for three weeks. Wdr13 knockout mice had enhanced beta cell proliferation as compared to that in their wild type littermates (Figure 4C, D). We have not analyzed the proliferation rates of other cell types in the pancreatic islets. Given the fact that beta cells contribute to 80–85% mass of pancreatic islets, it would appear that the increase in islet mass in Wdr13 knockout mice might be primarily due to the enhanced beta cell proliferation. This conclusion is further strengthened by reversal of the cell proliferation phenotype i.e. the growth retardation observed by us in the pancreatic MIN6 cells upon overexpression of Wdr13 gene (Figure 5A). The lack of difference between in vitro insulin secretion capacities of the wild type and knockout pancreatic islets (Figure 4F) provides further indirect support to the above conclusion. Various extrinsic and intrinsic factors PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38685