Sox9-Haploinsufficiency Causes Glucose Intolerance Figure 6. High-fat diet induces fasting hyperglycemia in Sox9-haploinsufficient mice. Sox9+/Dpan mice and control littermates were fed a 60% high-fat diet over 50 weeks. Body weight (A) and random fed blood glucose levels (B) do not significantly differ in Sox9+/Dpan and control mice (n = 14). (C) Fasting blood glucose levels are significantly higher in Sox9+/Dpan than control mice (n = 14). The dashed line indicates the threshold at which blood glucose levels are considered diabetic. (D) Following an intraperitoneal glucose challenge, Sox9+/Dpan mice exhibit elevated blood glucose levels compared to control mice (n = 14). Values are shown as mean 6 S.E.M.; * = P,0.05. doi:10.1371/journal.pone.0023131.g006 to find that glucose homeostasis in these mice was only mildly impaired. Therefore, we sought to characterize how Sox9- haploinsufficient mice lacking half the usual complement of beta-cells are able to remain relatively normoglycemic, even with age and under increased metabolic stress. Immunofluorescence analysis revealed that at six weeks of age, while smaller than those of their control siblings, islets of Sox9+/Dpan mice displayed normal cytoarchitecture, with a central core of insulin+ beta-cells surrounded by a mantle composed primarily of glucagon+ alpha-cells (Fig. 7A). Concordantly, islets isolated from Sox9-haploinsufficient mice exhibited normal insulin secretion when stimulated with glucose in vitro (Fig. 7B). We have previously reported that at e18.5, beta-cell mass in Sox9+/Dpan mice was 50% reduced compared to control mice [14]. However, by six weeks of age, beta-cell mass in Sox9 heterozygous mutant mice had risen to 68% that of control littermates, an increase that was maintained at 16 weeks of age (Fig. 7C). Consistent with our morphometric data, total pancreatic insulin content in six-week-old Sox9+/Dpan mice was 70% that in control animals (Fig. 7D). Fasting serum insulin levels in Sox9-haploinsufficient mice were not significantly different from those of controls (Fig. 7E). The increase in beta-cell mass from 50% to 68% of control mice between e18.5 and six weeks of age suggested that beta-cells of Sox9+/Dpan mice were capable of adaptive expansion to maintain normoglycemia. This inference was also supported by the fact that these mice did not become overtly diabetic even when metabol- ically stressed via maintenance on a high-fat diet. To determine how and when this increase in beta-cell mass occurred, we measured the proliferation rate of beta-cells in Sox9+/Dpan mice by assaying for BrdU incorporation at the age of two weeks, which is a dynamic period of islet remodeling and growth in rodents [29,30]. This analysis revealed that the beta-cell BrdU labeling index in Sox9+/Dpan mice was twice that in littermate controls, although by six weeks of age, proliferation rates returned to baseline levels (Fig. 7F,G). This is consistent with there being no further adaptive increase in beta-cell mass in Sox9-haploinsufficient mice beyond six weeks (Fig. 7C). Together, our data suggest that while Sox9-haploinsufficiency results in a reduced beta-cell complement, adaptive expansion of the beta-cell mass occurs through proliferation to partially restore glucose homeostasis in Sox9+/Dpan mice. Discussion Sox9 as a critical regulator of the endocrine differentiation program As we reported previously [14], Sox9-haploinsufficiency leads to a 50% decrease in the numbers of both Ngn3+ progenitors and the mature endocrine cells they give rise to. Studies by Lynn et al. [15] in the pancreatic ductal mPAC cell line suggested that in addition PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23131