Sox9-Haploinsufficiency Causes Glucose Intolerance Figure 4. Sox9 regulates Pdx1 mRNA and protein expression. Quantitative RT-PCR of pancreata from Sox9flox/fox or Sox9+/fox embryos injected with tamoxifen at e12.5 and analyzed at e15.5 (Sox9De13/De13 or Sox9+/De13) (A; n = 9) shows reduced levels of Pdx1 and Ngn3, but not of Hnf6, FoxA2, or Tcf2. In Sox9+/Dpan pancreata after Pdx1-Cre-mediated recombination of one Sox9-flox allele (B; n = 9), Pdx1, Ngn3, Hnf6, and FoxA2 mRNA levels are reduced at e15.5. Western Blot analysis (C, D; n = 4) shows diminished Pdx1 expression, but not Hnf6, Foxa2, or Tcf2 expression in pancreata from Sox9De13/De13 embryos at e15.5. Values are shown as mean 6 S.E.M.; * = P,0.05; ** = P,0.01. doi:10.1371/journal.pone.0023131.g004 Pancreatic Sox9 haploinsufficiency results in glucose intolerance Previous studies demonstrating that haploinsufficiency for regulators of beta-cell development can manifest in MODY-type diabetes [19,27] led us to next examine whether Sox9+/Dpan mice display a diabetic phenotype in adulthood. To ascertain the physiological effects of pancreatic Sox9-haploinsufficiency in adulthood, we monitored the body weight and blood glucose levels of ad libitum-fed Sox9+/Dpan and Cre2 control mice over a 60- week period. We found that, over the period of study, Sox9+/Dpan mice showed no difference in body weight (Fig. 5A), nor random- fed blood glucose levels (Fig. 5B) compared to littermate controls. However, while there was also no significant difference between fasted blood glucose levels of Sox9+/Dpan mice and control siblings, Sox9+/Dpan mice tended to exhibit higher blood glucose levels which in time progressed to diabetic levels (.7 mmol/l in the fasted state, as indicated by the broken line; Fig. 5C). Consistent with developing glucose intolerance, by six weeks of age, blood glucose levels were significantly elevated in Sox9+/Dpan mice compared with control littermates following an acute glucose challenge (Fig. 5D). Glucose intolerance persisted in Sox9+/Dpan mice at 12 weeks of age and later time points (Fig. 5E,F). In order to confirm that the glucose intolerance observed in Pdx1- Cre; Sox9+/flox versus Sox9+/flox control mice was not attributable to extraneous effects of the Pdx1-Cre transgene as reported in the RIP-Cre mouse line [28], we assayed glucose tolerance in Pdx1-Cre versus wild- type siblings on a Sox9+/+ background. The absence of any difference in blood glucose levels between the two genotypes in response to a glucose challenge (Fig. 5G) shows that the Pdx1-Cre transgene itself exerts no influence on glucose tolerance in the current studies. Thus, pancreatic Sox9-haploinsufficiency results in glucose intolerance. High-fat diet causes fasting hyperglycemia in Sox9-haploinsufficient mice Though the endocrine function of the reduced beta-cell complement in Sox9+/Dpan pancreata was sufficiently high to maintain normogycemia under basal conditions, we postulated that Sox9-haploinsufficiency might impair the ability to withstand additional metabolic stress. To test this, we maintained Sox9+/Dpan and littermate control mice on a 60% high-fat diet and monitored body weight and ad libitum fed blood glucose levels. Throughout the course of one year on this diet, no significant difference was detected in either body weight or blood glucose levels between Sox9-haploinsufficient and control siblings (Fig. 6A,B). However, fasting blood glucose levels in Sox9+/Dpan mice were significantly elevated over those of control animals, attaining levels considered diabetic (Fig. 6C). Concordantly, when high-fat diet-fed mice were subjected to intraperitoneal glucose challenge, Sox9+/Dpan mice displayed significantly elevated blood glucose levels over those of control littermates (Fig. 6D), mirroring the findings in normal diet-fed Sox9+/Dpan mice. Thus, the additional metabolic demand/stress of a high-fat diet mildly exacerbated the metabolic defect resulting from pancreatic Sox9-haploinsufficiency. Compensatory postnatal beta-cell proliferation in Sox9-haploinsufficient mice Given that heterozygous pancreatic deletion of Sox9 results in a two-fold decrease in beta-cell mass at birth [14], we were surprised PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23131