Distinct SERT Function in Feeding and Obesity Figure 4. Biochemical, histological and immunohistochemical analyses of insulin secretion and pancreatic beta-cell morphology in SERT-deficient mice. A and B. Quantification of basal levels of plasma insulin in 16 h fasted mice at the age of 3 months (A) and 6 months (B). SERT mutant mice exhibited elevated fasting insulin levels during ND and HFD feeding as compared to corresponding WT control mice, *p,0.05, **p,0.01, ***p,0.001, Student’s t-test. The number of mice analyzed is indicated in parentheses. C. Quantification of glucose-induced plasma insulin levels. 6-month old mice fasted for 16 h were injected with glucose (i.p., 1 g/kg). Insulin levels in plasma samples collected before and 15 and 30 min post glucose administration were analyzed. SERT2/2 and SERT+/2 mice exhibited higher insulin levels at all tested time points, compared to corresponding WT mice. Data represent mean 6 SEM. *p,0.05, **p,0.01, ***p,0.001, Student’s t-test. N, number of animal analyzed. D. Immunofluorescent staining of pancreata of WT mice. The left panel shows insulin (green), the middle panel shows SERT (red) co-stained with DAPI (blue), and the right panel shows merge of the fluorescence. Noticing SERT staining in both insulin-expressing cells (as pointed by a triangle) and non-insulin cells (as pointed by an arrow) in the islets. Scale bar, 50 mm. E and F. H&E staining of pancreatic tissue sections from WT and SERT2/2 mice. Noticing enlarged islet mass in the SERT2/2 background. Scale bar, 100 mm. G and H. Triple-staining of pancreas of WT and SERT2/2 mice for insulin (green), glucagon (red) and DAPI (blue). Scale bar, 200 mm. I and J. Quantification of islet mass in WT and SERT2/2 mice. I. Beta-cell density was determined by calculating the percentage of insulin-labeled area on serial sections of pancreata. J. Islet density was determined by calculating the average number of islets on the pancreatic sections. The value from SERT2/2 mice was compared with that of WT mice, *p,0.05, **p,0.01, Student’s t-test. The number of mice analyzed is indicated in parentheses. K and L. Representative photomicrographs showing BrdU incorporation (red) in insulin-labeled cells (green) in pancreatic tissue sections of WT and SERT2/2 mice. Scale bar, 50 mm. M. Beta-cell proliferation was estimated by calculating the number of beta cells incorporated BrdU relative to the islet area. Data represent mean 6 SEM, **P,0.01, Student’s t-test. The number of animals analyzes is indicated in parentheses. doi:10.1371/journal.pone.0032511.g004 Hyperinsulinemia frequently reflects an adaptive pancreatic response to increased metabolic demand. In the state of chronically increased nutrient load, obesity, and insulin resistance, pancreatic beta cells increase insulin secretion by expanding beta- cell mass [28]. Indeed, following three months of HFD feeding, fasting plasma insulin levels were increased in WT as well as in PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32511