and at indicated time after insulin injection were determined by the glucometer. To examine diet-induced changes in glucose metabo- lism, 3-month old mice were fed ND or the HFD for 3 months, and GTT and ITT were performed. For measuring corticosterone, blood was collected from mice with food ad libitum, and the concentrations of corticosterone in the plasma was determined using Corticosterone Enzyme Immunoassay Kit (Enzo life science). Lipid staining of hepatic tissues The liver was dissected from 3- and 6-month old mice and fixed in 4% paraformaldehyde solution overnight and placed in a 30% sucrose solution. Fixed hepatic tissues were embedded in OCT compound (Sakura Finetek) on dry ice, sectioned to slices at 9 mm thickness, and stained with Oil Red O. Histological and morphometric analysis of pancreatic tissues Pancreatic tissues from 6-month old mice were fixed in 4% paraformaldehyde solution overnight, embedded in OCT com- pound and sectioned into serial slices at 9 mm thickness. Sections were stained with hematoxylin-eosin (H&E) for morphological evaluation. For immunohistochemical analyses, the following antibodies were used in indicated dilution: rabbit anti-SERT (1:400; Chemicon International, Temecula, CA), guinea pig anti- human insulin (1:400; Abcam), rabbit anti-glucagon (1:500; gift of D. Cai), rat anti-BrdU (1:400; ABcam). Fixed tissues were incubated with primary antibodies overnight in a humid chamber, and labeled by Alexa Fluor conjugated secondary antibodies (Molecular Probe) in 1:800 dilution: Alexa Fluor 488 goat anti- guinea pig (for insulin), Alexa Fluor 594 goat anti-rabbit (for glucagon and SERT), and Alexa Fluor 594 goat anti-rat (for BrdU). Staining patterns were visualized under an AxioImager Z1 microscope equipped with proper filters, and images were captured using Axiocam MR digital camera (Zeiss). For islet morphometric analyses, 4–5 sections (9 mm) separated by 250 mm from serial sections of individual pancreas were analyzed. The islet areas on each section were evaluated. Islet areas with clustered insulin-positive cell .850 mm2, which contains estimated 5 cells (,170 mm2/b-cell) [53], were scored, using the Image-Pro Plus 5 Software (Media Cybernetics). The percentage of beta-cell area was calculated by dividing the value of analyzed insulin-positive areas on one section by the values of the total area of this section. The islets density was calculated by dividing the number of islets on one section by the total area of this section. The values from all analyzed sections of each pancreas were pooled. Beta-cell proliferation was evaluated by 5-bromo-29-deoxyur- idine (BrdU) incorporation. 6-month old mice were injected intraperitoneally with 100 mg/kg/day of BrdU (Sigma-Aldrich) for three days and sacrificed afterwards. Pancreatic tissues were removed, fixed and embedded as described above and stained with insulin and BrdU. Islet cells stained by both BrdU and insulin were scored as BrdU-positive beta cells. For every pancreas, at least 20 islets from 3 sections were counted. The proliferation rate was calculated as the total number of BrdU-positive beta cells over the value of the total area of islets analyzed. Apoptosis analysis was performed on pancreas sections from 6- month old mice by the terminal deoxynucleotidyltransferase- mediated dUTP-biotin nick end labeling (TUNEL) method using the In Situ Cell Death Detection Kit (Roche Applied Science). Immunoblot analysis Hepatic tissues and muscle were isolated from mice at indicated age. The tissues were homogenized in lysis buffer (30 mM HEPES Distinct SERT Function in Feeding and Obesity (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5.0 mM EGTA, 1.0 mM phenylmethylsulfonyl fluoride, 3.0 mM aprotinin, 10 mM leupeptin, 5.0 mM pepstatin A, 25 mM benzamidine, 2 mM sodium vanadate, 5.0 mM glycerol phosphate, 100 mM NaF, 1.0 mM ammonium molybdate and 30 mM tetrasodium pyro- phosphate). Following centrifugation at 14000 rpm for 20 minutes at 4uC, protein extracts were resolved by SDS-PAGE and transferred onto Polyvinylidene fluoride (PVDF) membrane. For IRS1 studies, IRS1 was first immunoprecipitated from liver lysate (2 mg) with anti-IRS1 antibody (Cell signaling Technology), and immunoprecipitates were washed three times with lysis buffer and used for western blotting. The following antibodies were used: anti- b-actin antibody, anti-phospho-Ser473 AKT antibody, anti-Akt antibody, anti-SAPK/JNK antibody, anti-phospho-Thr183/ Tyr185-SAPK/JNK antibody (Cell Signaling Technology), anti- p70 S6 Kinase antibody and anti-p38 MAP Kinase antibody (gifts from C. Chow), anti-phospho-p70 S6Kinase (Thr389) antibody and anti-phospho-Thr180/Tyr182-p38 MAP Kinase antibody (gifts of S. Horwitz), anti-phosphotyrosine antibody and anti-pSer307-IRS1 antibody (Millipore). Protein levels were quantified by densitometry using Image-Pro software (Media Cybernetics). The value of pAKT, pJNK, pS6K, pp38, pY-IRS1 and pSer307-IRS1 was normalized respectively to that of total AKT, JNK, S6K, p38 and IRS1. To measure insulin-induced AKT and IRS1 activation, mice fasted for 16 h were injected with insulin (1 unit/kg body weight), and the liver and muscle were collected 20 min post-injection, and western blot analyses were performed. Statistical analysis Data are presented as mean 6 SEM. Statistical significance was evaluated using 2-tailed, unpaired Student’s t-test. Differences were considered significant at P values of less than 0.05. Supporting Information Figure S1 Effects of SERT deficiency on food intake. A. Daily food intake of 3-month old WT, SERT2/2 and SERT+/2 mice. Amount of food consumed by individually housed mice and the body weight of each mouse were monitored for 20 consecutive days. Data represent the average of daily food intake adjusted for body weight 6 SEM. B. Summary of the entire monitoring period for each genotype. *, p,0.05 Student’s t-test. N, number of mice analyzed. (TIF) Figure S2 Quantification of plasma triglyceride levels in 6-month old mice fasted for 16 h. The differences between SERT mutant mice and WT mice fed ND are not statistically significant (SERT2/2, p = 0.58; SERT+/2, p = 0.66). HFD-fed SERT2/2 mice exhibited lower triglyceride levels than HFD-fed WT mice, *, p,0.05 Student’s t-test. The number of mice analyzed is indicated in parentheses. (TIF) Figure S3 Representative photomicrographs showing TUNEL staining (red) of sections of pancreata to visualize apoptotic cells. There was no appreciable difference detected between WT and SERT2/2 mice. All animals analyzed were 6-month old fed ND. Scale bar, 50 mm. (TIF) Figure S4 Western blot analysis of AKT activity in the muscle of 6-month old WT and SERT-deficient mice. AKT activity was evaluated by the levels of phosphorylation on Ser473 of AKT (pAKT) 6 SEM. Insulin (2), basal AKT activity determined from muscle collected from 16 h fasted mice, and PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e32511