mechanism enhancing leptin secretion. Both SERT deficiency and leptin have been shown to inhibit bone mass accrual [42,43]. Consequently, it is plausible that elevated circulatory leptin underscores the effects of SERT deficiency not only on feeding, but also on bone mass and perhaps other aspects of hypothalamic function. One interesting finding uncovered from our study is that SERT- deficient mice develop glucose intolerance, insulin resistance and obesity, despite reduced feeding. SERT deficiency has been associated with anxiety [18] and glucocorticoids play an important role in insulin sensitivity [44]. However, basal plasma corticoste- rone was not significantly altered in SERT-deficient mice (Figure S5), consistent with a previous report [45]. It has been reported that SERT2/2 mice were hypoactive during exploratory and social behavioral tests [46], although another study indicated that SERT2/2 rats did not reduce total distance traveled during behavioral assays [47]. Thus, hypoactivity could be a contributing factor to obesity of SERT-deficient mice. However, our studies of 3- and 6-month old SERT-deficient mice suggest that glucose intolerance and insulin resistance manifest prior to an appreciable increase in the adiposity. Previously, it was reported that in C. elegans 5-HT regulates feeding and fat metabolism through independent molecular mechanisms [48]. Thus, 5-HT is likely to regulate distinct targets to control the perception of satiety and energy metabolism of animals, and the dual action of 5-HT may represent an evolutionary ancient strategy for metabolic homeo- stasis conserved in disparate organisms. The impact of SERT deficiency on insulin signaling in the peripheral tissues Several lines of experimental results suggest that SERT- deficiency impairs insulin action on peripheral tissues. First, basal pAKT and pY-IRS1 levels were elevated, while insulin-induced increase of pAKT and pY-IRS1 was attenuated in the liver of SERT-deficient mice. Increased basal pAKT is considered as a trigger of insulin resistance by desensitizing insulin signaling [32]. Second, PTEN+/2 mutation enhanced AKT signaling in SERT- deficient mice and improved their glucose tolerance. Third, SERT2/2 mice constitutively increase JNK activity in the liver, coupled with enhanced the phosphorylation at the inhibitory sites of IRS1, a key regulator of IR signaling. It is interesting to note that the metabolic deficits in SERT-deficient mice, including glucose intolerance, insulin resistance, and increased JNK activity, all arise prior to a measurable increase in the adiposity. These data suggest that the metabolic deficits of SERT-deficient mice are not the consequence of obesity. Further studies are required to determine the mechanisms by which SERT function control glucose homeostasis and insulin sensitivity. Reduced SERT function has been implicated in anxiety-related disorders [18]. Low expression alleles of the human SERT gene are linked to post-traumatic syndrome and anxiety-related traits [49,50]. SERT+/2 mice exhibit elevated anxiety behavior [51]. We found that SERT+/2 mice develop significant metabolic deficits, showing that a partial reduction in SERT function may also perturb metabolic homeostasis and confer a risk factor for diabetes. Materials and Methods Animals All mice used in the experiments were bred on a C57BL/6 background and housed in facilities accredited by the American Association for Laboratory Animal Care (AALAC #30549) on a 12-h light/dark cycle with ad libitum access to normal diet Distinct SERT Function in Feeding and Obesity (D5053; Research Diets) or a high-fat diet (D12492; Research Diets). The C57BL/6J strain (Jackson Laboratory) was used as a WT. Targeted deletion of the SERT gene Slc6a42/2 (SERT2/2) has been described previously [20]. PTEN+/2 mice [52] were obtained from NCI. SERT2/2; PTEN+/2 and SERT+/2; PTEN+/2 double mutants were generated from this work. Mice were genotyped by PCR analyses of genomic DNA extracted from tail tissue. All studies were performed using male mice at the age of 3 and 6 months. The animal studies were approved by the Institute Animal Care and Use Committee (IACUC #20090508) of the Albert Einstein College of Medicine. SERT mRNA profiling Total RNA was extracted from tissues of 6-month old WT mice using TRIZOL reagents (Invitrogen). 2 mg of total RNA from each tissue were used for reverse transcription with the Superscript III reverse transcriptase (Invitrogen), and the resultant cDNA was amplified by PCR with the following primers: SERT: 59- CGCAGTTCCCAGTACAAGC-39 and 59-CGTGAAGGAG- GAGATGAGGT-39; GAPDH: 59-GCCTTCCGTGTTCCTA- CCC-39 and 59-TGAAGTCGCAGGAGACAACC-39. Food intake and body composition measurements To evaluate food intake, food consumption by individually housed mouse was recorded daily for a period of one week or 20 days. Daily HFD intake was monitored using 6-month old mice that had fed on the HFD for 10 weeks. Briefly, a measured amount of ND or the NFD was provided daily to individually housed mouse, and leftover and spillover were collected and weighed on the following day, and the difference was calculated as daily net food intake of the mouse. While potential error attributable to loss of food particles over spillover cannot be excluded, the bedding was carefully screened to minimize potential error. Body weight of test mice was recorded every day, and the amount of food consumed by each mouse adjusted for its bodyweight was calculated as daily food intake. Data represent the average of daily food intake of mice analyzed in parallel for each genotype. Body composition of mice at indicated age was determined by nuclear magnetic resonance using an EchoMRI analyzer (Echo Medical Systems). Metabolic measurements Metabolic parameters were measured in 3- and 6-month-old mice fasted for 16 h, unless specified otherwise. Glucose levels in the blood samples obtained by tail bleeding were measured using a glucometer (Bayer HealthCare). For measuring triglycerides, leptin and insulin levels, blood was collected from the tail vein and the plasma was isolated by centrifugation of the blood samples. Total plasma triglycerides were determined using the Triglyceride Determination Kit (Sigma-Aldrich). Plasma insulin and leptin levels were determined using the Rat/Mouse Insulin ELISA Kit (Millipore) and Mouse Leptin ELISA Kit (Crystal Chem), respectively. To measure insulin secretion in response to glucose, mice fasted for 16 h were injected with D-glucose (1 g/kg body weight), and blood was collected from the tail vein prior to the injection and at the indicated time points post injection, and the plasma insulin levels were determined. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were carried out using 3- and 6-month old mice. For GTT, 16 h fasted mice were injected intraperitoneally (i.p.) with D-glucose (1 g/kg body weight), and the blood glucose level was determined by tail bleeding immediately before and at indicated time points after glucose injection using the glucometer. ITT was performed on 6 h fasted mice by intraperitoneal injection of insulin (1 unit/kg body weight; Sigma-Aldrich), and the blood glucose concentrations before PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e32511