Distinct SERT Function in Feeding and Obesity Figure 7. Western blot analysis of insulin signaling component activity in SERT2/2;PTEN+/2 mice. A–F. AKT activity in the liver of 3- and 6-month old SERT2/2; PTEN+/2 and SERT2/2 mice was evaluated by AKT Ser473 phosphorylation (pAKT) before and after insulin injection. G– J. IRS1 activities in the liver of 3- and 6-month old SERT2/2;PTEN+/2 and SERT2/2 mice were evaluated by tyrosine-phosphorylated IRS1. IRS1 was first immunoprecipitated with anti-IRS1 antibody (IP-IRS1) and then blotted with anti-IRS1 (IB-IRS1) or anti-phosphotyrosine antibodies (IB-pY-IRS1). A, D, G and I show images of representative western blot results. B and E show densitometric quantification of the ratio of pAKT vs. total AKT, and H and J show densitometric quantification of the ratio of pY-IRS1 vs. total IRS1. Insulin (2), basal activity determined from the tissues collected from16 h fasted mice. Insulin (+), insulin-induced activity determined from the tissues collected from mice 20 min post insulin injection. The value of WT treated with insulin and mutants with and without insulin treatment is normalized to that of WT without insulin treatment. C and F show the ratio of pAKT before and after insulin injection for each genotype. The net increase in pAKT amounts following insulin injection was elevated in SERT2/2; PTEN+/2 mice relative to SERT2/2 mice, *p,0.05, Student’s t-test. K–N. Western blot analysis of phospho-JNK (pJNK) and phospho- Ser307-IRS1 in the liver of 16 h fasted 3- and 6-month old SERT2/2; PTEN+/2 and SERT2/2 mice. K and M show images of representative western blot results. L and N show densitometric quantification of pJNK and pSer307-IRS1. The amounts of phosphorylated proteins were normalized to that of total corresponding proteins. Shown are the relative values of SERT2/2; PTEN+/2 mice to SERT2/2 mice, with the average value from SERT2/2 mice defined as 1. Data represent mean 6 SEM. The number of mice analyzed is indicated in parentheses. doi:10.1371/journal.pone.0032511.g007 intolerance and insulin resistance of SERT-deficient mice are attributable, at least in part, to impaired PI3K and JNK signaling in the peripheral tissues. These findings suggest that food intake and glucose homeostasis may be regulated by separable 5-HT signaling pathways in multiple peripheral organs and are influenced by SERT function. The metabolic defects of SERT+/2 mice indicate that even a partial SERT deficiency can profoundly impair glucose homeostasis and insulin sensitivities. Impact of SERT function on metabolic homeostasis is multifactorial Our analyses of SERT-deficient mice revealed a new effect of SERT function on food intake. We found that basal levels of leptin, the major inhibitory hormone of food intake [25], were markedly increased in SERT2/2 and SERT+/2 mice. Since SERT deficiency presumably results in increased 5-HT signals [24], excessive 5-HT signaling may inhibit food intake via a PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e32511