Distinct SERT Function in Feeding and Obesity phosphotyrosine antibodies (IB-pY-IRS1). A, D, G and J show images of representative western blot results. B and E show densitometric quantification of the ratio of pAKT vs. total AKT, and H and K 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. I and L show the ratio of pY-IRS1 before and after insulin injection. The basal pAKT and pY-IRS1 were elevated, but the net increase in pAKT and pY-IRS1 following insulin injection was attenuated in SERT mutant mice. M–P. Western blot analysis of phospho-Ser307-IRS1, phospho-JNK (pJNK), phospho-S6K (pS6K), and phospho-p38 MAPK (pp38) in the liver of 16 h fasted 3- and 6-month old SERT mutant mice. M and O show images of representative western blot results. N and P show densitometric quantification of pSer307-IRS1, pJNK, pS6K and pp38. The amounts of phosphorylated proteins are normalized to that of total corresponding proteins. Shown are the relative values of SERT2/2 mice to WT, with the average value from WT mice defined as 1. Data represent mean 6 SEM, *p,0.05, **p,0.01, Student’s t-test. The number of mice analyzed is indicated in parentheses. doi:10.1371/journal.pone.0032511.g005 month old SERT2/2; PTEN+/2 mice compared to that of SERT2/2 mice. As a control, we analyzed insulin-induced phosphorylation of IRS1, an upstream regulator of PI3K. Figure 7G–J show that pY-IRS1 levels in the liver of SERT2/2; PTEN+/2 mice were similar to that in SERT2/2 mice. These data suggest that reduced PTEN phosphatase activity results in an increase in pAKT, thereby enhancing AKT downstream pathways, but it does not correct IRS1 response to insulin. Consistent with this idea, basal JNK activity in SERT2/2; PTEN+/2 mice was as high as in SERT2/2 mice, based on western blot analyses of pJNK and p-Ser307-IRS1 in the liver (Figure 7K–N). Discussion The data presented here show that SERT-deficient mice progressively develop obesity and hepatic steatosis, despite reduced food intake. We also show that SERT-deficient mice exhibit hyperglycemia and insulin resistance, both of which are charac- teristic features of diabetes. Our data suggest that glucose Figure 6. Reduced PTEN function corrected glucose tolerance in SERT mutants. A and B show GTT (i.p., 1 g/kg), and C and D show ITT (i.p., 1 U/kg). The SERT2/2; PTEN+/2 mice exhibited improved insulin sensitivity at 3 months of age and improved glucose tolerance at 6 months of age compared to age-matched SERT2/2 mice, *p,0.05, ***p,0.001. The SERT+/2; PTEN+/2 mice exhibited significantly improved glucose tolerance and insulin sensitivity at the age of 3- and 6-months, compared to age-matched SERT+/2, #p,0.05, ##p,0.01, ###p,0.001, Student’s t-test. N, number of mice analyzed. doi:10.1371/journal.pone.0032511.g006 PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32511