Western blot analysis of insulin signaling component activity in SERT-deficient mice.

<p><b>A–F.</b> AKT activity in the liver of 3- and 6-month old WT and SERT-deficient mice was evaluated by western blot analysis of AKT Ser473 phosphorylation (pAKT) before and after insulin injection. <b>G–L.</b> IRS1 activity in the liver of 3- and 6-month old WT and SERT-deficient mice was evaluated by western blot analysis of 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). <b>A, D, G and J</b> show images of representative western blot results. <b>B and E</b> show densitometric quantification of the ratio of pAKT vs. total AKT, and <b>H and K</b> show densitometric quantification of the ratio of pY-IRS1 vs. total IRS1. Insulin (−), 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. <b>C and F</b> show the ratio of pAKT before and after insulin injection for each genotype. <b>I and L</b> 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. <b>M–P.</b> 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. <b>M and O</b> show images of representative western blot results. <b>N and P</b> 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 SERT−/− mice to WT, with the average value from WT mice defined as 1. Data represent mean ± SEM, *p<0.05, **p<0.01, Student's t-test. The number of mice analyzed is indicated in parentheses.</p

Reduced PTEN function corrected glucose tolerance in SERT mutants.

<p><b>A and B</b> show GTT (i.p., 1 g/kg), and <b>C and D</b> show ITT (i.p., 1 U/kg). The SERT−/−; PTEN+/− mice exhibited improved insulin sensitivity at 3 months of age and improved glucose tolerance at 6 months of age compared to age-matched SERT−/− mice, *p<0.05, ***p<0.001. The SERT+/−; PTEN+/− mice exhibited significantly improved glucose tolerance and insulin sensitivity at the age of 3- and 6-months, compared to age-matched SERT+/−, #p<0.05, ##p<0.01, ###p<0.001, Student's t-test. N, number of mice analyzed.</p

SERT mRNA detected by RT-PCR from WT mice tissues.

<p>Intestine tissue from a SERT−/− mouse is presented as a negative control. 6-month old mice were analyzed.</p

Characterization of glucose homeostasis in SERT-deficient mice.

<p><b>A.</b> GTT (i.p., 1 g/kg) of 3-month-old mice after 16 h fast. <b>B.</b> GTT (i.p., 1 g/kg) of 6-month-old mice after 16 h fast. <b>C.</b> ITT (i.p., 1 U/kg) of 3-month-old mice after 6 h fast. <b>D.</b> ITT (i.p., 1 U/kg) of 6-month-old mice after 6 h fast. N, number of mice analyzed for each genotype. Data represent mean ± SEM. Student's t-test was used to analyze statistical significance of the difference between the following groups: SERT−/− vs. WT mice on ND feeding, *p<0.05, **p<0.01, ***p<0.001; SERT−/− vs. WT mice on HFD feeding, #p<0.05, ##p<0.01, Student's t-test. At 3 months of age, the differences in GTT of SERT+/− vs. WT mice were not statistically significant; however, there were significant differences between SERT+/− vs. WT mice in ITT. At 6 months of age, both SERT−/− and SERT+/− mice differed significantly from WT mice in GTT and ITT.</p