Exercise and Leptin Action microscopy was performed using IL-6 receptor (IL-6R)-specific antibody (green) and DAPI (blue), with 506 magnification (i). Co-localization of IL-6R (green) and AMPK (red) and IL-6R (green) and phospho-p70S6K (red) 60 minutes after i.c.v. saline or IL-6 infusion in the arcuate nuclei of rats, with 2006 magnification (j). Data are the means6SEM. *p,0.05, vs. control group; **p,0.01, vs. control group; # p,0.05, vs. other groups. doi:10.1371/journal.pone.0003856.g001 exercise), exercised animals showed a 33% reduction in 12-hour total food intake (Figure 3a). Consistent with food intake, AICAR increased AMPK threo- nine and ACC serine phosphorylation levels in the hypothalami of control rats, whilst in exercised animals, AICAR did not change AMPK/ACC phosphorylation status (Figures 3b and c). Com- paring AICAR treated groups (control vs. exercise), exercised animals showed reductions in AMPK threonine and ACC serine phosphorylation of 52% and 31%, respectively. AICAR reduced p70S6K and 4EBP1 threonine phosphorylation levels in the hypothalamus of control and exercised rats. However, comparing AICAR treated groups (control vs. exercise), exercised animals showed increases in p70S6K and 4EBP1 threonine phosphoryla- tion of 230% and 310%, respectively (Figures 3d and e). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 3b–e, lower panels). Similar results were observed after intraperitoneal treatment with 2-DG, another pharmacological activator of AMPK (Figure 3f–j). To be sure that the experiments represent a physiological condition we measured food intake in control and exercised animals after fasting. In the control animals, prolonged fasting (48 hours) increased ,35% of food intake during 12-hours of refeeding period compared to control group. However, in the fasted rats, acute exercise session prevented fasting-induced hyperphagic response (Figure 3k). Comparing fasting treated groups (control vs. exercise), exercised animals showed reductions in AMPK threonine and ACC serine phosphorylation of 65% and 41%, respectively (Figures 3l and m). Comparing fasting groups (control vs. exercise), exercised animals showed increases in p70S6K and 4EBP1 threonine phosphorylation of 261% and 240%, respectively (Figures 3n and o). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 3l–o, lower panels). Intracerebroventricular a-LA reduces food intake by modulating AMPK-mTOR hypothalamic phosphorylation levels to a greater extent in exercised animals a-LA is an essential cofactor of mitochondrial respiratory enzymes and exerts potent anti-obesity effects by suppressing hypothalamic AMPK activity [34]. The effects of a-LA (3 mg) intracerebroventricular administration, or its vehicle, on food intake control were studied by measuring the 12-hour total food intake after an acute exercise bout. In exercised rats, a-LA (3 mg) reduced food intake by 86% while control group showed a reduction of 58%. Comparing a-LA treated groups (control vs. exercise), exercised animals showed a 64% reduction in 12-hour total food intake (Figure 4a). To determine the effects of exercise on the AMPK-mTOR signaling pathway, a-LA was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus of all rats. a-LA reduced AMPK and ACC phosphorylation levels, in the hypothalami of control and exercised rats. Comparing a-LA treated groups (control vs. exercise), in exercised animals a-LA reduced both AMPK threonine phosphorylation and ACC serine phosphorylation of 39% and 57%, respectively (Figures 4b and c). a-LA induced p70S6K and 4EBP1 threonine phosphorylation in the hypothalami of control and exercised rats. Comparing a-LA treated groups (control vs. exercise), in exercised animals, a-LA increased p70S6K and 4EBP1 threonine phosphorylation of 19% and 11%, respectively (Figures 4d and e). a-LA did not change aAMPK, ACC, p70S6K and 4EBP1 protein expression (Figures 4b–e, lower panels). Intracerebroventricular leptin reduces food intake by modulating AMPK-mTOR hypothalamic phosphorylation levels to a greater extent in exercised animals The effects of leptin (1026 M) i.c.v. administration or its vehicle on food intake control were studied by measuring the 12-hour total food intake after an acute exercise bout. In exercised rats, leptin (1026 M) reduced food intake by 43%, when compared with exercised plus vehicle treated group, while the control group showed a reduction of 25%, when compared with vehicle treated group. Comparing leptin-treated groups (control vs. exercise), exercised animals showed a 31% reduction in 12-hour total food intake (Figure 5a). To determine the effects of exercise on AMPK-mTOR signaling pathway, leptin was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus of all rats. Leptin reduced AMPK and ACC phosphorylation levels in the hypothalami of control and exercised rats. Comparing leptin-treated groups (control vs. exercise), in exercised animals, leptin reduced both AMPK threonine phos- phorylation and ACC serine phosphorylation of 57% and 45%, respectively (Figures 5b and c). Leptin induced p70S6K and 4EBP1 threonine phosphorylation in the hypothalamus of control and exercised rats. Comparing leptin treated groups (control vs. exercise), in exercised animals, leptin increased both p70S6K and 4EBP1 threonine phosphorylation of 30% and 40% respectively (Figures 5d and e). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 5b–e, lower panels). Role of IL-6 in anorectic response to leptin Hypothalamic IL-6 expression was detected in control animals; however, a 420% increase was observed in the exercised group (Figure 6a). We tested whether the inhibitory effects of leptin on food intake depends on IL-6, by i.c.v. infusion of anti–IL-6 antibody into exercised rats. Treatment with leptin (1026 M) markedly reduced 12-h food intake in exercised rats pretreated with vehicle, although pretreatment with anti–IL-6 antibody blocked exercise-induced leptin responsiveness in a concentration- dependent manner (Figure 6b). Both AMPK and ACC phosphorylation levels, reduced by exercise, were reversed by anti–IL-6 antibody (Figure 6c and d). We also observed that the increased phosphorylations of p70S6K and 4EBP1, induced by exercise, were also reversed by anti–IL-6 infusion (Figures 6e and f). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 6c–f, lower panels). The role of IL-6 on leptin responsiveness in the hypothalamus of diet-induced obesity (DIO) rats We next investigated the effect of IL-6 on leptin responsiveness in the hypothalamus of diet-induced obesity (DIO) rats after acute exercise. Hypothalamic IL-6 expression was detected in the PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e3856