Exercise and Leptin Action Figure 3. AICAR, 2-DG and fasting effects on 12-h cumulative food intake and AMPK/mTOR signaling, in the hypothalami of control and exercised rats. (a) AICAR (2 mM) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (b, c, d, e) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. (f) 2-DG (500 mg/Kg) was administered in control and exercised rats. Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (g, h, i, j) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. (k) Exercise decreases food intake in the fasting state (48 h fasting). Animals were immediately exposed to food for a 12-hour period (n = 12–15 animals per group). (l, m, n, o) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK; phospho-ACCser79, serine phosphorylated ACC and total ACC; phospho- p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K; phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1. Data are the means6SEM. * p,0.05, ** p,0.01, vs. control group; # p,0.05, ## p,0.001 vs. AICAR- or 2-DG-stimulated control group. doi:10.1371/journal.pone.0003856.g003 hypothalamus of diet-induced obesity (DIO) rats; however, a 156% increase was observed in the DIO rats after acute exercise (Figure 7a). The effects of leptin (1026 M) i.c.v. administration on energy intake control were studied by measuring the 12-hour total food intake after an acute exercise bout in DIO rats. Comparing leptin-treated rats (control vs. DIO), the energy intake was 28% higher in DIO rats after leptin infusion. However, the i.c.v. infusion of leptin was able to reduce the energy intake by about 31% in DIO rats after the exercise protocol, compared to DIO rats at rest (Figure 7b). Interestingly, the i.c.v. pretreatment with anti-IL-6 antibody (25 ng) blunted the anorexigenic effects of leptin in exercised DIO rats. To determine the effects of exercise on the AMPK-mTOR signaling pathway in the hypothalamus of DIO rats, leptin was i.c.v. administered and AMPK, ACC, p70S6K and 4EBP1 phosphorylation levels were assessed in the hypothalamus. The suppressive effects of i.c.v. infusion of leptin on AMPK and ACC phosphorylation were impaired in the hypothalamus of DIO rats by about 58 and 54%, respectively, when compared to the control group (Figure 7c and d). In exercised DIO rats, leptin reduced the phosphorylation of AMPK by 65% and ACC by 37%, compared to DIO rats at rest and the i.c.v. pretreatment with anti-IL6 antibody before the exercise protocol, reversed the suppressive effects of leptin on AMPK/ACC pathway in the hypothalamus of exercised DIO rats (Figures 7c and d). The AMPK and ACC protein levels did not differ between the groups (Figures 7c and d, lower panels). In addition, p70S6K and 4EBP1 phosphorylation in the hypothalamus of DIO rats after i.c.v. leptin infusion was reduced by about 46% and 45%, respectively, when compared to the control group. In exercised DIO animals, leptin increased the phosphorylation of p70S6K by 62% and 4EBP1 by 59%, compared to DIO rats at rest. I.c.v. pretreatment with anti-IL6 antibody before the exercise protocol blocked these effects in the hypothalamus of exercised DIO rats (Figures 7e and f). The p70S6K and 4EBP1 protein levels were not different between the groups (Figures 7e and f, lower panels). Blocking effects of AICAR and Rapamycin on leptin- induced anorexia We tested whether the i.c.v. administration of AICAR or Rapamycin, 60 minutes before the administration of leptin (1026 M), prevents the anorexigenic effects of leptin. Leptin (1026 M) treatment markedly reduced 12-h food intake in both control and exercised groups, although leptin was much more effective in exercised rats. AICAR (0.5 mM) or Rapamycin (25 mg), at doses that do not alter ingestion, completely blocked the suppression of food intake induced by an i.c.v. injection of leptin (1026 M) (Figure 8a). The i.c.v. administration of leptin (1026 M) to exercised rats pretreated with vehicle reduced AMPK and ACC phosphorylation in the hypothalamus by 63% and 60% respectively, compared with the control group. The administration of AICAR increased AMPK threonine and ACC serine phosphorylation, although at this dose, AICAR was not sufficient to induce significant an increase in food intake in exercised animals (data not shown). Comparing exercised animals, i.c.v. administration of leptin (1026 M) to rats pretreated with AICAR increased both AMPK and ACC phosphorylation levels in the hypothalamus (Figures 8b and c). The i.c.v. administration of leptin (1026 M) to exercised rats pretreated with vehicle induced p70S6K and 4EBP1 phosphor- ylation in the hypothalamus of 60% and 70%, respectively, compared with the control group. Comparing exercised animals, i.c.v. administration of leptin (1026 M) to rats pretreated with AICAR reduced both p70S6K and 4EBP1 phosphorylation levels in the hypothalamus. Exercised animals pretreated with Rapamy- cin also reduced hypothalamic p70S6K and 4EBP1 phosphory- lation (Figures 8d and e). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 8b–e, lower panels). Discussion During the last decade, a substantial number of studies have investigated the role of physical activity in the control of energy intake in rodents [5,6,35] and in humans [36–38]. However, the molecular mechanisms by which exercise controls food intake are still unsolved. Several experimental studies have demonstrated that neither acute [5,36] nor chronic [6,39] exercise per se change food intake, on the other hand, accumulating evidence shows that both acute and chronic exercise potentiate the anorexigenic effects of leptin in the hypothalamus. Our data indicate that IL-6 signaling through AMPK and mTOR reduces food intake in a dose- dependent manner. Leptin, as well as a-LA infusion, reduced food intake in exercised rats to a greater extent than that observed in control animals. Conversely, AICAR, 2-DG and fasting increased food intake in exercised rats to a lower extent than that observed in control animals. Exercise was associated with the effects of leptin on the AMPK/mTOR pathway activity in the hypothalamus. In addition, we investigated the regulatory role of IL-6 in mediating the increase in leptin responsiveness in the hypothalamus. Treatment with leptin markedly reduced food intake, AMPK activity and increased mTOR activity in exercised rats that were pretreated with vehicle, although no increase in response to leptin- induced anorexia and modulation of AMPK/mTOR pathway were detected after i.c.v. pretreatment with anti-IL-6 antibody. Taken together, these results suggest that IL-6 is a major component of the effects of exercise on the control of food intake. Increasing evidence shows that leptin and IL-6 activates AMPK in the peripheral tissues, such as skeletal muscle and adipose tissue, increasing fatty acid oxidation and glucose uptake in these tissues [40–42], however, leptin has an opposing effect in hypothalamic tissue, reducing neuronal AMPK activity [12,43]. As well as in response to leptin, in the present study, we demonstrated that IL-6 alone reduced AMPK phosphorylation in the hypothalamus of rats. We also show that, IL-6 increased ATP levels and decreased PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e3856