hypothalamus is, however, not fully understood. In cells, binding IL-6 to the a subunit of its receptor triggers the recruitment of gp130, subsequently leading to the activation of the gp130- associated JAK [29–31]. JAK links cytokine receptor to the STAT3 and MAP kinase pathway [29,30,32]. In addition to JAK/ STAT and MAP kinase pathways, IL-6 also activates the PI(3)K/ Akt pathway [33]. In this study, we sought to determine whether the improved response of the AMPK and mTOR pathways to leptin could contribute to the increased molecular response of leptin in rats submitted to exercise in an IL-6-dependent manner. We therefore, examined hypothalamic modulation of AMPK/ACC and mTOR signaling pathways, induced by IL-6, as well as the role of IL-6 in those signaling pathways induced by leptin in rats after acute exercise. Results IL-6 decreases hypothalamic AMPK and increases mTOR signaling To determine whether IL-6 modulates hypothalamic AMPK/ ACC signaling, we injected IL-6 into the third ventricle of rats and evaluated food intake and AMPK signaling. IL-6 caused a significant reduction in food intake (Figure 1a). We next investigated whether the microinfusion of IL-6 modulates the hypothalamic ATP concentration. Figure 1b shows that IL-6 (200 ng) changed ATP, ADP and AMP concentrations in the hypothalamus of rats, whereas, sixty minutes after IL-6 injection, the ATP content increased by ,88% (Figure 1c) and decreased AMP:ATP ratio by ,54% in Wistar rats (Figure 1d). Consistent with the modulation of the AMP:ATP ratio, we observed reduced hypothalamic AMPK and ACC phosphorylation induced by IL-6 (Figures 1e and f); whilst IL-6 increases p70S6K and 4EBP1 phosphorylation (Figures 1g and h). The aAMPK, ACC, p70S6K and 4EBP1 protein levels were not different between the groups (Figures 1e–h, lower panels). If the effect of IL-6 on food intake is mediated by AMPK inhibition, AICAR infusion at doses that do not change food intake, but still increase AMPK and ACC phosphorylation, could be sufficient to block the effects of IL-6. Thus, to determine whether the effects of IL-6 on food intake are AMPK-dependent, we first identified a dose of AICAR that did not alter food intake (0.5 mM) when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of AICAR, or its vehicle, on the anorectic response to i.c.v. IL-6 (200 ng) and we observed that the anorectic response to i.c.v. IL-6 was reversed by AICAR at the time points studied (figure 1a). These findings indicate that inactivation of neuronal AMPK is necessary for some of the effects of IL-6 on food intake. Immunohistochemistry with anti-Interleukin-6 Receptor (IL- 6R)-specific antibody showed that IL-6R is expressed in a majority of neurons in the arcuate nucleus (Figure 1i). Double-staining confocal microscopy showed the positive immunoreactivity of IL- 6R and AMPK in the hypothalamic arcuate nucleus of rats 60 minutes after i.c.v. saline or IL-6 (200 ng) infusion. The immunoreactivity of IL-6R and pp70S6K was not detected in the hypothalamic arcuate nucleus of rats after i.c.v. saline infusion. However, we observed the positive double-staining of IL-6R and pp70S6K 60 minutes after i.c.v. IL-6 (200 ng) infusion (Figure 1j). IL-6 induction of hypothalamic mTOR does not require changes in PI(3)K signaling If the effect of IL-6 on food intake is mediated by mTOR activation, Rapamycin infusion at doses that do not change food Exercise and Leptin Action intake, but still decrease p70S6K and 4EBP1 phosphorylations could be sufficient to block the effects of IL-6. Thus to determine whether IL-6 modulates hypothalamic mTOR signaling, we injected IL-6 (200 ng) into the third ventricle of rats and evaluated food intake and mTOR signaling. IL-6 caused a significant reduction in food intake (Figure 2a) and induced hypothalamic p70S6K and 4EBP1 phosphorylation (Figures 2b and c). The p70S6K and 4EBP1 protein levels were not different between the groups (Figures 2b and c, lower panels). To determine whether the effects of IL-6 on food intake are mTOR-dependent, we first identified a dose of Rapamycin that did not alter food intake (25 mg) when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of Rapamycin or its vehicle, on the anorectic response to i.c.v. IL-6 and we observed that IL-6 reduction of food intake was reversed by Rapamycin. We next examined whether PI(3)K signaling is required for the IL-6-dependent reduction of food intake, by IL-6 administration in LY294002 pretreated (i.c.v.) animals. Pretreatment with LY294002 at a dose that did not alter food intake (1 nmol) had no effect on anorectic response to i.c.v. IL-6 (Figure 2d). Consistent with these data, we observed that a single IL-6 i.c.v. injection did not change Akt phosphorylation status in the hypothalamus (Figure 2e). The Akt protein levels were not different between the groups (Figure 2e, lower panel). These findings indicates that activation of neuronal mTOR is necessary for some of the effects of IL-6 on food intake and that these effects of IL-6 do not require any change in PI(3)K signaling. We also injected IL-6 (200 ng) into the third ventricle of rats and evaluated food intake and JAK2/STAT3 signaling. IL-6 caused a significant reduction in food intake (Figure 2f) and induced hypothalamic JAK2 and STAT3 phosphorylation (Figures 2g and h). The JAK2 and STAT3 protein levels were not different between the groups (Figures 2g and h, lower panels). To determine whether the effects of IL-6 on food intake are also JAK2/STAT3-dependent, we first identified a dose of AG490 that did not alter food intake when administered at the onset of the dark cycle. We then evaluated the effect of i.c.v. pretreatment with this dose of AG490 or its vehicle, on the anorectic response to i.c.v. IL-6 and we observed that IL-6 reduction of food intake was reversed by AG490 (Figure 2f). Physiological parameters measured in basal conditions after exercise protocol The plasma glucose level was lower in the exercised group compared to the control group (3.660.8 vs 4.660.5 mmol/L; n = 5; p,0.05) and the insulin levels were also lower (88612 vs 193617 pmol/L, n = 5; p,0.05). Exercise did not, however, reduce plasma leptin (2.660.5 vs 2.360.7 ng/ml). Insulinemia and leptinemia were not altered by third ventricle microinjection of leptin (data not shown). Exercise partially reverses the effects of AMPK agonists and fasting on food intake through modulation of the AMPK-mTOR signaling pathway in the hypothalamus To test the role of a single session of exercise on AICAR- increased food intake, AICAR (2 mM) or its vehicle were administered (i.c.v.) to control and exercised animals. 12-hour total food intake was measured after exercise. In exercised rats, AICAR (2 mM) did not cause any acute change in food intake but, in the control group, AICAR (2 mM) increased food intake by 32% (Figure 3a), suggesting that AICAR is not effective in exercised rats. Comparing AICAR-treated groups (control vs. PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3856