Exercise and Leptin Action Figure 8. Blocking effects of AICAR and Rapamycin on leptin-induced anorexia. (a) Effect of i.c.v. administration of leptin on exercised rats; pretreatment with AICAR or Rapamycin (n = 8–10 animals per group). (b, c, d, e) Representative Western blots of five independent experiments showing hypothalamic lysates from Wistar rats. Phospho-AMPKthr172, threonine-phosphorylated AMPK and total AMPK (b); phospho-ACCser79, serine phosphorylated ACC and total ACC (c); phospho-p70S6Kinasethr389, threonine phosphorylated p70S6Kinase and total p70S6K (d); phospho-4EBP1thr70, threonine phosphorylated 4EBP1 and total 4EBP1 (e). Data are the means6SEM. *p,0.05, vs. other groups. doi:10.1371/journal.pone.0003856.g008 the AMP/ATP ratio in the hypothalamus (figures 1b–d), but the mechanisms by which IL-6 modulates the ATP levels require further investigations. A number of recent studies have shown that AMPK plays a key role in regulating both energy intake and expenditure [12,34,44,45]. In peripheral tissues, such as skeletal muscle, activation of AMPK switches on energy producing pathways and switches off energy consuming pathways. In the hypothalamus, activation of AMPK leads to increased feeding, thereby increasing energy intake. Conversely, inhibition of AMPK in the hypothalamus reduces food intake. These dual functions of AMPK suggest that it may act to coordinate energy expenditure with energy intake. There is already some evidence that this may be the case in one situation. More recently, Gao and colleagues [10] demonstrated that the hypotha- lamic ACC activation makes an important contribution to the anorexigenic effects of leptin that are mediated by AMPK. The aim of this study was to investigate whether IL-6 could affect AMPK activity in the hypothalamus, thereby providing a potential mechanism for the coordination of energy expenditure and energy intake during, or following exercise. Beyond STAT3 activation, we detected changes in the hypothalamic AMPK activity in rats after i.c.v. infusion of IL-6; we show that, IL-6 markedly decreased phospho-AMPK abundance (an index of activity) in the hypothal- amus. In accordance with the reduction in AMPKthr172 phosphor- ylation, we observed that, after IL-6 administration, hypothalamic AMP:ATP ratio was decreased. Wallenius et al [28] elegantly showed that long-term peripheral IL-6 treatment to Il62/2 mice caused a decrease in body weight. In addition to increasing energy expenditure, IL-6 may prevent obesity by inhibiting feeding as obese IL-62/2 mice had increased absolute food intake. However, central IL-6 treatment at the same dose that we used does not influence food intake in mice. In concordance with our results another study of the same group showed a reduced daily food intake over a two-week ICV treatment period with IL-6 in rats indicating a different pattern of response between rats and mice [46]. The mTOR, an evolutionary conserved serine-threonine kinase, central to integrating similar signals to control food intake, has now emerged as a detector of hormonal and nutritional signals in the hypothalamus [13,15]. In this study, we investigated whether IL-6 activates mTOR. IL-6 increased mTOR activity; moreover inhibition of central mTOR reversed the anorectic effect of IL-6. In addition, the anorexigenic effect of IL-6 was absent in AICAR- and Rapamycin-pretreated rats, however, pretreatment with LY294002 - a PI(3)K inhibitor - had no effect on IL-6 induced anorexia, indicating that, in the hypothalamus, the effect of IL-6 is independent of the PI(3)K pathway. Signaling through gp130 commonly results in activation of PI(3)K, and IL-6 can activate PI(3)K [47] and its downstream target Akt [48–51], but it should be noted that this effect has not been observed in all studies [52], suggesting a tissue-dependent effect. Next, we investigated whether the increased sensitivity of the leptin action on food intake induced by exercise, could be due to the modulation of AMPK activity. As previously shown [43], exercise, per se, does not alter AMPK activity in the hypothal- amus; however, we observed that the normal inhibition of AMPK phosphorylation and activity in the hypothalamus, induced by leptin administration, was improved in both lean and diet-induced obesity rats after acute protocol of exercise. In addition, we did not observe any normal stimulation of AMPK activity by AICAR in the hypothalamus of exercised rats, indicating that AMPK pathway is disrupted. This observation agrees with data from aging studies in which acute stimulation with AICAR was blunted in skeletal muscle of old rats [53]. Furthermore, fasting and the use of another activator of AMPK (2- DG) in exercising rats resulted in a lower activation of AMPK when compared to the control animals. In contrast, the pharmacological inhibition of AMPK by a-LA results in a greater inhibition of AMPK activity, compared to control animals. The mechanism by which exercise inhibits AMPK-induced food intake in the hypothalamus is not clear. Several lines of evidence point to a possible link between inhibited AMPK-induced food intake in the hypothalamus and IL-6 signaling through the AMPK/mTOR pathway. Firstly, we found that the leptin- inhibited food intake enhanced by exercise was blunted by anti- IL-6 antibody. Secondly, exercise induced increased response of leptin-inhibited AMPK signaling was reverted by AICAR. Finally, exercise induced increased responsiveness of leptin stimulated mTOR signaling was reverted by rapamycin. Our data are in accordance with earlier studies demonstrating that IL-6 treatment enhances energy expenditure in both rodents and humans [28,54–56]. In exercising rats, hypothalamic leptin and insulin responsiveness are increased in an IL-6-dependent manner [5,6]. It has been previously shown that IL-6 treatment stimulates energy expenditure at the level of the brain in rodents [28,55,57], and it might be assumed that endogenous IL-6 also acts on the brain during exercise. The IL-6 exerting this effect during exercise could be produced by the brain itself, which has been shown to have increased IL-6 production during exercise [58]. Alternatively, the large quantities of endocrine IL-6 produced from working skeletal muscle [58] might reach appropriate sites in the brain [21,59,60]. Interestingly, we did not observe any difference in food intake over a 12-h period, although the levels of hypothalamic IL-6 dramatically increased after exercise. At first glance, these data appear to be contradictory. However, a large decrease in insulin level was observed after exercise, and we have previously shown a synergic effect of IL-6 on the insulin and leptin signaling pathway, in the rat hypothalamus [5]. Since we did not observe modifications in the phosphorylation of JAK2 and the downstream targets of mTOR after exercise, these data suggest that the cross-talk between insulin, IL-6 and leptin have an essential role in controlling food intake after exercise. In this case, it is possible that increases in IL-6 levels were counterbalanced by the reduction in insulin levels. However, the present study has certain limitations. Exercise per se did not evoke any meaningful effect in terms of food intake; rather, it seemed to enhance the anorectic effect of exogenous leptin. Thus, the data presented herein may suggest but do not establish the mechanism by which long term exercise decreases leptin levels; whilst increases the response to leptin, contributing to its food-suppressive actions. Furthermore, settings of activation of AMPK or inactivation of mTOR were selected to induce changes in target protein phosphorylation, but not food intake. Such dissociation does not preclude a pharmacological rather than physiological effect of our data. PLoS ONE | www.plosone.org 13 December 2008 | Volume 3 | Issue 12 | e3856