Increased responsiveness of leptin action in the hypothalamus, through modulation of the AMPK-mediated pathway by exercise, could be pathophysiologically important in the prevention of obesity. Recent studies have shown that modulation of leptin signaling through the AMPK pathway could be involved in the development of obesity [61]. Taken together, these data indicate that the anti-obesity actions, induced by leptin, could be increased due to the more pronounced inhibition of the AMPK pathway observed after leptin infusion in the hypothalamus of both lean and diet-induced obesity rats after acute exercise. If the mechanism used by IL-6 to reduce food intake is AMPK- dependent, as our results suggest, the defective activation of AMPK in the hypothalamic neurons induced by exercise may increase the ability of leptin to reduce food intake. In conclusion, exercise improved the AMPK and mTOR responses to leptin administration and contributed to appetite- suppressive actions. This increased dynamic responsiveness of the AMPK/mTOR pathway to leptin could provide information regarding the molecular mechanism underlying the biological sensitivity to leptin in exercise. Furthermore, these findings provide support to the hypothesis that AMPK and mTOR interact in the hypothalamus to control feeding in exercised rats, in an IL-6- dependent manner. Methods Antibodies and Chemicals Reagents for SDS-polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Richmond, CA, USA). Tris[hydroxymethyl]amino-methane (Tris), aprotinin, ATP, di- thiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and bovine serum albumin (fraction V) were from Sigma Aldrich (St. Louis, MO, USA). Protein A-Sepharose 6 MB and Nitrocellulose paper (Hybond ECL, 0.45 mm) from Amer- sham Pharmacia Biotech United Kingdom Ltd. (Buckingham- shire, United Kingdom). Ketamin was from Parke-Davis (Sa˜o Paulo, SP, Brazil) and diazepam and thiopethal were from Crista´lia (Itapira, SP, Brazil). Anti-phospho-[Ser79] ACC (rabbit polyclonal, #07-184) and anti-phospho- [Tyr1007/1008] JAK2 (rabbit polyclonal, AB3805) antibodies were from Upstate Biotechnology (Charlottesville, VA, USA). Anti-ACC (goat polyclonal, sc-26816), anti-JAK2 (rabbit polyclonal, sc 278), anti- STAT3 (rabbit polyclonal, sc 483) and anti-IL-6 (rabbit polyclon- al, sc-7920) antibodies were from Santa Cruz Biotechnology, Inc. Anti-phospho-[Thr172] AMPKa (rabbit polyclonal, #2531), anti- AMPKa (rabbit polyclonal, #2532), anti-phospho- [tyr705] STAT3 (rabbit polyclonal, #9135), anti-phospho- [Thr389] p70S6K (rabbit polyclonal, #9205), anti-p70S6K (rabbit poly- clonal, #9202), anti-phospho- [Thr70]4EBP1 (rabbit polyclonal, #9455), anti-4EBP1 (rabbit polyclonal, #9452), anti-phospho- [Ser 473]Akt (rabbit polyclonal, #9271), and anti-Akt (rabbit polyclonal, #9272) were from Cell Signalling Technology (Beverly, MA, USA). Leptin, LY294002, and Interleukin-6 were from Calbiochem (San Diego, CA, USA); 5-Aminoimidazole-4- carboxamide 1-b-D-ribofuranoside (AICAR), 2-Deoxy-D-glucose and a-lipoic acid were from Sigma Chemical Co. (St. Louis, MO). Rapamycin was from LC Laboratories (Woburn, MA, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Serum insulin and leptin quantification Plasma was separated by centrifugation (1100 g) for 15 minutes at 4uC and stored at 280uC until assayed. RIA was employed to measure serum insulin, according to a previous description [62]. Exercise and Leptin Action Leptin concentrations were determined using a commercially available Enzyme Linked Immuno Sorbent Assay (ELISA) kit (Crystal Chem Inc, Chicago, IL). Experimental Animals Male Wistar rats (8 weeks old/250–300 g) obtained from the University of Campinas Animal Breeding Center were used in the experiments. The investigation was approved by the ethics committee and followed the University guidelines for the use of animals in experimental studies and conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. 85-23 revised 1996). The animals were maintained on 12h:12h artificial light–dark cycles and housed in individual cages. Diet induced obesity (DIO) Male 4-wk-old Wistar rats from the University of Campinas Breeding Center were randomly divided into two groups, control, fed standard rodent chow (3948 kcal.Kg21) and DIO, fed a fat- rich chow (5358 kcal.Kg21) ad libitum for 3 months and then submitted to the different experimental protocols. This diet composition has been previously used [63]. Intracerebroventricular (i.c.v.) cannulation The animals were stereotaxically instrumented under intraper- itoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body weight) with a chronic 26-gauge stainless steel indwelling guide cannula, aseptically placed into the third ventricle (0.5 mm posterior and 8.5 mm ventral to bregma), as previously described [64]. After a 1-wk recovery period, cannula placement was confirmed by a positive drinking response after administration of Angiotensin II (40 ng/2 mL); animals that did not drink 5 mL of water within 15 minutes after treatment were not included in the experiment. Exercise Protocol Rats were acclimated to swimming for 2 days (10 min per day). On the day of the experiment, animals swam in groups of four, in plastic barrels of 45 cm in diameter, filled to a depth of 50 cm. Water temperature was maintained at 34–35uC. They performed two 3-h exercise bouts, separated by one 45-min rest period, as previously described [65]. After the last exercise bout, some rats were injected into the cannula and food intake was determined over the next 4 and/or 12 h; the other rats were injected into the cannula and then anesthetized with intraperitoneal injection of sodium thiopethal (5 mg/100 g body weight) and hypothalamus was removed. Treatments For acute treatments, rats were deprived of food for 6 h with free access to water and i.p. injected (200 ml bolus injection) with either vehicle or 2-DG (500 mg/kg) or i.c.v. injected (3 ml bolus injection) with either vehicle, IL-6 (100 ng or 200 ng), AICAR (0.5 or 2.0 mM), Rapamycin (25 mg), a-LA (3 mg), leptin (1026 M), LY294002 (50 mM) or anti-IL-6 antibody. Similar studies were carried out in rats that were initially pre-treated with i.c.v. microinjection of vehicle, AICAR, Rapamycin, anti-IL-6 antibody or LY294002, and after 60 min with i.c.v. microinjection of IL-6 or leptin. Thereafter, standard chow was given and food intake was determined by measuring the difference between the weight of chow given and the weight of chow at the end of 4 and/or 12-h periods. All acute treatments were performed at 5:00 and 6:00 p.m. PLoS ONE | www.plosone.org 14 December 2008 | Volume 3 | Issue 12 | e3856