Western Blot Analysis After exercise and i.c.v. treatments, rats were anaesthetized with intraperitoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body weight), and used as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The rats were killed, and hypothalamus was quickly removed, minced coarsely and homogenized immediately in a freshly prepared ice-cold buffer (1% Triton X-100, 100 mmol/l Tris pH 7.4, 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium vanadate, 2 mmol/l phenyl methylsulphonyl fluoride and 0.1 mg aprotinin) suitable for preserving phosphoryla- tion states of enzymes and Western blot was performed, as previously described [66]. Insoluble material was removed by centrifugation (50 000 g) for 25 minutes at 4uC. Total extracts of hypothalamus were prepared and 0.25 mg total proteins were separed by SDS- PAGE. After SDS-PAGE (15% resolving gels for phospho-4EBP1 and 4EBP1; 12% resolving gels for phospho-AMPK, AMPKa2; phospho-p70S6K, p70S6K, phospho-Akt, Akt, phospho-STAT3 and STAT3; 8% resolving gels for phosphor-JAK2 and JAK2; 6.5% resolving gels for ACC and phospho-ACC), proteins were transferred from gel to nitrocellulose membrane. Membranes were blocked in 5% nonfat dried milk in PBST (139 mM NaCl, 2.7 mM KH2PO4, 9.9 mM Na2HPO4, and 0.1% Tween 20) for 2 hours and then incubated overnight with specific antibodies. After incubation with the relative second antibody, immune complexes were detected using the ECL method. Results were visualized by autoradiography using preflashed Kodak XAR film (Eastman Kodak, Rochester, N.Y., USA) with Cronex Lightning Plus intensifying screens at 280uC for 12–48 h. (Mass., USA). Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software - Scion Corporation, Frederick, Md., USA). Confocal microscopy Paraformaldehyde-fixed hypothalami were sectioned (5 mm). The sections were obtained from hypothalamus of six rats per group in the same localization (antero-posterior = 21.78 from bregma) and used in regular single- or double-immunofluores- cence staining using DAPI, anti-IL6 receptor (IL6R), anti-AMPK, anti-phospho-p70S6K (1:200; Santa Cruz Biotechnology), anti- bodies, according to a previously described protocol [67]. Analysis and photodocumentation of results were performed using a LSM 510 laser confocal microscope (Zeiss, Jena, Germany). The anatomical correlations were made according to the landmarks given in a stereotaxic atlas [68]. Chromatography After exercise, i.c.v. infusion of IL-6 or vehicle, rats were anaesthetized, and used as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The animals were killed and, within 40 seconds, the cranium was opened, the brain was References 1. 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Chromatographic analyses were carried out on a Waters Alliance equipment series 2695 (Milford, MA, USA) equipped with a quaternary pump, an autosampler, a degasser, and a Waters 2475 fluorescence detector model. The fluorescence of derivatized compounds (ATP, ADP, AMP, and ADO) was monitored with excitation and emission wavelengths set at 280 and 420 nm, respectively. Chromatographic separations of the compounds were achieved at room temperature, using a reversed-phase Cosmosil 5C18-MS column (15064.6 mm i.d.; 5 mm particle size) with a Cosmosil guard column (5C18-MS 1064.6 mm) purchased from Phenomenex (Torrance, CA, USA). The mobile phase composition was 50 mmol/L KH2PO4, 25 mmol/L citric acid (pH 4.5), and methanol (90:10, v/v), which was prepared immediately before use and filtered through a 0.45 mm filter (Millipore, Milford, MA, USA). The column was equilibrated and eluted under isocratic conditions using a flow rate of 1.0 ml/min. The chromatographic run time for each analysis was 20 min. Aliquots of 25 ml were injected into the HPLC system. System control, data acquisition, and processing were performed with a PC-Pentium IV Processor personal computer from Dell, operated with Microsoft Windows XP version 2003 and Waters Empower 2002 chromatography software. A validation chromatographic run included a set of calibration samples assayed in duplicate and quality control samples at four levels in triplicate. The standard calibration curves for known amounts of ATP, ranging from 0.025 to 10.0 mmol/L, were linear (R.0.999) and could be described by the linear regression equation: y = 0.4992*x20.0463 (n = 4, P,0.0001, r = 0.9997), in which y is the ATP concentration in micromoles and x is the chromatogram peak area. Statistical Analysis All numeric results are expressed as the means6SEM of the indicated number of experiments. 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