a freshly prepared ice-cold buffer suitable for preserving phosphorylation states of enzymes and Western blot was performed, as previously described. Insoluble material was removed by centrifugation for 25 minutes at 4uC. Total extracts of hypothalamus were prepared and 0.25 mg total proteins were separed by SDSPAGE. After SDS-PAGE, proteins were transferred from gel to nitrocellulose membrane. Membranes were blocked in 5% nonfat dried milk in PBST 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 with Cronex Lightning Plus intensifying screens at 280uC for 1248 h.. Band intensities were quantified by optical densitometry of developed autoradiographs. removed and the hypothalamus was quickly dissected and frozen in liquid N2 for Thiazovivin web chromatographic analyses. Chromatographic analyses were carried out on a Waters Alliance equipment series 2695 equipped with a quaternary pump, an autosampler, a degasser, and a Waters 2475 fluorescence detector model. The fluorescence of derivatized compounds 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 with a Cosmosil guard column purchased from Phenomenex. The mobile phase composition was 50 mmol/L KH2PO4, 25 mmol/L citric acid, and methanol, which was prepared immediately before use and filtered through a 
0.45 mm filter. 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 and could be described by the linear regression equation: y = 0.4992×20.0463, in which y is the ATP concentration in micromoles and x is 22967846 the chromatogram peak area. Confocal microscopy Paraformaldehyde-fixed hypothalami were sectioned. The sections were obtained from hypothalamus of six rats per group in the same localization and used in regular single- or double-immunofluorescence staining using DAPI, anti-IL6 receptor, anti-AMPK, anti-phospho-p70S6K, 14530216 antibodies, according to a previously described protocol. Analysis and photodocumentation of results were performed using a LSM 510 laser confocal microscope. The anatomical correlations were made according to the landmarks given in a stereotaxic atlas. Statistical Analysis All numeric results are expressed as the means6SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands or spots in autoradiographs and quantified by optical densitometry. Statistical analysis was performed by employing the ANOVA test with Bonferroni post test. Significance was established at the p,0.05 level. Acknowledgments We thank Dr Nicola Conran for English language editing. A