The Scientific World Journal / 2012 / Article / Fig 4

Review Article

Metabolic Basis for Thyroid Hormone Liver Preconditioning: Upregulation of AMP-Activated Protein Kinase Signaling

Figure 4

Effect of L-3,3′,5-triiodothyronine (T3) administration on rat liver AMP-activated protein kinase (AMPK). Male Sprague-Dawley rats (Animal facility of the Institute of Biomedical Sciences, Faculty of Medicine, University of Chile) weighing 180–200 g were housed on a 12-hour light/dark cycle and were provided with rat chow and water ad libitum. Animals received a single intraperitoneal dose of 0.1 mg of T3/kg body weight or equivalent volumes of hormone vehicle (0.1 N NaOH, controls shown at time zero). Liver samples (100–500 mg) frozen in liquid nitrogen were homogenized and suspended in a buffer solution pH 7.9 containing 10 mM HEPES, 1 mM EDTA, 0.6% Nonidet P-40, 150 mM NaCl, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM orthovanadate). Soluble protein fractions (50 μg) were separated on 12% polyacrylamide gels using SDS-PAGE [85] and transferred to nitrocellulose membranes [86], which were blocked for 1 hour at room temperature with TBS-containing 5% bovine serum albumin. The blots were washed with TBS containing 0.1% Tween 20 and hybridized with either rabbit polyclonal antibody for phospho-AMPK (62 kDa) and mouse monoclonal antibodies for AMPK (62 kDa) (Abcam, Inc., Cambrige, MA) or β-actin (43 kDa) (ICN Biomedicals, Inc., Aurora, OH). In all determinations, anti-β-actin was used as internal control for cytosolic fractions. After extensive washing, the antigen-antibody complexes were detected using horseradish peroxidase goat anti-rabbit IgG or goat anti-mouse IgG and a SuperSignal West Pico Chemiluminescence kit detection system (Pierce, Rockford, IL). Values shown correspond to the means ± SEM for 3 to 4 separate animals. aP < 0.05 compared to control values at time zero; bP < 0.05 compared to T3-treated rats at 1, 2, 4, 6, 8, 12, 36, and 48 h, assessed by one-way ANOVA and the Newman-Keuls, test.