Protein extraction and Western blot analysis

Proteins from 50 mg of liver samples were extracted, separated in gradient SDS-PAGE gels and electroblotted onto nitrocellulose membranes and western blot analysis was performed as described⁴⁹. Specific blotted proteins were detected with the corresponding primary rabbit antibody: anti-AMPK, anti-phospho-AMPK, anti-Akt, -anti-phospho-Akt (Cell Signaling Technology Inc. MA.; cat. number: #2532, #2535, #9272 and #9271, respectively); mouse anti-βactin (Sigma-Aldrich; cat. number: # A5316) and anti-Adaptin γ (Abcam, Cambridge, UK.;, cat. number #ab167153) followed by 1 h incubation with the corresponding secondary antibody: HRP-conjugated anti-rabbit IgG (H+L) or HRP-conjugated anti-mouse IgG (H+L) antibodies (Promega; cat. number W4011 and W4021, respectively). Specific protein bands were revealed using the enhanced chemiluminiscence detection system (Santa Cruz, Biotechnology Inc. CA, USA), in accordance with the manufacturer’s instructions. Images were visualized in an Autochemi-UVP Bioimaging System. Finally, bands were quantified by densitometry performed by ImageJ software (http://imagej.nih.gov/ij). Levels of specific proteins were normalized to actin levels for liver samples or adaptin levels for skeletal muscle samples.