4.9. AMPK Activity Assay

AMP-activated protein kinase (AMPK) activity was performed as described previously [18], using an AMPK Kinase Assay kit in accordance with the manufacturer’s instructions. Samples were briefly incubated for 30 min at 30 °C, on a precoated plate with a substrate peptide that corresponded to mouse IRS-1. AMPK activity was measured by monitoring phosphorylation of Ser 789 in IRS-1, using an anti-mouse phospho-Ser 789 IRS-1 monoclonal antibody and peroxidase-coupled anti-mouse IgG. Conversion of the chromogenic substrate tetramethylbenzidine was quantified by measuring absorbance at 450 nm. Protein was determined using a BCA protein assay kit. Values for AMPK activity were expressed as a fold-increase of the untreated control.