in a plate pre-coated with a substrate peptide, corresponding to mouse IRS-1. AMPK activity in cell lysates was measured by monitoring the phosphorylation of Ser789 in IRS-1 using an anti-mouse phospho-Ser-789 IRS-1 monoclonal antibody and peroxidase-coupled anti-mouse IgG antibody. Conversion of the chromogenic substrate tetramethylbenzidine was quantified by measuring changes in absorbance at 450 nm. AMPK activity was calculated as the difference between the absorbance measured in the absence or in the presence of Compound C. Activity was measured in duplicates, normalized to total protein in the cell lysates, and expressed as fold induction with respect to non-stimulated cells. Culture and cell surface labeling of L6 cells expressing brown trout GLUT4 In order to determine if the cell surface levels of brown trout GLUT4 increase in response to AMPK activators in skeletal muscle cells, we used a rat skeletal muscle 14726663” cell line L6 stably expressing brown trout GLUT4 harboring an exofacial myc epitope that has been used previously by our group to study the traffic characteristics of brown trout GLUT4. Briefly, L6-btGLUT4myc myoblasts were maintained in aMEM supplemented with 10% FBS and 1% antibiotic/ antimycotic solution in a humidified atmosphere of air and 5% CO2 at 37uC. MedChemExpress LY-411575 Myotubes obtained by differentiating L6-btGLUT4myc myoblasts in media supplemented with 2% FBS within five days after seeding were serum-starved for two hours and subsequently treated with AICAR or metformin for 18 h. The doses of the AMPK activators used for these experiments were chosen based of the effective doses reported in the literature to affect GLUT4 traffic in mammalian cells. As a positive control, cells were treated for 20 min with 100 nM human insulin, which stimulates btGLUT4 translocation in this cell line. Media was removed 
and cells were washed three times with ice-cold PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 at 4uC. Cells were then fixed with 3% paraformaldehyde for 15 min on ice and quenched with 100 mM glycine for 10 min. To label cell surface btGLUT4myc, cells were blocked in 5% goat serum in PBS+ for 10 min, and then incubated with a-myc antibody solution for 1 h at room temperature. Following labeling, excess anti-myc antibodies were removed by extensive washing in ice-cold PBS+. Cell surface GLUT4-bound anti-myc antibodies were probed Identification of AMPK subunits in trout skeletal muscle cells by Western blotting Western blot analyses were conducted using lysates from trout myotubes and from C2C12 ” myotubes, used as a positive control. Myotube lysates were diluted in Laemmli sample buffer, heated for 5 min at 95uC, and centrifuged at 12000 g for 5 min. Proteins from the supernatant and protein standards were loaded and separated on 12% SDS-PAGE precast gels using a Mini-Protean system for 12 h at 100 V and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer containing 5% non-fat dry milk for 1 h. The membrane was washed several times in blocking buffer and incubated overnight at 4uC with rabbit antibodies against the different subunits of human AMPK AMPK Subunit Antibody Sampler Kit, Cell Signaling, Barcelona, Spain), diluted to 1:1000 in blocking buffer containing 5% Bovine Serum Albumin under continuous shaking. After three washes, the membrane was incubated with a secondary antibody against rabbit IgG conjugated with horseradish peroxidase diluted 1:2000 in TBST buffer containing
