Mice (TGwt
n = 4, TGmut
n = 4) were euthanized by decapitation with hearts dissected out, then placed immediately into liquid nitrogen for storage at −80°C. Tissue was ground using a mortar and pestle under liquid nitrogen. Glycogen content was determined using an amyloglucosidase digestion method as previously described [
18]. Briefly, 10 to 50 mg of ground cardiac tissue was homogenized in a 1:9 (
wt./vol.) ratio of PBM buffer (20 mM KH
2PO
4, 10 μM CaCl
2, 1 mM MgCl
2, pH 6.1). Tissue homogenate (50 μL) was boiled for 20 min in 30% KOH (
wt./vol.) saturated with anhydrous Na
2SO
4. The remainder of tissue homogenate was used to determine protein concentration with a bicinchoninic acid (BCA) assay. Glycogen was precipitated with 95% ethanol (
vol./vol.), centrifuged at 4,000 ×
g for 15 min at 4°C. The glycogen pellet was dissolved in double distilled H
2O and incubated at 100°C for 20 min with 0.2% anthrone (
wt./vol.) in H
2SO
4. Absorbance was read at 595 nm to determine glycogen concentration of samples relative to an oyster glycogen standard curve (Sigma Aldrich, Canada) and normalized to protein content (microgram of glycogen per microgram of protein). Ground tissue was homogenized in buffer (10 mM Tris–HCl, 50 mM NaF, 1 mM EDTA, 10 mM dithiothreitol, 10% glycerol (
vol./vol.), pH 7.5 at 4°C) and a BCA assay performed to determine protein concentration. Tissue lysate (20 μg) was applied to each lane of an 8% SDS-polyacrylamide gel with separated bands of proteins transferred to a polyvinylidene fluoride membrane. Blocked membranes were incubated with either AMPK α (1:1,000), phosphorylated-AMPK α (Thr 172) (1:500), or insulin receptor β (1:1,000) (Cell Signaling Technology, Danvers, MA, USA). A mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) was used as a loading control. Membranes were analyzed using a Fluor Chem HD image system (Alpha Innotech, San Leandro, CA, USA). Data were expressed as a ratio of target protein to GAPDH protein band intensities, normalized to the mean of TGwt controls for each membrane.