Measuring Muscle Fatty Acid Oxidation

Rates of fatty acid oxidation in muscle tissues were measured by degradation of ¹⁴C-palmitate (American Radiolabeled Chemicals; St. Louis, MO, USA) into ¹⁴C acid soluble metabolites (ASM) and ¹⁴C-labeled CO₂^{46 (link),48} . Briefly, gastrocnemius muscle strips were collected from 6 h fasted mice. Tissues were kept on ice no longer than 30 min. Muscle tissues were minced and homogenized in a Dounce homogenizer followed by centrifugation for 10 min at 420 × g. Supernatants were transferred to microtubes containing 0.4 μCi ¹⁴C-palmitate/500 μM palmitate conjugated with 0.7% fatty acid-free BSA and incubated at 37 °C for 30 min. ¹⁴C-labeled CO₂ produced by TCA cycle was captured onto filter paper soaked with 1 M NaOH and ¹⁴C-labeled ASM were separated with 1 M perchloric acid. ¹⁴C-labeled CO₂ in the filter paper and ¹⁴C-labeled ASM in the supernatant were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA) and were counted using a liquid scintillation counter (Beckman Coulter; Danvers, MA).