Hepatocytes were serum fasted overnight and incubated with DMEM containing 3H-oleic acid (2 μCi/ml) (PerkinElmer) and 0.2 mM oleic acid bound to BSA (Sigma) at basal or insulin (100 nM)–stimulated conditions for 3 hours. Subsequently, the supernatant was collected, and the cells were washed twice with PBS and scraped in 0.1 N HCl to determine the protein concentration. Then, 1 volume of MeOH/CHCl3 (2:1, v/v) and 1 volume of 2 M KCl/2 M HCl (1:1, v/v) were added to 0.5 volume of the supernatant and mixed stepwise. The phases were separated by centrifugation (3000g, 10 min), and the upper phase was transferred to a new tube and the procedure was repeated once more. Last, the upper phase was transferred to a scintillation tube, and 4 ml of scintillation liquid was used for scintillation counting. The FA oxidation rate was calculated as the amount of tritium incorporated into 3H2O (measured in dpm) per total protein (Bradford, Bio-Rad) per hour.

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