Measurement of the oxygen consumption rate

The mitochondrial oxygen consumption rate (OCR) was measured using a Seahorse XF-24 analyzer (Seahorse Bioscience Inc., North Billerica, MA, USA) in 24-well plates. Hepatocytes were seeded at 2 × 10⁴ cells per well 24 h before the analysis. On the day before the OCR measurement, the sensor cartridge was placed into calibration buffer (Seahorse Bioscience) and incubated in a non-CO₂ incubator at 37 °C. Hepatocytes were washed and incubated in DMEM without sodium bicarbonate. The medium and mitochondrial OXPHOS inhibitors were adjusted to pH 7.4 on the day of the OCR assay. The basal OCR was measured three times, and three readings were taken after the addition of each mitochondrial OXPHOS inhibitor [oligomycin (2 µg/mL) and rotenone (1 µM)]. The basal and post-oligomycin OCRs were calculated by averaging the last three measurements after maintaining a steady state. Coupled respiration was expressed as the percent decrease from basal respiration. Additionally, carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 µM) was used to measure maximal mitochondrial respiration of the cells. OCR was automatically calculated and recorded by the sensor cartridge and the Seahorse XF-24 software.