2.1. Isolation of rat liver mitochondria

Rat liver mitochondria were isolated accordance the standard protocol [2]. Male rat was decapitated and the liver was quickly extracted and placed into ice-cold isolation buffer containing 250 mM sucrose, 3 mM Tris–HCl (pH 7.3), and 0.5 mM ethylene glycol tetraacetic acid (EGTA). The decapitation procedure of fasted animals is mandatory in isolating rat liver mitochondria. Then the liver was minced with scissors, washed out by the medium, transferred into a Potter-Elvehjem glass homogenizer and homogenized using a teflon pestle. The liver homogenate was centrifuged at 800×g for 7.5 min, then the pellet has been thrown out and the supernatant was centrifuged at 10,000×g for 10 min. The mitochondrial pellet was twice washed out with a buffer containing 250 mM sucrose and 3 mM Tris–HCl (pH 7.3) and centrifuged at 10,000×g for 10 min. The final pellet was resuspended in 950 μl of the wash buffer and kept on ice during the experiment. The whole process of mitochondrial isolation was carried out on ice. The mitochondrial protein content was determined by Bradford [3] and was within the range of 50–60 mg/ml.