Mitochondrial Function Assay in Tissues

Immediately after harvest liver, quadriceps muscle, and kidneys were placed in ice-cold STE1 buffer (250 mmol/L sucrose, 5 mmol/L Tris/HCl, 2 mmol/L EGTA, pH 7.4). Tissues were minced and either incubated in 2.5 mL STE2 buffer (STE1 containing [wt/vol] 0.5% BSA, 5 mmol/L MgCl₂, 1 mmol/L ATP, and 2.5 U/mL protease Subtilisin A) for 4 min (quadriceps muscle) or immediately proceeded to homogenization (liver and kidney tissue). All tissues were homogenized using a Teflon pistil in a Potter-Elvejhem homogenizer. Quadriceps muscle homogenates were further diluted with 2.5 mL STE1 buffer containing Complete Mini protease inhibitor cocktail (Roche, Mannheim, Germany), centrifuged at 8,000 g for 10 min, and the resulting pellet was resuspended in 4 mL STE1 buffer. Next, all tissues homogenates were centrifuged at 800 g for 10 min and supernatants were centrifuged at 8,000 g for 10 min. Pellets obtained from mitochondrial isolation were resuspended in 200 µL STE1 buffer each. State III oxygen consumption rates (V_ADP) were determined in 300 µg of mitochondria by using a Clark-type oxygen electrode (Strathkelvin, North Lanarkshire, Scotland) with 20 µmol/L palmitoyl-carnitine (PC)/2 mmol/L malate or 10 mmol/L pyruvate/5 mmol/L malate as substrates as previously described (14 (link)).