4.2. Measurement of Mitochondrial Oxygen Consumption

Mitochondria were isolated from mouse liver (ICR; 7–10-week-old females) by differential centrifugation as described previously [17,51]. Mitochondrial oxygen consumption was measured using a Clark-type oxygen electrode (Strathkelvin Instruments Ltd., North Lanarkshire, Scotland, UK) as described previously [52,53]. Briefly, the mitochondria-enriched fraction was incubated at 30 °C in an oxygen measurement buffer (225 mM mannitol, 75 mM sucrose, 5 mM succinate, 5 mM glutamate, 10 mM KCl, 0.1 mM EDTA, 3 mM phosphate, and 20 mM Tris-HCl, pH 7.4) in the presence of a vehicle (DMSO or EtOH) or various concentrations of DIF-1 and its derivatives. After recording “State 4” (resting) respiration reaction, an aliquot of ADP was added to a final concentration of 200 μM to induce “State 3” respiration reaction [17,51].