Mitochondrial respiration was evaluated as O2 consumption in isolated liver as previously described (28 (link)). Mitochondria were supplemented with substrates 10 mM glutamate/2 mM malate (Sigma-Aldrich) and 10 mM succinate/0.5 mM rotenone (Sigma-Aldrich), to measure adenosine diphosphate (ADP)–independent respiration activity (state 4). After addition of 1 mM ADP (Sigma-Aldrich), state 3 respiration activity was measured. Respiration was uncoupled by successive addition of carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) up to 3 mM to reach maximal respiration.
Fatty acid oxidation was measured from isolated liver mitochondria from mice fasted for 5 hours before sacrifice using the Oxygraph 2K respirometer (Oroboros Oxygraph-2K, Oroboros Instruments) as described in (51 (link)) and reviewed in (52 ). State 4 respiration was measured using 5 mM malate (Sigma-Aldrich) and 1 mM palmitoyl carnitine (Sigma-Aldrich), and state 3 respiration was measured using 1 mM K-ADP (Sigma-Aldrich) and 10 mM K-succinate (Sigma-Aldrich). Inhibitors used included 0.5 mM oligomycin (Sigma-Aldrich) and 2.5 μM antimycin. FCCP was used as a measure of uncoupling and maximal respiration rate in titrations up to 3 μM.
Fatty acid oxidation was measured from isolated liver mitochondria from mice fasted for 5 hours before sacrifice using the Oxygraph 2K respirometer (Oroboros Oxygraph-2K, Oroboros Instruments) as described in (51 (link)) and reviewed in (52 ). State 4 respiration was measured using 5 mM malate (Sigma-Aldrich) and 1 mM palmitoyl carnitine (Sigma-Aldrich), and state 3 respiration was measured using 1 mM K-ADP (Sigma-Aldrich) and 10 mM K-succinate (Sigma-Aldrich). Inhibitors used included 0.5 mM oligomycin (Sigma-Aldrich) and 2.5 μM antimycin. FCCP was used as a measure of uncoupling and maximal respiration rate in titrations up to 3 μM.
