Palmitoylcarnitine

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.

Tissue respiration was performed using an O2K high-resolution respirometer (Oroboros, Austria). Freshly isolated tissues were rapidly weighed (BAT 2–5 mg), minced, and homogenized using a PBI-shredder PBI set (Oroboros, Austria). Homogenized tissue was further re-suspended in 2 mL of MiR05 medium (0.5 mM of EGTA, 3 mM of MgCl2, 60 mM of K-lactobionate, 20 mM of Taurine, 10 mM of KH₂PO₄, 20 mM of HEPES, 110 mM of Sucrose, and 1 g/L of BSA (essentially fatty acid free)). Respiratory oxygen flux was measured in real time and expressed as picomoles of O₂ per second per mg of tissue. 5 mM of pyruvate (Sigma), 2 mM of malate (Sigma), and 1 mM of ADP (Sigma) were added to stimulate complex I respiration. 1 µM of rotenone (Sigma) and 5 mM of succinate (Sigma) were added to test complex II respiration. 2 mM of ascorbate and 0.5 mM of N, N, N, N-tetramethyl-p-phenylenediamine (TMPD) (Sigma) were used to test complex IV respiration. To measure uncoupled respiration, succinate (Sigma) was used as the substrate, and 1 μM of oligomycin (Sigma) was added to determine the coupled respiration. Uncoupled respiration was calculated using the respiration rate after oligomycin deducted by respiration rate after antimycin A (1 μM) (Sigma) inhibition. To measure mitochondrial β-oxidation, palmitoyl-carnitine (5 μM) (Sigma) was added to the chamber.