Mitochondrial respiration assays

Mitochondrial respiration of liver homogenate was measured at each time-point. Respiration was measured in 2-mL chambers using an OROBOROS Oxygraph 2K (Anton Paar, Graz, Austria) at 37  °C in mitochondrial respiration media, with a calculated saturated oxygen concentration of 190 nmol O₂ per milliliter at 100 kPa barometric pressure, and oxygen flux calculated using the DatLab 5 analysis software. Liver homogenate (50 μL) was added to each chamber, and the remainder was stored at -80  °C for later analysis. To account for potential variations in mitochondrial mass, citrate synthase (CS)-normalized[16] oxygen flux (pmol O₂.s^-1.U CS^-1) was calculated.

A multiple substrate-inhibitor titration protocol was used to explore relative contributions of complex I (CI), complex II (CII), and combined CI+CII in the electron transport system (ETS). Respiration states were defined according to Gnaiger[17], where leak respiration and oxidative phosphorylation (OXPHOS) were the flux measured before and after addition of adenosine diphosphate (ADP), respectively. The assay protocol steps are described in Table ​Table3.3. The integrity of tissue preparations and comparison of coupling efficiencies were made from the respiratory control ratio (RCR).

Mitochondrial respiration assay protocol