Mitochondrial measurement of oxygen consumption rates (OCR)

To examine hepatic mitochondrial function in this model, we used a novel technology that is capable of measuring dynamic changes in mitochondrial function, not easily attainable by other technologies [17]. The Seahorse XF24 extracellular flux analyser (Seahorse Bioscience, MA, USA) uses a piston to reversibly enclose a small volume (7 μl) above the mitochondria that can monitor oxygen uptake in that volume for 2–5 min, then raises the piston, allowing the bulk incubation medium (~1 ml) to re-equilibrate. The ability to make up to four additions during the experiment allows mitochondrial respiration to be measured under various metabolic conditions. This piece of equipment has never been used to measure mitochondrial function in SS.

In this protocol, we sequentially measured mitochondrial function under various metabolic conditions [17]. To obtain basal OCR, we first added a substrate to the preparation that contained pyruvate (10 mM) and malate (2 mM). We then added ADP (adenosine- diphosphate; 4 mM) that allows ATP synthase to function, producing an increase in OCR (coupled OCR). When the ATP/ADP ratio approaches equilibrium, pmf rises, after which proton reentry through the synthase stops and respiration slows. We terminated coupled respiration by the addition of the ATP synthase inhibitor oligomycin (2.0 μM) where ATP recycling cannot contribute and there is a stable decrease in OCR. We then added the protonophore, FCCP (4 μM; carbonyl cyanide p-triflouromethoxy-phenylhydrazone). FCCP leads to uncoupled respiration in which we previously titrated the concentration of FCCP to yield a maximal effect. In mitochondria in which ATP synthase activity is a limiting factor, uncoupled respiration may be greater than coupled respiration. This indicates that the mitochondria have reached maximal respiration in uncoupled respiration in which this respiration is not limited by ATP synthase activity (i.e., ATP accumulation feedbacks on OCR inhibition). We finally added rotenone (2 μM; R) and antimycin A (2 μM; AA) which are inhibitors of the electron transport chain (ETC) Complexes, I and III, respectively. When added to the mitochondrial preparation, these inhibitors produce a marked decline in mitochondrial respiration since the electron transport chain is inhibited.

With each treatment group, we obtained measurements of basal OCR, coupled OCR, maximal OCR, and spare respiratory capacity from (uncoupled respiration minus basal OCR). We obtained two measurements during each condition in which we reported the average of the two measurements in the analysis.