Mitochondrial respiration was studied in saponin-skinned fibers (35). Fiber bundles were mechanically separated with tongs and permeabilized with saponin (60 mg/l, 20 minutes). Bundles were then washed three times for 10 minutes to remove ADP, creatine phosphate, soluble enzymes and metabolites. Fiber respiration rates were measured at 25°C using an oxygraph system (Hansatech Instruments). Different substrates were used as follows: glutamate 5mM + malate 2mM as complex 1 substrates; succinate 5mM + rotenone 2.2µM as a complex 2 substrates with inhibition of complex 1 by rotenone; octanoyl-carnitine (110µM) or palmitoyl-carnitine (55µM) in presence of 1mM malate, as -oxydation substrates. State 3 was measured in the presence of respiratory substrates after the addition of 1mM ADP and state 4 was measured after the addition of 60µM of atractyloside, a potent inhibitor of the ATP/ADP carrier. State 4 was considered as the control state of respiration. Finally, fibers were dried for 24 hours at 100°C and weighted. Respiration rates were expressed as nanoatoms (nat) O/(min.mg dried fiber). Respiratory control ratio was calculated by dividing state 3 by state 4 respiration rates.
