SUMMARYFlying birds couple a high daily energy turnover with double-digit millimolar blood glucose concentrations and insulin resistance. Unlike mammalian muscle, flight muscle predominantly relies on lipid oxidation during locomotion at high fractions of aerobic capacity, and birds outlive mammals of similar body mass by a factor of three or more. Despite these intriguing functional differences, few data are available comparing fuel oxidation and free radical production in avian and mammalian skeletal muscle mitochondria. Thus we isolated mitochondria from English sparrow pectoralis and rat mixed hindlimb muscles. Maximal O 2 consumption and net H 2 O 2 release were measured in the presence of several oxidative substrate combinations. Additionally, NADand FAD-linked electron transport chain (ETC) capacity was examined in sonicated mitochondria. Sparrow mitochondria oxidized palmitoyl-L-carnitine 1.9-fold faster than rat mitochondria and could not oxidize glycerol-3-phosphate, while both species oxidized pyruvate, glutamate and malate-aspartate shuttle substrates at similar rates. Net H 2 O 2 release was not significantly different between species and was highest when glycolytic substrates were oxidized. Sonicated sparrow mitochondria oxidized NADH and succinate over 1.8 times faster than rat mitochondria. The high ETC catalytic potential relative to matrix substrate dehydrogenases in sparrow mitochondria suggests a lower matrix redox potential is necessary to drive a given O 2 consumption rate. This may contribute to preferential reliance on lipid oxidation, which may result in lower in vivo reactive oxygen species production in birds compared with mammals.Key words: skeletal muscle mitochondria, substrate oxidation, aging, reactive oxygen species, bird. THE JOURNAL OF EXPERIMENTAL BIOLOGY 2040to complete substrate oxidation pathways would be coupled with lower superoxide production (measured as net H 2 O 2 release) in mitochondria from English sparrow (Passer domesticus) pectoralis compared with rat (Rattus norvegicus) mixed hindlimb muscle. Thus, one purpose of the present study was to measure the maximum (state 3) mitochondrial O 2 consumption rate (J . O2 ), ETC flux capacity in sonicated mitochondria from three sites of electron entry, and ROS production by intact 'resting' (oligomycin-inhibited) mitochondria. To our knowledge, no investigation into ROS production from avian muscle mitochondria currently exists.Fuel selection by contracting muscles during locomotion differs markedly between birds and mammals. Humans and rats run at 75% of their maximum rate of O 2 uptake (V O2,max ) with a respiratory quotient (RQ) at or above 0.90 (Brooks and Donovan, 1983;Brooks and Gaesser, 1980; O'Brien et al., 1993), which reflects a general pattern of carbohydrate dependence in mammals exercising at or above moderate intensity (Roberts et al., 1996). In contrast, pigeons fly at this intensity with an RQ of 0.73 (Rothe, 1987), indicating that pigeon flight muscle supports locomotion with the almost exclusive oxidatio...

Birds represent an anomaly as they couple a long life span with a high metabolic rate, hyperglycemia, and insulin resistance. Despite the strong links between mitochondria (mito) and aging/longevity, no comprehensive investigation has assessed avian mitochondrial function. Mito were isolated from English sparrow pectoralis and rat mixed hindlimb muscles. Maximal O2 consumption (Jo) and reactive oxygen species (ROS) production were measured in the presence of several oxidative substrates. NAD‐ and FAD‐linked electron transport chain (ETC) capacity was examined in sonicated mito. Compared to rat, sparrow muscle mito oxidize palmitoyl‐l‐carnitine 1.9‐fold faster (211.9 ± 25.6 vs 396.3 ± 36.4 nmol/mg/min), cannot oxidize glycerol‐3‐phosphate, and produce 60% less ROS with complex II linked substrates and both cytosolic‐mitochondrial electron shuttles. Despite similar maximal Jo for the majority of substrates, sonicated sparrow mito oxidize NADH and succinate 3‐ to 5‐fold faster than rat mito, 1809.34 ± 272.05 vs. 647.53 ± 73.07 and 416.93 ± 49.12 vs. 86.83 ± 9.23 nmol/mg/min, respectively. High ETC catalytic potential relative to matrix substrate dehydrogenases in sparrow mito suggests lower matrix redox potential necessary to drive a given O2 consumption rate, which may contribute to the lower ROS production observed in birds compared to mammals. Research supported by a grant from the NSF (IBN‐0116997).

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Birds are able to fuel flight, a moderate‐high intensity exercise, with fat, while mammals depend on carbohydrates for fuel at the same relative intensity. This difference in fuel selection persists in mitochondria (MITO) isolated from the skeletal muscle of these species, suggesting fuel selection can be controlled at the level of the MITO matrix. To compare the capacity rat and sparrow MITO to harvest electrons to the ability of the electron transport chain (ETC) to oxidize them, we assessed the activities of CAC and related enzymes, the Vmax of NADH oxidation from Complex I → IV, and cytochrome b content via myxothiazol titration. The Vmax of NADH oxidation (1074 ± 118 vs 516 ± 42 nmol O2·mg−1·min−1), as well as the cytochrome b content (1.50 vs 1.06 nmol·mg mitochondrial protein−1), were greater in sparrow compared to rat. These data suggests a greater capacity of sparrow ETC to oxidize electrons compared to the capacity of the dehydrogenases to produce them. This balance may result in a lower oxidation‐reduction potential in sparrow MITO, decreasing the inhibition of NADH on β‐oxidation and allowing fatty acid oxidation to proceed. Rat Sparrow nmol·mg mito protein−1·min−1 Isocitrate Dehydrogenase (IDH) 276 ± 48 366 ± 51 Malate Dehydrogenase (MDH) 20600 ± 2628 22209 ± 2309 Citrate Synthase (CS) 2748 ± 258 4343 ± 600 Glutamate Dehydrogenase (GDH) 399 ± 51 51 ± 9 Aspartate Aminotransferase (AspAT) 5667 ± 233 19326 ± 1214