Regulation of muscle pyruvate dehydrogenase activity and exercise fuel use in high-altitude deer mice.

Adult, lab-reared, highland deer mice acclimate to hypoxia by increasing reliance on carbohydrates to fuel exercise. Yet neither the underlying mechanisms for this shift in fuel use pattern, nor the impact of lifetime hypoxia exposure experienced in the high alpine, are fully understood. Thus, we assessed exercise fuel use in wild highland deer mice running in their native environment. We examined a key step in muscle carbohydrate oxidation, pyruvate dehydrogenase (PDH) and its regulation with exercise at altitude in wild highlanders and using first generation (G1) lab born and raised highlanders acclimated to normoxia or hypoxia. PDH activity was also determined in the gastrocnemius of G1 highlanders using an in situ muscle preparation. We found wild highlanders had a high reliance on carbohydrates while running in their native environment, consistent with data from hypoxia acclimated G1 highlanders. PDH activity in the gastrocnemius was similar post exercise between G1 and wild highlanders. However, when the gastrocnemius was stimulated at a light work rate in situ, PDH activity was higher in hypoxia acclimated G1 highlanders, and associated with a lower intramuscular lactate. These findings were supported by lower PDH kinase 2 protein expression in hypoxia acclimated G-1 mice. Our findings indicate that adult phenotypic plasticity in response to low oxygen is sufficient to increase exercise carbohydrate reliance in highland deer mice. Additionally, variation in PDH regulation with hypoxia acclimation contributes to shifts in whole-animal fuel use patterns and are likely to improved exercise performance via elevated energy yield per mole O2.

Migration increases mitochondrial oxidative capacity without increasing reactive oxygen species emission in a songbird.

Birds remodel their flight muscle metabolism prior to migration to meet the physiological demands of migratory flight, including increases in both oxidative capacity and defence against reactive oxygen species. The degree of plasticity mediated by changes in these mitochondrial properties is poorly understood but may be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or in individual mitochondrial function. We tested these hypotheses using yellow-rumped warblers (Setophaga coronata), a Nearctic songbird which biannually migrates 2000-5000 km. We predicted higher flight muscle mitochondrial abundance and substrate oxidative capacity, and decreased reactive oxygen species emission in migratory warblers captured during autumn migration compared with a short-day photoperiod-induced non-migratory phenotype. We assessed mitochondrial abundance via citrate synthase activity and assessed isolated mitochondrial function using high-resolution fluororespirometry. We found 60% higher tissue citrate synthase activity in the migratory phenotype, indicating higher mitochondrial abundance. We also found 70% higher State 3 respiration (expressed per unit citrate synthase) in mitochondria from migratory warblers when oxidizing palmitoylcarnitine, but similar H2O2 emission rates between phenotypes. By contrast, non-phosphorylating respiration was higher and H2O2 emission rates were lower in the migratory phenotype. However, flux through electron transport system complexes I-IV, II-IV and IV was similar between phenotypes. In support of our hypotheses, these data suggest that flight muscle mitochondrial abundance and function are seasonally remodelled in migratory songbirds to increase tissue oxidative capacity without increasing reactive oxygen species formation.

Mitochondrial techniques for physiologists.

Mitochondria serve several important roles in maintaining cellular homeostasis, including adenosine triphosphate (ATP) synthesis, apoptotic signalling, and regulation of both reactive oxygen species (ROS) and calcium. Therefore, mitochondrial studies may reveal insights into metabolism at higher levels of physiological organization. The apparent complexity of mitochondrial function may be daunting to researchers new to mitochondrial physiology. This review is aimed, therefore, at such researchers to provide a brief, yet approachable overview of common techniques used to assess mitochondrial function. Here we discuss the use of high-resolution respirometry in mitochondrial experiments and common analytical platforms used for this technique. Next, we compare the use of common mitochondrial preparation techniques, including adherent cells, tissue homogenate, permeabilized fibers and isolated mitochondria. Finally, we outline additional techniques that can be used in tandem with high-resolution respirometry to assess additional aspects of mitochondrial metabolism, including ATP synthesis, calcium uptake, membrane potential and reactive oxygen species emission. We also include limitations to each of these techniques and outline recommendations for experimental design and interpretation. With a general understanding of methodologies commonly used to study mitochondrial physiology, experimenters may begin contributing to our understanding of this organelle, and how it affects other physiological phenotypes.

Function of left ventricle mitochondria in highland deer mice and lowland mice.

To gain insight into the mitochondrial mechanisms of hypoxia tolerance in high-altitude natives, we examined left ventricle mitochondrial function of highland deer mice compared with lowland native deer mice and white-footed mice. Highland and lowland native deer mice (Peromyscus maniculatus) and lowland white-footed mice (P. leucopus) were first-generation born and raised in common lab conditions. Adult mice were acclimated to either normoxia or hypoxia (60 kPa) equivalent to ~ 4300 m for at least 6 weeks. Left ventricle mitochondrial physiology was assessed by determining respiration in permeabilized muscle fibers with carbohydrates, lipids, and lactate as substrates. We also measured the activities of several left ventricle metabolic enzymes. Permeabilized left ventricle muscle fibers of highland deer mice showed greater rates of respiration with lactate than either lowland deer mice or white-footed mice. This was associated with higher activities of lactate dehydrogenase in tissue and isolated mitochondria in highlanders. Normoxia-acclimated highlanders also showed higher respiratory rates with palmitoyl-carnitine than lowland mice. Maximal respiratory capacity through complexes I and II was also greater in highland deer mice but only compared with lowland deer mice. Acclimation to hypoxia had little effect on respiration rates with these substrates. In contrast, left ventricle activities of hexokinase increased in both lowland and highland deer mice after hypoxia acclimation. These data suggest that highland deer mice support an elevated cardiac function in hypoxia, in part, with high ventricle cardiomyocyte respiratory capacities supported by carbohydrates, fatty acids, and lactate.

Plasticity of non-shivering thermogenesis and brown adipose tissue in high-altitude deer mice.

High altitude environments challenge small mammals with persistent low ambient temperatures that require high rates of aerobic heat production in face of low O2 availability. An important component of thermogenic capacity in rodents is non-shivering thermogenesis (NST) mediated by uncoupled mitochondrial respiration in brown adipose tissue (BAT). NST is plastic, and capacity for heat production increases with cold acclimation. However, in lowland native rodents, hypoxia inhibits NST in BAT. We hypothesize that highland deer mice (Peromyscus maniculatus) overcome the hypoxic inhibition of NST through changes in BAT mitochondrial function. We tested this hypothesis using lab born and raised highland and lowland deer mice, and a lowland congeneric (Peromyscus leucopus), acclimated to either warm normoxia (25°C, 760 mmHg) or cold hypoxia (5°C, 430 mmHg). We determined the effects of acclimation and ancestry on whole-animal rates of NST, the mass of interscapular BAT (iBAT), and uncoupling protein (UCP)-1 protein expression. To identify changes in mitochondrial function, we conducted high-resolution respirometry on isolated iBAT mitochondria using substrates and inhibitors targeted to UCP-1. We found that rates of NST increased with cold hypoxia acclimation but only in highland deer mice. There was no effect of cold hypoxia acclimation on iBAT mass in any group, but highland deer mice showed increases in UCP-1 expression and UCP-1-stimulated mitochondrial respiration in response to these stressors. Our results suggest that highland deer mice have evolved to increase the capacity for NST in response to chronic cold hypoxia, driven in part by changes in iBAT mitochondrial function.