No abstract

In response to hypoxic stress, many animals compensate for a reduced cellular O 2 supply by suppressing total metabolism, thereby reducing O 2 demand. For small endotherms that are native to high-altitude environments, this is not always a viable strategy, as the capacity for sustained aerobic thermogenesis is critical for survival during periods of prolonged cold stress. For example, survivorship studies of deer mice (Peromyscus maniculatus) have demonstrated that thermogenic capacity is under strong directional selection at high altitude. Here, we integrate measures of whole-organism thermogenic performance with measures of metabolic enzyme activities and genomic transcriptional profiles to examine the mechanistic underpinnings of adaptive variation in this complex trait in deer mice that are native to different elevations. We demonstrate that highland deer mice have an enhanced thermogenic capacity under hypoxia compared with lowland conspecifics and a closely related lowland species, Peromyscus leucopus. Our findings suggest that the enhanced thermogenic performance of highland deer mice is largely attributable to an increased capacity to oxidize lipids as a primary metabolic fuel source. This enhanced capacity for aerobic thermogenesis is associated with elevated activities of muscle metabolic enzymes that influence flux through fatty-acid oxidation and oxidative phosphorylation pathways in high-altitude deer mice and by concomitant changes in the expression of genes in these same pathways. Contrary to predictions derived from studies of humans at high altitude, our results suggest that selection to sustain prolonged thermogenesis under hypoxia promotes a shift in metabolic fuel use in favor of lipids over carbohydrates.functional genomics | RNA-seq | thermoregulation | transcriptomics D uring cold stress, homeothermic endotherms maintain a constant body temperature by increasing metabolic heat production. In small endotherms like mice that have high thermoregulatory demands, thermogenic capacity influences survival in cold environments and therefore has a clear connection to Darwinian fitness (1, 2). Indeed, thermogenic capacity in freeranging deer mice (Peromyscus maniculatus) is subject to strong directional selection at high altitude (3).Sustaining maximal thermogenic capacities during prolonged periods of cold stress requires a high rate of O 2 flux through oxidative pathways, and this requirement presents a unique challenge for endothermic animals that live under conditions of chronic O 2 deprivation at high altitude. The reduced partial pressure of O 2 (PO 2 ) at high altitude imposes well-documented constraints on aerobic metabolism (4-8), thereby exacerbating the increased thermoregulatory demands faced by endothermic animals that are native to cold, alpine environments.In rodents, aerobic thermogenesis is accomplished through both shivering and nonshivering mechanisms, and in deer mice, shivering accounts for roughly 35-50% of total thermogenic capacity (9). As with other forms of strenuous exerci...

The hypoxic and cold environment at high altitudes requires that small mammals sustain high rates of O2 transport for exercise and thermogenesis while facing a diminished O2 availability. We used laboratory-born and -raised deer mice (Peromyscus maniculatus) from highland and lowland populations to determine the interactive effects of ancestry and hypoxia acclimation on exercise performance. Maximal O₂consumption (V̇o(2max)) during exercise in hypoxia increased after hypoxia acclimation (equivalent to the hypoxia at ∼4,300 m elevation for 6-8 wk) and was consistently greater in highlanders than in lowlanders. V̇o(2max) during exercise in normoxia was not affected by ancestry or acclimation. Highlanders also had consistently greater capillarity, oxidative fiber density, and maximal activities of oxidative enzymes (cytochrome c oxidase and citrate synthase) in the gastrocnemius muscle, lower lactate dehydrogenase activity in the gastrocnemius, and greater cytochrome c oxidase activity in the diaphragm. Hypoxia acclimation did not affect any of these muscle traits. The unique gastrocnemius phenotype of highlanders was associated with higher mRNA and protein abundances of peroxisome proliferator-activated receptor γ (PPARγ). Vascular endothelial growth factor (VEGFA) transcript abundance was lower in highlanders, and hypoxia acclimation reduced the expression of numerous genes that regulate angiogenesis and energy metabolism, in contrast to the observed population differences in muscle phenotype. Lowlanders exhibited greater increases in blood hemoglobin content, hematocrit, and wet lung mass (but not dry lung mass) than highlanders after hypoxia acclimation. Genotypic adaptation to high altitude, therefore, improves exercise performance in hypoxia by mechanisms that are at least partially distinct from those underlying hypoxia acclimation.

In species that are distributed across steep environmental gradients, adaptive variation in physiological performance may be attributable to transcriptional plasticity in underlying regulatory networks. Here we report the results of common-garden experiments that were designed to elucidate the role of regulatory plasticity in evolutionary adaptation to hypoxic cold-stress in deer mice (Peromyscus maniculatus). We integrated genomic transcriptional profiles with measures of metabolic enzyme activities and whole-animal thermogenic performance under hypoxia in highland (4350 m) and lowland (430 m) mice from three experimental groups: (1) wild-caught mice that were sampled at their native elevations; (2) wild-caught/lab-reared mice that were deacclimated to low-elevation conditions in a common-garden lab environment; and (3) the F1 progeny of deacclimated mice that were maintained under the same low-elevation common-garden conditions. In each experimental group, highland mice exhibited greater thermogenic capacities than lowland mice, and this enhanced performance was associated with upregulation of transcriptional modules that influence several hierarchical steps in the O2 cascade, including tissue O2 diffusion (angiogenesis) and tissue O2 utilization (metabolic fuel use and cellular oxidative capacity). Most of these performance-related transcriptomic changes occurred over physiological and developmental timescales, suggesting that regulatory plasticity makes important contributions to fitness-related physiological performance in highland deer mice.

Both exercise training and cold acclimatization induce muscle remodelling in vertebrates, producing a more aerobic phenotype. In ectothermic species exercise training and cold-acclimatization represent distinct stimuli. It is currently unclear if these stimuli act through a common mechanism or if different mechanisms lead to a common phenotype. The goal of this study was to survey responses that represent potential mechanisms responsible for contractionand temperature-induced muscle remodelling, using an ectothermic vertebrate. Separate groups of adult zebrafish (Danio rerio) were either swim trained or cold acclimatized for 4 weeks. We found that the mitochondrial marker enzyme citrate synthase (CS) was increased by 1.5× in cold and by 1.3× with exercise (P < 0.05). Cytochrome c oxidase (COx) was increased by 1.2× following exercise training (P < 0.05) and 1.2× (P = 0.07) with cold acclimatization. However, only cold acclimatization increased β-hydroxyacyl-CoA dehydrogenase (HOAD) compared to exercise-trained (by 1.3×) and pyruvate kinase (PK) relative to control zebrafish. We assessed the whole-animal performance outcomes of these treatments. Maximum absolute sustained swimming speed (U crit ) was increased in the exercise trained group but not in the cold acclimatized group. Real-time PCR analysis indicated that increases in CS are primarily transcriptionally regulated with exercise but not with cold treatments. Both treatments showed increases in nuclear respiratory factor (NRF)-1 mRNA which was increased by 2.3× in cold-acclimatized and 4× in exercise-trained zebrafish above controls. In contrast, peroxisome proliferator-activated receptor (PPAR)-α mRNA levels were decreased in both experimental groups while PPAR-β 1 declined in exercise training only. Moreover, PPAR-γ coactivator (PGC)-1α mRNA was not changed by either treatment. In zebrafish, both temperature and exercise produce a more aerobic phenotype, but there are stimulus-dependent responses (i.e. HOAD and PK activities). While similar changes in NRF-1 mRNA suggest that common responses might underlie aerobic muscle remodelling there are distinct changes (i.e. CS and PPAR-β 1 mRNA) that contribute to specific temperature-and exercise-induced phenotypes.