We measured metabolic enzyme activities and genomic transcriptional profiles of skeletal muscle from both highland and lowland deer mice to investigate the mechanistic underpinnings of adaptive population differences in shivering thermogenesis. The functional genomic analysis revealed significant among-treatment variation in transcript abundance in 11.7% of measured genes (Table 2), but only 0.7% of the genes exhibited constitutive expression differences between highland and lowland mice across the three experimental groups. In comparisons between highland and lowland mice, transcriptional modules that are associated with both thermogenic capacity and endurance (P4 and T16) tended to have similar expression profiles in the deacclimation and F1 groups (Fig. 4). These results suggest that much of the observed plasticity in whole-animal thermogenic performance may stem from plasticity in a small number of discrete transcriptional modules. A similar study of a broadly distributed Andean bird species (Zonotrichia capensis) also documented a large degree of transcriptomic plasticity in skeletal muscle in response to changes in elevation (Cheviron et al. 2008). Together, these studies suggest that regulatory plasticity may make significant contributions to the niche breadth of species that have broad altitudinal distributions.
TRANSCRIPTOMIC CORRELATES OF HYPOXIC THERMOGENIC PERFORMANCE
Although many genes exhibited significant expression differences between populations and among acclimation treatments, only a small subset of these genes was associated with variation in thermogenic performance. Genes that exhibited different expression levels between populations or among treatments clustered into a relatively small number of highly intercorrelated transcriptional modules (Fig. 2). Many of these modules were enriched for genes that play a role in one or more steps of the O2 transport cascade (Tables S3, S4), but only five modules were significantly associated with one or both aspects of thermogenic performance. These performance-associated modules were significantly enriched for genes that influence tissue vascularization, skeletal muscle growth, metabolic fuel use, and mitochondrial oxidative capacity, suggesting that elevated thermogenic performance under hypoxia is associated with simultaneous modifications of multiple, intersecting pathways (Fig. 3).
Our previous analyses suggested that the elevated thermogenic capacities of highland mice were largely attributable to changes in gene expression that enhance lipid oxidation and mitochondrial oxidative capacities (Cheviron et al. 2012). The objective of this study was to assess the extent to which these performance-associated transcriptomic differences are attributable to regulatory plasticity. Highland mice in the in situ and deacclimation treatment group exhibited elevated HOAD and COX enzyme activities, suggesting greater cellular lipid oxidation and mitochondrial oxidative capacities in skeletal muscle, and these differences were mirrored by the concomitant upregulation of genes across the lipid oxidation and OXPHOS pathways. These results suggest that variation in enzyme activities mainly stems from variation in enzyme concentration, which is largely a function of mRNA transcript abundance (i.e., transcriptional hierarchical regulation; Suarez et al. 2005; Table S6). Interestingly, traditional biomarkers of mitochondrial biogenesis (e.g., PGC-1α and Tfam) did not exhibit significant expression differences between populations or across acclimation groups after genome-wide FDR correction, suggesting that the elevated oxidative capacities of highland mice may largely stem from gene expression changes that influence the concentration of mitochondrial oxidative enzymes rather than changes in mitochondrial production. However, direct measures of mitochondrial abundance are required to confirm this. Although constitutive expression may not differ between highland and lowland deer mice, populations may exhibit differences in inducible expression in response to energetic and environmental cues.
In the case of the F1 mice, neither HOAD nor COX activities were significantly different between the progeny of highland natives and the progeny of lowland natives that were born and reared under common-garden, low-elevation conditions. Although there was a trend toward upregulation of gene expression across the lipid oxidation pathway in the highland F1 mice, the direction of gene expression change was random across the OXPHOS pathway. These results suggest a degree of developmental plasticity in the regulation of genes that influence cellular lipid oxidation and mitochondrial oxidative capacities, and this plasticity likely makes important contributions to thermogenic capacity at high elevation. Consistent with this idea, there was no difference in thermogenic capacity between the in situ lowland mice and the F1 highland mice, nor was there a difference between these two groups in the activities of any of the aerobic enzymes (Table S5).
Despite the distinct patterns of phenotypic plasticity for thermogenic capacity and endurance (Fig. 1), environmentally induced changes in both performance measures were associated with expression changes in the same transcriptional modules (Table 3). Although smaller modules were unique to either thermogenic capacity or thermogenic endurance, these performance-associated modules were enriched for similar metabolic gene ontology terms (Fig. 3). Thus, there is little evidence that functional uncoupling of thermogenic capacity and endurance stems from differences in the transcriptional program in skeletal muscle. Instead, functional uncoupling of these performance measures may stem from plasticity in other steps of the O2 transport cascade, differences in substrate availability, or nonshivering components of thermogenic performance.
Our combined results suggest that high-altitude adaptation and acclimatization in deer mice involves the maintenance of a highly aerobic phenotype in the face of reduced O2 availability. Elite endurance athletes and highly aerobic nonhuman mammals are characterized by an enhanced capacity for fatty acid oxidation during exercise in normoxia (Bjorntorp 1991; McClelland et al. 1994; Henriksson and Hickner 1996; Bangsbo et al. 2006; Weber 2011; Templeman et al. 2012), and the changes in lipid oxidation and aerobic capacities across the acclimation treatments mirror physiological changes associated with cold exposure and winter acclimatization in rodents (Wickler 1981; Vaillancourt et al. 2009). Effectively allocating fuel substrates for oxidative metabolism is especially critical at high elevation. Relative to carbohydrates, the oxidation of lipids produces a higher overall yield of ATP per unit of fuel at the expense of increased O2 consumption. The stoichiometric advantage of carbohydrate metabolism under O2 deprivation has led to the suggestion that a shift in metabolic fuel selection in favor of carbohydrates may represent a general feature of high-altitude adaptation (Hochachka 1985). Indeed, high-altitude human populations (Sherpas and Andean Quechuas) exhibit enhanced glucose uptake, and a greater reliance on glucose for ATP production in cardiac muscle while at rest (Holden et al. 1995; Hochachka et al. 1996). Similarly, high-elevation leaf-eared mice (Phyllotis andium and Phyllotis xanthopygus) also use proportionally more carbohydrates while resting and during submaximal exercise compared to lowland congeners (P. amicus and P. limatus; Schippers et al. 2012). Our results, however, indicate the highland deer mice employ the opposite strategy to enhance thermogenic capacity under hypoxia, increasing their capacities to oxidize lipids during aerobic thermogenesis (Cheviron et al. 2012). These differences in fuel use strategies may stem from other energetic tradeoffs beyond the efficient use of O2. Although the oxidation of glucose yields ∼15% more ATP per mole of O2 (Brand 2005), lipids make up more than 80% of the total energy reserves in mammals, and the energy density of lipids is an order magnitude greater than that of carbohydrates (Weber 2011). These energetic advantages of lipids make them the preferred fuel source during periods of sustained submaximal exercise at low elevation (McClelland et al. 1994; McClelland 2004; Weber 2011), and during high-intensity shivering thermogenesis (Weber and Haman 2005; Vaillancourt et al. 2009). Together these studies of highland mammals suggest that optimal fuel use strategies at high elevation may depend on the intensity and nature of different aerobic activities (i.e., exercise vs. thermogenesis).
As with winter-acclimatized lowland rodents, an elevated capacity for fatty acid oxidation could enhance thermogenic performance, but under hypoxic conditions at high altitude, this would require additional physiological changes to ensure adequate O2 flux through oxidative pathways. The elevated hemoglobin-O2 affinity of highland deer mice safeguards arterial O2 saturation at low PO2, thereby preserving an adequate level of tissue O2 delivery in spite of hypoxia (Storz et al. 2009; Storz et al. 2010a; Natarajan et al. 2013). Highland mice exhibit plasticity in hematological traits such as hemoglobin concentration that enhance blood O2 carrying capacity (Tufts et al. 2013), and several regulatory modules were enriched for genes involved in angiogenesis that may promote an increased capacity for tissue O2 diffusion. Changes in tissue O2 oxygenation may help to power an enhanced capacity for lipid oxidation, underscoring the importance of integrated physiological responses to the challenges of life at high elevation.
Although we have focused on mechanisms that can enhance the capacity for shivering thermogenesis in skeletal muscle, deer mice also rely heavily on nonshivering mechanisms (Van Sant and Hammond 2008). Brown adipose tissue (BAT) is the primary site of nonshivering thermogenesis, and the size of BAT depots decreases dramatically with warm acclimation and seasonal acclimatization (Didow and Hayward 1969; Himms-Hagen 1985; Rafael et al. 1985; Klaus et al. 1988; Cannon and Nedergaad 2004). Regression of BAT depots across our acclimation treatments could lead to an increased reliance on shivering thermogenesis in the warm-acclimated mice, which would not only reduce total thermogenic capacity, but would also compound the effects of muscular atrophy associated with inactivity in captivity (Cheviron et al. 2013). Consistent with this idea, several modules associated with thermogenic performance were enriched for genes that influence muscle growth, and these modules were generally downregulated across the acclimation treatments (Figs. 3, 4). Because limb muscles like the gastrocnemis play a primary role in locomotion, the extent to which these transcriptomic changes are associated with reduced shivering thermogenesis or reduced activity in captivity is not known. Nonetheless, a similar integrative analysis of plasticity in nonshivering thermogenic performance would likely be fruitful avenue for future research.