Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas.

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb-O2 affinity. These experiments revealed that the effects of mutations on Hb-O2 affinity are highly dependent on the temporal order in which they occur: Each of three ß-chain substitutions produced a significant increase in Hb-O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb-O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

Phenotypic plasticity in blood-oxygen transport in highland and lowland deer mice.

In vertebrates living at high altitude, arterial hypoxemia may be ameliorated by reversible changes in the oxygen-carrying capacity of the blood (regulated by erythropoiesis) and/or changes in blood-oxygen affinity (regulated by allosteric effectors of hemoglobin function). These hematological traits often differ between taxa that are native to different elevational zones, but it is often unknown whether the observed physiological differences reflect fixed, genetically based differences or environmentally induced acclimatization responses (phenotypic plasticity). Here, we report measurements of hematological traits related to blood-O2 transport in populations of deer mice (Peromyscus maniculatus) that are native to high- and low-altitude environments. We conducted a common-garden breeding experiment to assess whether altitude-related physiological differences were attributable to developmental plasticity and/or physiological plasticity during adulthood. Under conditions prevailing in their native habitats, high-altitude deer mice from the Rocky Mountains exhibited a number of pronounced hematological differences relative to low-altitude conspecifics from the Great Plains: higher hemoglobin concentrations, higher hematocrits, higher erythrocytic concentrations of 2,3-diphosphoglycerate (an allosteric regulator of hemoglobin-oxygen affinity), lower mean corpuscular hemoglobin concentrations and smaller red blood cells. However, these differences disappeared after 6 weeks of acclimation to normoxia at low altitude. The measured traits were also indistinguishable between the F1 progeny of highland and lowland mice, indicating that there were no persistent differences in phenotype that could be attributed to developmental plasticity. These results indicate that the naturally occurring hematological differences between highland and lowland mice are environmentally induced and are largely attributable to physiological plasticity during adulthood.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges.

Semi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapnia of underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation, such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing. Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinal distributions. Regardless of their native altitude, all species have high Hb-O2 affinities, mainly due to suppressed sensitivities to allosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given that all canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, have Hb-O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and other mammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that are known to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacity for pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficient O2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that the effect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood-O2 affinity in hibernating ground squirrels.

Regulation of blood oxygen transport in hibernating mammals.

Along with the periodic reductions in O2 requirements of mammalian hibernators during winter, the O2 affinity of the blood of mammalian hibernators is seasonally regulated to help match O2 supply to consumption, contributing to limit tissue oxidative stress, particularly at arousals. Specifically, mammalian hibernators consistently show an overall increase in the blood-O2 affinity, which causes a decreased O2 unloading to tissues, while having similar or lower tissue O2 tensions during hibernation. This overview explores how the decreased body temperature and concentration of red blood cell 2,3-diphosphoglycerate (DPG) that occur in hibernation contribute separately or in combination to the concurrent increase in the O2 affinity of the hemoglobin, the O2 carrier protein of the blood. Most mammalian hemoglobins are responsive to changes in DPG concentrations, including that of the hibernating brown bear, although the smaller hibernators, such as golden-mantled ground squirrel, chipmunks, and dormice, have hemoglobins with low sensitivity to DPG. While the effect of DPG on oxygenation may vary, the decrease in body temperature invariably increases hemoglobin's O2 affinity in all hibernating species. However, the temperature sensitivity of hemoglobin oxygenation is low in hibernators compared to human, apparently due in part to endothermic allosteric quaternary transition in ground squirrels and dissociation of chloride ions in brown bears. A low heat of blood oxygenation in temporal heterotherms, like hibernators, may thus contribute to reduce heat loss, as found in regional heterotherms, like polar mammals, although the significance would be low in winter hibernation.

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation.

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.