Microbial urea-nitrogen recycling in arctic ground squirrels: the effect of ambient temperature of hibernation.

Energy conservation associated with hibernation is maximized at the intersection of low body temperature (Tb), long torpor bouts, and few interbout arousals. In the arctic ground squirrel (Urocitellus parryii), energy conservation during hibernation is best achieved at ambient temperatures (Ta) around 0 °C; however, they spend the majority of hibernation at considerably lower Ta. Because arctic ground squirrels switch to mixed fuel metabolism, including protein catabolism, at extreme low Ta of hibernation, we sought to investigate how microbial urea-nitrogen recycling is used under different thermal conditions. Injecting squirrels with isotopically labeled urea (13C/15N) during hibernation at Ta's of - 16 °C and 2 °C and while active and euthermic allowed us to assess the ureolytic activity of gut microbes and the amount of liberated nitrogen incorporated into tissues. We found greater incorporation of microbially-liberated nitrogen into tissues of hibernating squirrels. Although ureolytic activity appears higher in euthermic squirrels, liberated nitrogen likely makes up a smaller percentage of the available nitrogen pool in active, fed animals. Because non-lipid fuel is a limiting factor for torpor at lower Ta in this species, we hypothesized there would be greater incorporation of liberated nitrogen in animals hibernating at - 16 °C. However, we found higher microbial-ureolytic activity and incorporation of microbially-liberated nitrogen, particularly in the liver, in squirrels hibernating at 2 °C. Likely this is because squirrels hibernating at 2 °C had higher Tb and longer interbout arousals, a combination of factors creating more favorable conditions for gut microbes to thrive and maintain greater activity while giving the host more time to absorb microbial metabolites.

Stable isotope analysis of CO2 in breath indicates metabolic fuel shifts in torpid arctic ground squirrels.

Stable carbon isotope ratios (δ13C) in breath show promise as an indicator of immediate metabolic fuel utilization in animals because tissue lipids have a lower δ13C value than carbohydrates and proteins. Metabolic fuel consumption is often estimated using the respiratory exchange ratio (RER), which has lipid and carbohydrate boundaries, but does not differentiate between protein and mixed fuel catabolism at intermediate values. Because lipids have relatively low δ13C values, measurements of stable carbon isotopes in breath may help distinguish between catabolism of protein and mixed fuel that includes lipid. We measured breath δ13C and RER concurrently in arctic ground squirrels (Urocitellus parryii) during steady-state torpor at ambient temperatures from -2 to -26°C. As predicted, we found a correlation between RER and breath δ13C values; however, the range of RER in this study did not reach intermediate levels to allow further resolution of metabolic substrate use with the addition of breath δ13C measurements. These data suggest that breath δ13C values are 1.1‰ lower than lipid tissue during pure lipid metabolism. From RER, we determined that arctic ground squirrels rely on nonlipid fuel sources for a significant portion of energy during torpor (up to 37%). The shift toward nonlipid fuel sources may be influenced by adiposity of the animals in addition to thermal challenge.

A test of alternative models for increased tissue nitrogen isotope ratios during fasting in hibernating arctic ground squirrels.

We describe two models explaining the increase in tissue nitrogen isotope ratios (δ(15)N) that occurs during fasting in animals. The catabolic model posits that protein breakdown selectively removes the lighter isotope of nitrogen ((14)N) from catabolized tissues, causing an increase in the proportion of heavy nitrogen isotope ((15)N). The anabolic model posits that protein synthesis during fasting results in elevated δ(15)N values, as the unreplaced loss of (14)N to urea results in a higher proportion of (15)N in plasma amino acids used for protein synthesis. We effected a range of lean mass loss in arctic ground squirrels (Urocitellus parryii) fasting during hibernation and then collected organ and muscle tissues for analysis of δ(15)N values. The catabolic model predicts increased δ(15)N values in both liver and muscle, as these tissues undergo significant catabolism during hibernation. The anabolic model predicts no change in muscle, but an increase in δ(15)N values in liver, which has high levels of protein synthesis during euthermic phases of hibernation. We found a significant increase in liver δ(15)N values and no change in muscle δ(15)N values with lean mass loss, which supports the anabolic model. Heart, small intestine and brown adipose tissue also showed an increase in δ(15)N values, indicating protein synthesis in these organ tissues during hibernation. Urine was 3.8% lighter than plasma, and both urine and plasma increased in δ(15)N values with lean mass loss. This study helps clarify the mechanisms causing δ(15)N change during nutritional stress, thus increasing its utility for physiological research and reconciling previously contradictory results.

Estimating lean mass over a wide range of body composition: a calibration of deuterium dilution in the arctic ground squirrel.

Calculating body water through isotope dilution has become a useful way to nondestructively estimate body composition in many species. The most accurate estimates using this method require calibration against proximate chemical analysis of body composition for individual species, but no studies to our knowledge have calibrated this method on a hibernating mammal that seasonally undergoes dramatic changes in body composition. We use deuterium oxide to estimate total body water in captive arctic ground squirrels, Urocitellus parryii, and compare two approaches of calculating lean mass from total body water, both calibrated against lean mass based on proximate analysis. The first method uses a single tissue hydration constant to calculate lean mass from total body water; the second method uses a predictive equation to calculate lean mass from total body water and body mass. We found that the predictive equation performs better over the large range of body composition common to this species. Distillation of blood samples did not affect lean mass estimates from either calculation method. These findings indicate that isotope dilution using a predictive equation should work well as an alternative to destructive methods in other small mammals that undergo radical changes in body composition across their annual cycle.

Hibernating above the permafrost: effects of ambient temperature and season on expression of metabolic genes in liver and brown adipose tissue of arctic ground squirrels.

Hibernating arctic ground squirrels (Urocitellus parryii), overwintering in frozen soils, maintain large gradients between ambient temperature (T(a)) and body temperature (T(b)) by substantially increasing metabolic rate during torpor while maintaining a subzero T(b). We used quantitative reverse-transcription PCR (qRT-PCR) to determine how the expression of 56 metabolic genes was affected by season (active in summer vs hibernating), metabolic load during torpor (imposed by differences in T(a): +2 vs -10°C) and hibernation state (torpid vs after arousal). Compared with active ground squirrels sampled in summer, liver from hibernators showed increased expression of genes associated with fatty acid catabolism (CPT1A, FABP1 and ACAT1), ketogenesis (HMGCS2) and gluconeogenesis (PCK1) and decreased expression of genes associated with fatty acid synthesis (ACACB, SCD and ELOVL6), amino acid metabolism, the urea cycle (PAH, BCKDHA and OTC), glycolysis (PDK1 and PFKM) and lipid metabolism (ACAT2). Stage of hibernation (torpid vs aroused) had a much smaller effect, with only one gene associated with glycogen synthesis (GSY1) in liver showing consistent differences in expression levels between temperature treatments. Despite the more than eightfold increase in energetic demand associated with defending T(b) during torpor at a T(a) of -10 vs +2°C, transcript levels in liver and brown adipose tissue differed little. Our results are inconsistent with a hypothesized switch to use of non-lipid fuels when ambient temperatures drop below freezing.

Phenological variation in annual timing of hibernation and breeding in nearby populations of Arctic ground squirrels.

Ecologists need an empirical understanding of physiological and behavioural adjustments that animals can make in response to seasonal and long-term variations in environmental conditions. Because many species experience trade-offs between timing and duration of one seasonal event versus another and because interacting species may also shift phenologies at different rates, it is possible that, in aggregate, phenological shifts could result in mismatches that disrupt ecological communities. We investigated the timing of seasonal events over 14 years in two Arctic ground squirrel populations living 20 km apart in Northern Alaska. At Atigun River, snow melt occurred 27 days earlier and snow cover began 17 days later than at Toolik Lake. This spatial differential was reflected in significant variation in the timing of most seasonal events in ground squirrels living at the two sites. Although reproductive males ended seasonal torpor on the same date at both sites, Atigun males emerged from hibernation 9 days earlier and entered hibernation 5 days later than Toolik males. Atigun females emerged and bred 13 days earlier and entered hibernation 9 days earlier than those at Toolik. We propose that this variation in phenology over a small spatial scale is likely generated by plasticity of physiological mechanisms that may also provide individuals the ability to respond to variation in environmental conditions over time.