Frank van Breukelen

Proteolysis is depressed during torpor in hibernators at the level of the 20S core protease

Protein synthesis is depressed during mammalian hibernation in concordance with metabolic demands. In the absence of significant protein synthesis, continued proteolysis would rapidly deplete protein pools. Since ubiquitin-dependent proteolysis is implicated in the turnover of most regulatory proteins, we examined the fate of this system during hibernation. Ubiquitin-dependent proteolysis consists of two major steps: (1) the tagging of a protein substrate by ubiquitin and (2) the protein substrate’s subsequent degradation by the 26S proteasome. An earlier study revealed a two to threefold elevation of ubiquitin conjugate concentrations during hibernation: an unexpected result that seemingly would suggest increased proteolytic activity. A more likely explanation for these data would be that proteolysis per se was depressed and that the increased levels of ubiquitylated proteins reflect an inability to degrade tagged proteins. We employed an assay based on the cleavage of fluorogenic substrates to address the well characterized proteolytic activities of the proteasome. All activities show little to no activity at temperatures associated with deep torpor. Coordinated depression of proteolytic activities by low temperature supports the hypothesis that the increased levels of ubiquitylated proteins during hibernation is explained by a net accumulation due to an inability to degrade the tagged proteins.

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates.

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.

Bone strength is maintained after 8 months of inactivity in hibernating golden-mantled ground squirrels, Spermophilus lateralis.

SUMMARY Prolonged inactivity leads to disuse atrophy, a loss of muscle and bone mass. Hibernating mammals are inactive for 6–9 months per year but must return to full activity immediately after completing hibernation. This necessity for immediate recovery presents an intriguing conundrum, as many mammals require two to three times the period of inactivity to recover full bone strength. Therefore, if hibernators experience typical levels of bone disuse atrophy during hibernation, there would be inadequate time available to recover during the summer active season. We examined whether there were mechanical consequences as a result of the extended inactivity of hibernation. We dissected femur and tibia bones from squirrels in various stages of the annual hibernation cycle and measured the amount of force required to fracture these bones. Three groups were investigated; summer active animals were captured during the summer and immediately killed, animals in the 1 month detraining group were captured in the summer and killed following a 1-month period of restricted mobility, hibernating animals were killed after 8 months of inactivity. A three-point bend test was employed to measure the force required to break the bones. Apparent flexural strength and apparent flexural modulus (material stiffness) were calculated for femurs. There were no differences between groups for femur fracture force, tibia fracture force, or femur flexural strength. Femur flexural modulus was significantly less for the 1 month detraining group than for the hibernation and summer active groups. Thus, hibernators seem resistant to the deleterious effects of prolonged inactivity during the winter. However, they may be susceptible to immobilization-induced bone loss during the summer.

A systems-level approach to understanding transcriptional regulation by p53 during mammalian hibernation

Presumably to conserve energy, many mammals enter into hibernation during the winter. Homeostatic processes such as transcription and translation are virtually arrested. To further elucidate transcriptional regulation during hibernation, we studied the transcription factor p53. Here, we demonstrate that changes in liver mRNA and protein concentrations of known regulators of p53 are consistent with activation. p53 mRNA and protein concentrations are unrelated. Importantly, p53 protein concentration is increased ~2-fold during the interbout arousal that punctuates bouts of torpor. As a result, both the interbout arousal and the torpid state are characterized by high levels of nuclear-localized p53. Chromatin immunoprecipitation assays indicate that p53 binds DNA during the winter. Furthermore, p53 recruits RNA polymerase II, as indicated by nuclear run-on data. However, and consistent with previous data indicating an arrest of transcriptional elongation during torpor, p53 ‘activity’ does not result in expected changes in target gene transcripts. These data demonstrate the importance of using a systems level-approach in understanding a complex phenotype such as mammalian hibernation. Relying on interpretations of data that are based on steady-state regulation in other systems may be misleading in the context of non-steady-state conditions such as torpor.

One year in the life of Bufo punctatus: annual patterns of body temperature in a free-ranging desert anuran.

The Mojave Desert is characterized by hot dry summers and cold winters. The red-spotted toad (Bufo (Anaxyrus) punctatus) is the predominant anuran species; yet little is known of their thermal histories and strategies to avoid temperature extremes. We measured body temperature (Tb) in free-ranging adult toads across all four seasons of a year using implanted data loggers. There is marked individual variation in the temperatures experienced by these toads. As expected, toads generally escape extreme seasonal and diel temperature fluctuations. However, our data demonstrate a much wider estimated Tb range than was previously assumed. Though often for short periods, red-spotted toads do experience Tb as low as 3.1°C and as high as 39.1°C. All animals showed periods of prolonged thermal stability in cooler months and wider diel oscillations in warmer months. Red-spotted toad thermal history is likely a function of site choice; the exploitation of different refuges results in diverse thermal experiences. These data represent the most complete record of thermal experiences for a desert anuran and reveal greater extremes in body temperature than previously suggested.

Extreme physiological plasticity in a hibernating basoendothermic mammal, Tenrec ecaudatus

ABSTRACT Physiological plasticity allows organisms to respond to diverse conditions. However, can being too plastic actually be detrimental? Malagasy common tenrecs, Tenrec ecaudatus, have many plesiomorphic traits and may represent a basal placental mammal. We established a laboratory population of T. ecaudatus and found extreme plasticity in thermoregulation and metabolism, a novel hibernation form, variable annual timing, and remarkable growth and reproductive biology. For instance, tenrec body temperature (Tb) may approximate ambient temperature to as low as 12°C even when tenrecs are fully active. Conversely, tenrecs can hibernate with Tb of 28°C. During the active season, oxygen consumption may vary 25-fold with little or no change in Tb. During the austral winter, tenrecs are consistently torpid but the depth of torpor may vary. A righting assay revealed that Tb contributes to but does not dictate activity status. Homeostatic processes are not always linked, e.g. a hibernating tenrec experienced a ∼34% decrease in heart rate while maintaining constant body temperature and oxygen consumption rates. Tenrec growth rates vary but young may grow ∼40-fold in the 5 weeks until weaning and may possess indeterminate growth as adults. Despite all of this profound plasticity, tenrecs are surprisingly intolerant of extremes in ambient temperature (<8 or >34°C). We contend that while plasticity may confer numerous energetic advantages in consistently moderate environments, environmental extremes may have limited the success and distribution of plastic basal mammals. Highlighted Article: Common tenrecs, Tenrec ecaudatus, which may be representative of ancestral placental mammals, demonstrate extreme physiological plasticity.

Does the Road Traveled Matter? Natural Versus Prematurely Induced Arousal from Torpor

Although hibernating animals spontaneously arouse from torpor at regular intervals, the practice of prematurely inducing arousal is common. Herein we review the many differences between natural and prematurely induced arousal to address the question of whether these two paths to euthermy are truly synonymous events. We present data demonstrating that the duration of the interbout arousal (IBA) is significantly reduced following a prematurely induced arousal and that the time required to respond to the induction stimulus is influenced by the duration of time spent in torpor. There are numerous alterations in intracellular and whole animal physiology when arousal is prematurely induced; thus we recommend that careful consideration be given to experiments utilizing this type of arousal mechanism.

Living in a seasonal world: 15th international hibernation symposium

A brief history of the 15 times the international hibernation community has come together is warranted. The first meeting was held May 13–15, 1959 at the Massachusetts Institute of Technology Endicott House (see Table 1). The 26 presented papers in that first meeting focused greatly on the physiology of hibernation and has set the tone for the ensuing meetings. One of the highlights of the meeting and subsequent symposium volume were the candid discussions (Lyman and Dawe 1960). Members of the hibernation community shared insight after each presentation and discussed the future directions of the field. Some of this discussion is seemingly humorous in retrospective. In a discussion as to the ‘validity of the term, hibernation’, one participant debated the meaning of ‘validity’—perhaps highlighting the historical difficulty, the field has had with nomenclature that suitably describes the extraordinary diversity of torpor use. The community was trying to separate out hypothermia from hibernation—a problem that we grappled with even in the 2016 meeting. Indeed, many of the topics pursued in the first meeting are still relevant 58 years later. We know much more than we did then, but we still struggle with the basic questions as to why do animals hibernate or why do almost all hibernators experience periodic euthermic arousals during the winter. Every 3–6 years, meetings were held. Proceedings were published a year or two after the meeting. The impact of these meetings was tremendous even early on. As far as we can tell, the first suggestion that brown adipose tissue was thermogenic came from Robert E. Smith in the 2nd symposium in 1962. Smith discussed how no satisfactory physiological role had yet been assigned to brown adipose tissue and then presented data that supported a role for thermogenesis and homeothermy. The ideas presented at the meeting in 1962 led to the landmark paper in Science demonstrating the role of brown adipose tissue in thermogenesis This special issue of the Journal of Comparative Physiology B is dedicated to papers arising from the 15th International Hibernation Symposium held in Las Vegas, NV, USA from July 31–August 4th, 2016. These symposia occur every 4 years and bring together virtually all of the world’s experts on mammalian hibernation. The 2016 program was primarily focused on mammalian hibernation with presentations on topics such as the evolution or plasticity of torpor use, how hibernation impacts on physiological systems such as the immune system, muscles, or bones, the molecular underpinnings of hibernation, and biomedical implications and prospects from hibernation. Mammals such as ground squirrels, bats, tenrecs, bears, echidnas, dormice, and lemurs were discussed. However, there was also discussion about birds, fish, lizards, and snakes and even gut microbial communities. Approaches towards solving problems spanned from ecological to physiological to biochemical and molecular strategies. Importantly, the program extended beyond hibernation and included the basic science of thermoregulation, hypoxia tolerance, brown adipose tissue function, and cold adaptation. Perhaps, most extraordinary about these meetings is the lack of formal rules or programming guidelines. The meeting was organized based on contributions and truly reflects the diversity of evolving research paradigms. Posters were encouraged to reflect the latest projects and allowed for insightful discussion.

A refined technique for sciatic denervation in a golden-mantled ground squirrel (Callospermophilus lateralis) model of disuse atrophy.

Disuse atrophy of both muscle and bone can occur rapidly during periods of inactivity. In several rodent models developed for the study of disuse atrophy, immobilization is induced by prolonged cage restraint, hind limb unloading, tenotomy, sciatic nerve block or sciatic denervation. In less tractable species such as wild-caught hibernating rodents, the sciatic denervation model is superior in terms of both animal welfare and applicability to the characteristics of natural cases of disuse atrophy. The authors describe a refined surgical approach to sciatic denervation in golden-mantled ground squirrels (Callospermophilus lateralis), a hibernating species, that improves animal welfare and reduces the incidence of post-operative complications such as autotomy.