Hibernation Season Research Articles

Tricolored Bat (Perimyotis subflavus) microsite use throughout hibernation

Abstract White-nose syndrome (WNS) has caused dramatic population declines in several bat species, including the Tricolored Bat (Perimyotis subflavus). Several studies have documented bats using colder roosting temperatures after infection; however, this strategy may have costs such as increased freezing risks or greater predation risks and it is unknown when during hibernation bats begin to utilize these colder temperatures. Our aim was to examine Tricolored Bat roost locations in a WNS-positive site in relation to roost microclimate and other environmental conditions throughout the hibernation season. We conducted monthly censuses of tricolored bats across 2 hibernation seasons (November to March 2020 to 2021 and October to March 2021 to 2022) in a WNS-positive hibernaculum in northwestern South Carolina and recorded skin and adjacent wall temperature, tunnel section, and distance from the entrance for each bat species. We continuously measured hibernacula temperature and relative humidity during both hibernation seasons. Most bats roosted in the back part of the tunnel where temperatures were warmer and more stable, and vapor pressure deficit (VPD) and human disturbance were low. However, >20% of bats roosted in the front, where roost temperatures were significantly colder and VPD was higher but more variable; human disturbance was also higher in this section. The proportion of bats in each tunnel section did not vary among months and we did not find evidence of significant movement to the front section of the tunnel as hibernation progressed based on marked bats; however, bats in the front section roosted higher on the wall suggesting that they may be avoiding human disturbance or predators. Our results support the notion that no optimum hibernation temperature exists for tricolored bats and that high VPD and disturbance are likely important factors driving microsite use. Protection of Tricolored Bat hibernacula that offer a range of microclimates or a network of sites in close proximity that offer different microclimates may be helpful for recovery of this species.

The interplay of temperature and circadian periodicity in winter activity of non-cavernous hibernator, Nyctalusnoctula

Winter activity of hibernating mammals is likely to be influenced by climate change. Our study focuses on Nyctalus noctula, a non-cavernous hibernator using artificial roosts in a recently colonized winter region. Using continuous acoustic monitoring and temperature measurements inside and outside the roosts, we found that bats exhibit a circadian cycle (active at night, resting during the day) even during hibernation season. Activity duration and intensity changed in response to ambient temperature, photoperiod, and hibernation progression. Warm ambient temperatures led to increased nighttime activity, extending the duration of the active phase. As photoperiod increased, the rest phase lengthened, while the overall magnitude of activity decreased from the beginning to the end of the hibernation period. Below 0 °C vocal activity was nearly zero indicating a minimal probability of bat activity during both day and night. The species recent success in extending its hibernation range northward may be attributed to its flexible adjustment to prevailing environmental conditions. Nevertheless, it remains uncertain whether engaging in daily activity at temperatures above 0 °C confers any advantages at northern latitudes to prevent premature energy depletion. The persistence of circadian activity during winter could be a relic behavior, adapted from historical patterns of wintering in insect-rich and warm southern latitudes.

Paternal behavior in captive fat-tailed dwarf lemurs (Cheirogaleus medius) is preserved under socially relevant conditions.

Fat-tailed dwarf lemurs (Cheirogaleus medius), primates endemic to Madagascar, are obligate hibernators that form stable, lifelong pairs in the wild. Given the temporal constraints imposed by seasonal hibernation, infant dwarf lemurs must grow, develop, and wean within the first two months of life. Maternal as well as paternal infant care, observed in the wild, has been deemed critical for infant survival. Given the importance of fathers' involvement in early infant care, we expect this behavior to persist even under captive conditions. At the Duke Lemur Center, in Durham NC, we observed two families of fat-tailed dwarf lemurs and focused on the behavior of adult males within the first two months of the infants' lives. We report evidence of paternal involvement, including babysitting, co-feeding, grooming, accompanying, and leading infants, consistent with observations from the wild. As expected, paternal babysitting decreased as infants gained independence, while co-feeding increased. Supplemental anecdotes, video recorded by observers, also highlight clear cases of involvement by both parents, and even older siblings, in safeguarding and socializing new infants. We argue that maintaining captive fat-tailed dwarf lemur populations under socially and ecologically relevant conditions facilitates the full expression of physiological and behavioral repertoires. Most importantly, it also allows dwarf lemurs to realize their species' potential and become robust proxies of their wild kin.

Hypothalamic hormone deficiency enables physiological anorexia in ground squirrels during hibernation

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5–7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24–48-h periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

Torpor induces reversible tau hyperphosphorylation and accumulation in mice expressing human tau

Tau protein hyperphosphorylation and aggregation are key pathological events in neurodegenerative tauopathies such as Alzheimer’s disease. Interestingly, seasonal hibernators show extensive tau hyperphosphorylation during torpor, i.e., the hypothermic and hypometabolic state of hibernation, which is completely reversed during arousal. Torpor-associated mechanisms that reverse tau hyperphosphorylation may be of therapeutic relevance, however, it is currently not known to what extent they apply to human tau. Here we addressed this issue using daily torpor in wildtype mice that express mouse tau (mtau) and in mice that lack mtau expression and instead express human tau (htau). AT8, AT100 and Ser396 immunoblotting and immunohistochemistry were used to assess tau (hyper)phosphorylation at clinically relevant phosphorylation sites. We found that torpor robustly and reversibly increases the levels of phosphorylated tau in both mtau and htau mice. Immunohistochemistry revealed four brain areas that show prominent tau phosphorylation: the hippocampus, posterior parietal cortex, piriform cortex and cortical amygdala. Whereas wildtype mice primarily showed increased levels of diffusely organized hyperphosphorylated tau during torpor, htau mice contained clear somato-dendritic accumulations of AT8 reactivity resembling tau pre-tangles as observed in the Alzheimer brain. Interestingly, AT8-positive accumulations disappeared upon arousal, and tau phosphorylation levels at 24 h after arousal were lower than observed at baseline, suggesting a beneficial effect of torpor-arousal cycles on preexisting hyperphosphorylated tau. In conclusion, daily torpor in mice offers a quick and standardized method to study tau phosphorylation, accumulation and clearance in mouse models relevant for neurodegeneration, as well as opportunities to discover new targets for the treatment of human tauopathies.

C-fos induction in the choroid plexus, tanycytes and pars tuberalis is an early indicator of spontaneous arousal from torpor in a deep hibernator.

Hibernation is an extreme state of seasonal energy conservation, reducing metabolic rate to as little as 1% of the active state. During the hibernation season, many species of hibernating mammals cycle repeatedly between the active (aroused) and hibernating (torpid) states (T-A cycling), using brown adipose tissue (BAT) to drive cyclical rewarming. The regulatory mechanisms controlling this process remain undefined but are presumed to involve thermoregulatory centres in the hypothalamus. Here, we used the golden hamster (Mesocricetus auratus), and high-resolution monitoring of BAT, core body temperature and ventilation rate, to sample at precisely defined phases of the T-A cycle. Using c-fos as a marker of cellular activity, we show that although the dorsomedial hypothalamus is active during torpor entry, neither it nor the pre-optic area shows any significant changes during the earliest stages of spontaneous arousal. Contrastingly, in three non-neuronal sites previously linked to control of metabolic physiology over seasonal and daily time scales - the choroid plexus, pars tuberalis and third ventricle tanycytes - peak c-fos expression is seen at arousal initiation. We suggest that through their sensitivity to factors in the blood or cerebrospinal fluid, these sites may mediate metabolic feedback-based initiation of the spontaneous arousal process.

Changes in microRNA expression related to ischemia-reperfusion injury in the kidney of the thirteen-lined ground squirrel during torpor

During the hibernation season, the thirteen-lined ground squirrel undergoes cyclical torpor and arousal periods. The decrease and restoration of metabolic rate and oxygen delivery during torpor and arousal, respectively, may cause reperfusion-ischemia injury in the kidneys. In order to maintain the structural integrity of the kidneys necessary for renal function resumption during arousal, the thirteen-lined ground squirrel has developed adaptive methods to prevent and repair kidney injury. In this present study, computational methods were used to clean and analyze sequenced kidney RNA samples. Significantly differentially expressed microRNAs and enriched gene sets were also determined. From the gene set analysis, the results showed an increase in ubiquitin-related processes and p53 signaling pathways which suggested the occurrence of kidney damage during torpor. There was also an observed increase in cell cycle processes and the anchoring junction cellular compartment which may lend to the prevention of kidney injury. From the differentially expressed microRNAs, miR-27a (log2FC = 1.639; p-value = 0.023), miR-129 (log2FC = 2.516; p-value = 0.023), miR-let-7b (log2FC = 2.360; p-value = 0.025), miR-let-7c (log2FC = 2.291; p-value = 0.037) and miR-let-7i (log2FC = 1.564; p-value = 0.039) were found to be significantly upregulated. These biochemical adaptations may allow the thirteen-lined ground squirrel to maintain kidney structure and function during hibernation.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P > 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Timing of seasonal events is correlated with social network position in a wild mammal

Across animal systems, abiotic environmental features, including timing of seasonal events and weather patterns, affect fitness. An individual’s degree of social integration also has fitness consequences, but we lack an understanding of how abiotic features relate to patterns of individual sociality. A deeper understanding of this relationship could be developed from studying systems where these two links with fitness have already been identified. We explored the relationship between individual social behavior and seasonal timing, seasonal length, and weather patterns. We used social network analysis on a sixteen-year dataset of a wild population of hibernating yellow-bellied marmots (Marmota flaviventer). We fit a series of generalized linear mixed models and found that longer growing seasons before winter hibernation and longer winters were associated with increased individual sociality in the following spring. However, later snowmelt was associated with decreased sociality that spring. We found no relationship between individual sociality and various measures of precipitation and temperature. This suggests that seasonal timing and length may be a more important driver of sociality than weather patterns in this system, both as a lag and contemporary effect. Seasonal timing and length may mediate the opportunity or intensity of social interactions. The entwined relationships between the seasonal schedule and weather, and the seemingly contradictory role of winter length and snowmelt, suggests the timing of seasons and its relationship with sociality is complex and further exploration of environment-sociality relationships is required across taxa.Significance statementWhile the adaptive benefits of social behavior are well studied, less is known about how features of the abiotic environment drive variation in individual social behavior. Given increasing stochasticity in the timing of seasonal events and weather patterns, mapping the environment-sociality relationship will provide important insights to the drivers of sociality in the wild. This is particularly salient for species most vulnerable to climate and environmental change, such as seasonal hibernators, like yellow-bellied marmots (Marmota flaviventer). We found that features of seasonal duration were positively associated with increased sociality, whereas the timing of seasonal onset was negatively associated. This work provides empirical evidence towards an important gap in the behavioral ecology literature.

تقدير اعداد المجاميع السكانية لترسة الفرات ملساء الصدفة Rafetus euphraticus في الاهوار الوسطى ( موقع تراث عالمي)

تم تصنيف ترسة الفرات ملساء الصدفة Rafetus euphraticus على أنها مهددة بخطر الانقراض ضمن القائمة الحمراء للاتحاد الدولي لحفظ الطبيعة، ويُعتقد أنها تعرضت لانخفاض كبير في أعدادها مؤخرًا. تقتصر معلومات الأنواع في العراق على عدد قليل من المسوحات السريعة مع القليل من المعلومات التفصيلية عن التربية والتوزيع. هدفت الدراسة الى تقدير اعداد سلاحف الفرات ملساء الصدفة في الأهوار الوسطى باستخدام استقراء بسيط للعدد. تم استخدام طريقة المربع (بحجم 5 كم2 لكل منهما) لتسجيل توزيع سلحفاة الفرات ملساء الصدفة داخل موقع الدراسة وتم إجراء عشرة مسوحات ابتداءا من شهر تشرين الاول 2021 إلى ايلول 2022 باستثناء موسم السبات (كانون الاول وكانون الثانيو شباط). تم تسجيلها داخل المياه عن طريق وضع الشباك في مناطق معينة بالأهوار الوسطى. اقترح الاستقراء البسيط لتعداداتنا لكامل الأهوار الوسطى أن يبلغ الحد الأقصى لحجم المجاميع السكانية 2526.55 فردًا /المساحة الكلية) 219.700 هكتار.( تعتبر الأهوار الوسطى موقعًا مهمًا لـ R. euphraticus حيث تم تسجيل إجمالي 46 فردًا.

Hibernating at High Temperatures Increases Variability in Arousal Dynamics

In the winter, and presumably due to low resource availability, many mammals enter into a state of torpor wherein body temperature approaches that of ambient temperature and oxygen consumption rates can be as low as 1/100th of active rates. The canonical hibernation season is not static. Rather periods of torpor are interrupted by predictable periods of euthermy called interbout arousals which allows for a resetting of homeostatic processes. Here, we exposed golden-mantled ground squirrels to a range of ambient temperatures including high temperatures (4, 12, 20, 25, and 30°C). Preliminary analyses demonstrated higher temperatures resulted in decreased maximum rates of rewarming. On average, at 4°C ambient temperature rewarming was 0.14 °C per minute, while at 30°C ambient temperature the average maximum rate of rewarming was 0.012°C per minute. In addition, higher temperatures had a 2-fold higher relative time index which is defined as the time required to reach maximum rate of rewarming divided by the total arousal duration. Lastly, the interbout arousals increased 5-fold at warmer temperatures, lasting 12 hours at 4°C and 66 hours at 30°C. We interpret these data to suggest high temperature torpor leads to homeostatic issues wherein key processes are poorly coordinated. The result is an inherent increase in variability between individual squirrels and a loss of predictability of key hibernation aspects. NASA TRISH Department of Education NSF. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Telomere dynamics during hibernation in a tropical primate.

Hibernation is a widespread metabolic strategy among mammals for surviving periods of food scarcity. During hibernation, animals naturally alternate between metabolically depressed torpor bouts and energetically expensive arousals without ill effects. As a result, hibernators are promising models for investigating mechanisms that buffer against cellular stress, including telomere protection and restoration. In non-hibernators, telomeres, the protective structural ends of chromosomes, shorten with age and metabolic stress. In temperate hibernators, however, telomere shortening and elongation can occur in response to changing environmental conditions and associated metabolic state. We investigate telomere dynamics in a tropical hibernating primate, the fat-tailed dwarf lemur (Cheirogaleus medius). In captivity, these lemurs can hibernate when maintained under cold temperatures (11-15°C) with limited food provisioning. We study telomere dynamics in eight fat-tailed dwarf lemurs at the Duke Lemur Center, USA, from samples collected before, during, and after the hibernation season and assayed via qPCR. Contrary to our predictions, we found that telomeres were maintained or even lengthened during hibernation, but shortened immediately thereafter. During hibernation, telomere lengthening was negatively correlated with time in euthermia. Although preliminary in scope, our findings suggest that there may be a preemptive, compensatory mechanism to maintain telomere integrity in dwarf lemurs during hibernation. Nevertheless, telomere shortening immediately afterward may broadly result in similar outcomes across seasons. Future studies could profitably investigate the mechanisms that offset telomere shortening within and outside of the hibernation season and whether those mechanisms are modulated by energy surplus or crises.

Pre-hibernation diet alters skeletal muscle relaxation kinetics, but not force development in torpid arctic ground squirrels.

During the hibernation season, Arctic ground squirrels (AGS) experience extreme temperature fluctuations (body temperature, Tb, as low as - 3°C), during which they are mostly physically inactive. Once Tb reaches ~ 15°C during interbout arousals, hibernators recruit skeletal muscle (SkM) for shivering thermogenesis to reach Tb of ~ 35°C. Polyunsaturated fatty acids (PUFA) in the diet are known to influence SkM function and metabolism. Recent studies in the cardiac muscle of hibernators have revealed that increased levels of ω-6 and the ω-6:ω-3 PUFA ratio correlate with sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and hibernation status. We hypothesized that diet (increased ω-6:ω-3 PUFA ratio) and torpor status are important in the regulation of the SERCA pump and that this may improve SkM performance during hibernation. Ex vivo functional assays were used to characterize performance changes in SkM (diaphragm) from AGS fed the following diets. (1) Standard rodent chow with an ω-6:ω-3 ratio of 5:1, or (2) a balanced diet with an ω-6:ω-3 ratio of 1:1 that roughly mimics wild diet. We collected diaphragms at three different stages of hibernation (early torpor, late torpor, and arousal) and evaluated muscle function under hypothermic temperature stress at 4°C, 15°C, 25°C, and 37°C to determine functional resilience. Our data show that torpid animals fed standard rodent chow have faster SkM relaxation when compared to the balanced diet animals. Furthermore, we discovered that standard rodent chow AGS during torpor has higher SkM relaxation kinetics, but this effect of torpor is eliminated in balanced diet AGS. Interestingly, neither diet nor torpor influenced the rate of force development (rate of calcium release). This is the first study to show that increasing the dietary ω-6:ω-3 PUFA ratio improves skeletal muscle performance during decreased temperatures in a hibernating animal. This evidence supports the interpretation that diet can change some functional properties of the SkM, presumably through membrane lipid composition, ambient temperature, and torpor interaction, with an impact on SkM performance.

Comparison of Spermophilus dauricus gastrointestinal phenotypic flexibility between hibernating and non-hibernating seasons

Traits of the gastrointestinal (GI) tract are phenotypically flexible and seem to reflect the ecological features of animals. The Daurian ground squirrel (Spermophilus dauricus) is a typical fat-storing, hibernating animal. However, information on seasonal GI plasticity in this species is limited. The aim of the present study was to characterize the morphology, histology, and genetic regulation involved in GI motility in the ground squirrel during non-hibernating and hibernating seasons. The morphological and histological results showed that the body weight, total fresh weight, and total net fresh weight of the GI tract in summer and autumn were higher than those in spring and the winter hibernation season, although the total length of the gut did not change seasonally. The fresh weight and net fresh weight of the small intestine, cecum, and large intestine were significantly greater in autumn and summer than in winter, but the lengths of these segments showed no significant seasonal differences. The mucosal and muscular layer thicknesses of the stomach, illum, cecum, and colon were greater in autumn or summer than in winter, and the small intestine diameter and villus height were decreased in winter compared to those in the spring or summer. Moreover, the expression of nNOS and NPY genes in the small intestine in autumn and winter was higher than that in spring or summer. VIP expression in winter was the highest across seasons; in contrast, CHRM2 expression in autumn was the highest across seasons. These results indicate that morphological and histological traits of the gut, as well motility adjustments, exhibit seasonal plasticity in the ground squirrel, and these are especially affected by the hibernating cycle. This work will expand our knowledge of GI phenotypic plasticity in hibernating rodent species.

High latitude Northern bats (Eptesicus nilssonii) reveal adaptations to both high and low ambient temperatures.

Insectivorous bats at northern latitudes need to cope with long periods of no food for large parts of the year. Hence, bats which are resident at northern latitudes throughout the year will need to undergo a long hibernation season and a short reproductive season where foraging time is limited by extended daylight periods. Eptesicus nilssonii is the northernmost occurring bat species worldwide and hibernates locally when ambient temperatures (Ta) limit prey availability. Therefore, we investigated the energy spent maintaining normothermy at different Tas, as well as how much bats limit energy expenditure while in torpor. We found that, despite being exposed to Ta as low as 1.1°C, bats did not increase torpid metabolic rate, thus indicating that E. nilssonii can survive and hibernate at low ambient temperatures. Furthermore, we found a lower critical temperature (Tlc) of 27.8°C, which is lower than in most other vespertilionid bats, and we found no indication of any metabolic response to Tas up to 37.1°C. Interestingly, carbon dioxide production increased with increasing Ta above the Tlc, presumably caused by a release of retained CO2 in bats that remained in torpor for longer and aroused at Ta above the Tlc. Our results indicate that E. nilssonii can thermoconform at near-freezing Ta, and hence maintain longer torpor bouts with limited energy expenditure, yet also cope with high Tawhen sun-exposed in roosts during long summer days. These physiological traits will likely enable the species to cope with ongoing and predicted climate change.

Cell cycle parameters and ornithine decarboxylase activity in the red bone marrow of hibernating ground squirrels Urocitellus undulatus

During the hibernation season, the values for the parameters of the cell cycle of red bone marrow cells in the hibernating ground squirrels Urocitellus undulatus, when they return to an active-like state between periods of torpor and interbout arousal, do not differ from those observed in summer-active animals. In animals that enter a state of torpor, the cumulative percentage of cells in the resting phase (G0 phase) and pre-synthesis phase (G1 phase) increased from 71.8 to 76.0%, the percentage of cells in the synthesis phase (S phase) decreased from 19.3 to 15.3% compared to those animals that return to an active-like state between periods of torpor and interbout arousal. The cumulative percentage of cells in the post DNA synthesis phase (G2 phase) and mitosis (M) does not change, but (G2 + M)/S ratio increases. When animals enter a state of torpor, changes in parameter values are observed when the animal’s body temperature drops below 25°C, this effect refers to a system whose thermal relaxation time is a nonmonotonic function of the initial temperature. The activity of the key enzyme of polyamine synthesis ornithine decarboxylase, a marker of cell activation and proliferation during interbout arousal does not significantly differ from that observed in summer-active animals; the enzymatic activity decreases sharply, when animals decrease their body temperature below 25°C and enter a state of torpor, and this activity remains at a low level during hibernation and arousal until body temperature reaches 30°C. The role of changes in the parameter values associated with proliferative activity in adaptation of hematopoietic tissue during hibernation of the Yakutian ground squirrel is discussed.

Seasonal variation in glucose and insulin is modulated by food and temperature conditions in a hibernating primate.

Feast-fast cycles allow animals to live in seasonal environments by promoting fat storage when food is plentiful and lipolysis when food is scarce. Fat-storing hibernators have mastered this cycle over a circannual schedule, by undergoing extreme fattening to stockpile fuel for the ensuing hibernation season. Insulin is intrinsic to carbohydrate and lipid metabolism and is central to regulating feast-fast cycles in mammalian hibernators. Here, we examine glucose and insulin dynamics across the feast-fast cycle in fat-tailed dwarf lemurs, the only obligate hibernator among primates. Unlike cold-adapted hibernators, dwarf lemurs inhabit tropical forests in Madagascar and hibernate under various temperature conditions. Using the captive colony at the Duke Lemur Center, we determined fasting glucose and insulin, and glucose tolerance, in dwarf lemurs across seasons. During the lean season, we maintained dwarf lemurs under stable warm, stable cold, or fluctuating ambient temperatures that variably included food provisioning or deprivation. Overall, we find that dwarf lemurs can show signatures of reversible, lean-season insulin resistance. During the fattening season prior to hibernation, dwarf lemurs had low glucose, insulin, and HOMA-IR despite consuming high-sugar diets. In the active season after hibernation, glucose, insulin, HOMA-IR, and glucose tolerance all increased, highlighting the metabolic processes at play during periods of weight gain versus weight loss. During the lean season, glucose remained low, but insulin and HOMA-IR increased, particularly in animals kept under warm conditions with daily food. Moreover, these lemurs had the greatest glucose intolerance in our study and had average HOMA-IR values consistent with insulin resistance (5.49), while those without food under cold (1.95) or fluctuating (1.17) temperatures did not. Remarkably low insulin in dwarf lemurs under fluctuating temperatures raises new questions about lipid metabolism when animals can passively warm and cool rather than undergo sporadic arousals. Our results underscore that seasonal changes in insulin and glucose tolerance are likely hallmarks of hibernating mammals. Because dwarf lemurs can hibernate under a range of conditions in captivity, they are an emerging model for primate metabolic flexibility with implications for human health.

Treatment with gut-specific nonsteroidal anti-inflammatory drug attenuates metabolic inflammation but not body mass in fattening ground squirrels.

The active season of hibernators corresponds to rapid adiposity in preparation for the next hibernation season. We have previously shown that this dramatic increase in adipose mass is associated with metabolic inflammation similar to what is seen in obesity and metabolic disease. We next sought to determine whether curbing this inflammation at its source (i.e., the gut) would attenuate weight gain in fattening 13-lined ground squirrels (Ictidomys tridecemlineatus). We fed active yearling ground squirrels a diet containing the gut-specific nonsteroidal anti-inflammatory drug mesalazine (5-aminosalicylic acid) for 10 wk. Mesalazine treatment had slight effects on microbial community diversity in the cecum and colon. Not surprisingly, mesalazine treatment decreased inflammatory cytokine levels in the ileum and colon. Mesalazine also decreased proinflammatory and increased anti-inflammatory cytokines in omental white adipose tissue (oWAT). Despite this, body mass was unaffected, and caloric intake increased in mesalazine-treated squirrels, mainly in males. Mass of the primary WAT depot, intra-abdominal WAT (iaWAT), or the highly metabolic oWAT were unaltered by treatment, as was adiposity index. Together, these results suggest that mesalazine treatment has some effects on adiposity in fattening ground squirrels, but this treatment needs to be modified to overcome the strong drive to fatten in this species.NEW & NOTEWORTHY Adiposity and obesity are caused, at least in part, by inflammation of metabolic tissues. Hibernators, like ground squirrels, undergo this same metabolic inflammation during their summer fattening period. We attempted to curb this inflammation, and thus fattening, using mesalazine. We found that mesalazine did curb the inflammation but did not affect fattening, likely due to the strong drive to fatten in hibernators.

Microbial gene expression during hibernation in arctic ground squirrels: greater differences across gut sections than in response to pre-hibernation dietary protein content.

Obligate seasonal hibernators fast for 5-9months depending on species yet resist muscle atrophy and emerge with little lean mass loss. The role of the gut microbiome in host nitrogen metabolism during hibernation is therefore of considerable interest, and recent studies support a role for urea nitrogen salvage (UNS) in host-protein conservation. We were interested in the effect of pre-hibernation diet on UNS and the microbial provision of essential amino acids (EAAs) during hibernation; therefore, we conducted a study whereby we fed arctic ground squirrels (Urocitellus parryii) pre-hibernation diets containing 9% vs. 18% protein and compared the expression of gut bacterial urease and amino acid (AA) metabolism genes in 4 gut sections (cecum mucosa, cecum lumen, small intestine [SI] mucosa, and SI lumen) during hibernation. We found that pre-hibernation dietary protein content did not affect expression of complete bacterial AA pathway genes during hibernation; however, several individual genes within EAA pathways were differentially expressed in squirrels fed 18% pre-hibernation dietary protein. Expression of genes associated with AA pathways was highest in the SI and lowest in the cecum mucosa. Additionally, the SI was the dominant expression site of AA and urease genes and was distinct from other sections in its overall microbial functional and taxonomic composition. Urease expression in the gut microbiome of hibernating squirrels significantly differed by gut section, but not by pre-hibernation dietary protein content. We identified two individual genes that are part of the urea cycle and involved in arginine biosynthesis, which were significantly more highly expressed in the cecum lumen and SI mucosa of squirrels fed a pre-hibernation diet containing 18% protein. Six bacterial genera were responsible for 99% of urease gene expression: Cupriavidus, Burkholderia, Laribacter, Bradhyrizobium, Helicobacter, and Yersinia. Although we did not find a strong effect of pre-hibernation dietary protein content on urease or AA metabolism gene expression during hibernation, our data do suggest the potential for pre-hibernation diet to modulate gut microbiota function during hibernation, and further investigations are warranted.

Non-shivering thermogenesis is differentially regulated during the hibernation season in Arctic ground squirrels.

Arctic ground squirrels are small mammals that experience physiological extremes during the hibernation season. Body temperature rises from 1°C to 40°C during interbout arousal and requires tight thermoregulation to maintain rheostasis. Tissues from wild-caught Arctic ground squirrels were sampled over 9months to assess the expression of proteins key to thermogenic regulation. Animals were sacrificed while aroused, and the extensor digitorum longus, diaphragm, brown adipose tissue, and white adipose tissue were probed using Western blots to assess protein expression and blood was sampled for metabolite analysis. Significant seasonal expression patterns emerged showing differential regulation. Contrary to our prediction, white adipose tissue showed no expression of uncoupling protein 1, but utilization of uncoupling protein 1 peaked in brown adipose tissue during the winter months and began to taper after terminal arousal in the spring. The opposite was true for muscular non-shivering thermogenesis. Sarco/endoplasmic reticulum calcium ATPase 1a and 2a expressions were depressed during the late hibernation season and rebounded after terminal arousal in diaphragm tissues, but only SERCA2a was differentially expressed in the extensor digitorum longus. The uncoupler, sarcolipin, was only detected in diaphragm samples and had a decreased expression during hibernation. The differential timing of these non-shivering pathways indicated distinct functions in maintaining thermogenesis which may depend on burrow temperature, availability of endogenous resources, and other seasonal activity demands on these tissues. These results could be impacted by fiber type makeup of the muscles collected, the body weight of the animal, and the date of entrance or exit from hibernation.