Hibernating bears as a model for preventing disuse osteoporosis

The hibernating bear is an excellent model for disuse osteoporosis in humans because it is a naturally occurring large animal model. Furthermore, bears and humans have similar lower limb skeletal morphology, and bears walk plantigrade like humans. Black bears (Ursus americanus) may not develop disuse osteoporosis during long periods of disuse (i.e. hibernation) because they maintain osteoblastic bone formation during hibernation. As a consequence, bone volume, mineral content, porosity, and strength are not adversely affected by annual periods of disuse. In fact, cortical bone bending strength has been shown to increase with age in hibernating black bears without a significant change in porosity. Other animals require remobilization periods 2-3 times longer than the immobilization period to recover the bone lost during disuse. Our findings support the hypothesis that black bears, which hibernate for as long as 5-7 months annually, have evolved biological mechanisms to mitigate the adverse effects of disuse on bone porosity and strength.     

Bending properties, porosity, and ash fraction of black bear (Ursus americanus) cortical bone are not compromised with aging despite annual periods of disuse

In many species, including humans, disuse causes an imbalance in bone remodeling that leads to increased bone porosity as a result of increased bone resorption and decreased bone formation. However, black bears (Ursus americanus) may not develop disuse osteopenia, to the extent that other animals do, during long periods of disuse (i.e. hibernation) because they maintain osteoblastic bone formation during hibernation, even though bone resorption is increased during hibernation. Black bears may also have a mechanism to rapidly and completely recover the bone lost (by increased resorption during hibernation) during their remobilization period. Our findings suggest that cortical bone bending strength (211-328 MPa), bending modulus (16.0-29.5 MPa), fracture energy (0.0118-0.0205 J mm(-2)), porosity (2.3-7.1%), and ash fraction (0.638-0.672) are not compromised with age in black bears, despite annual periods of disuse. In fact, the ultimate strength (p=0.01), modulus (p=0.04), and ash fraction (p=0.03) of cortical bone were shown to significantly increase with age (2-14 yrs). Female bears give birth and nurse during hibernation; however, we found no significant (p>0.16) differences between male and female bone properties. Other animals require remobilization periods 2-3 times longer than the immobilization period to recover the bone lost during disuse. Our findings support the idea that black bears, which hibernate 5-7 months annually, have evolved a biological mechanism to mitigate the adverse effects of disuse on bone porosity and mechanical behavior.     

Mammalian hibernation as a model of disuse osteoporosis: the effects of physical inactivity on bone metabolism, structure, and strength

Reduced skeletal loading typically leads to bone loss because bone formation and bone resorption become unbalanced. Hibernation is a natural model of musculoskeletal disuse because hibernating animals greatly reduce weight-bearing activity, and therefore, they would be expected to lose bone. Some evidence suggests that small mammals like ground squirrels, bats, and hamsters do lose bone during hibernation, but the mechanism of bone loss is unclear. In contrast, hibernating bears maintain balanced bone remodeling and preserve bone structure and strength. Differences in the skeletal responses of bears and smaller mammals to hibernation may be due to differences in their hibernation patterns; smaller mammals may excrete calcium liberated from bone during periodic arousals throughout hibernation, leading to progressive bone loss over time, whereas bears may have evolved more sophisticated physiological processes to recycle calcium, prevent hypercalcemia, and maintain bone integrity. Investigating the roles of neural and hormonal control of bear bone metabolism could give valuable insight into translating the mechanisms that prevent disuse-induced bone loss in bears into novel therapies for treating osteoporosis.     

Vitamin D status and bone and connective tissue turnover in brown bears (Ursus arctos) during hibernation and the active state

Background

Extended physical inactivity causes disuse osteoporosis in humans. In contrast, brown bears (Ursus arctos) are highly immobilised for half of the year during hibernation without signs of bone loss and therefore may serve as a model for prevention of osteoporosis.

Aim

To study 25-hydroxy-vitamin D (25OHD) levels and bone turnover markers in brown bears during the hibernating state in winter and during the active state in summer. We measured vitamin D subtypes (D₂ and D₃), calcitropic hormones (parathyroid hormone [PTH], 1,25-dihydroxy-vitamin D [1,25(OH)₂D]) and bone turnover parameters (osteocalcin, ICTP, CTX-I), PTH, serum calcium and PIIINP.

Material and Methods

We drew blood from seven immobilised wild brown bears during hibernation in February and in the same bears while active in June.

Results

Serum 25-hydroxy-cholecalciferol (25OHD₃) was significantly higher in the summer than in the winter (22.8±4.6 vs. 8.8±2.1 nmol/l, two tailed p - 2p = 0.02), whereas 25-hydroxy-ergocalciferol (25OHD₂) was higher in winter (54.2±8.3 vs. 18.7±1.7 nmol/l, 2p<0.01). Total serum calcium and PTH levels did not differ between winter and summer. Activated 1,25(OH)₂D demonstrated a statistically insignificant trend towards higher summer levels. Osteocalcin levels were higher in summer than winter, whereas other markers of bone turnover (ICTP and CTX-I) were unchanged. Serum PIIINP, which is a marker of connective tissue and to some degree muscle turnover, was significantly higher during summer than during winter.

Conclusions

Dramatic changes were documented in the vitamin D₃/D₂ ratio and in markers of bone and connective tissue turnover in brown bears between hibernation and the active state. Because hibernating brown bears do not develop disuse osteoporosis, despite extensive physical inactivity we suggest that they may serve as a model for the prevention of this disease.     

Plasma levels of ursodeoxycholic acid in black bears, Ursus americanus: seasonal changes

To date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 micromol/L (0.9 +/- 0.2 micromol/L, mean +/- SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 +/- 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 +/- 0.1 and 1.2 +/- 0.4 micromol/L, respectively; p < 0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 +/- 3.2% and 22.2 +/- 4.5%, p < 0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for >67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears.     

Six months of disuse during hibernation does not increase intracortical porosity or decrease cortical bone geometry,strength, or mineralization in black bear (Ursus americanus) femurs

Disuse typically uncouples bone formation from resorption, leading to bone loss which compromises bone mechanical properties and increases the risk of bone fracture. Previous studies suggest that bears can prevent bone loss during long periods of disuse (hibernation), but small sample sizes have limited the conclusions that can be drawn regarding the effects of hibernation on bone structure and strength in bears. Here we quantified the effects of hibernation on structural, mineral, and mechanical properties of black bear (Ursus americanus) cortical bone by studying femurs from large groups of male and female bears (with wide age ranges) killed during pre-hibernation (fall) and post-hibernation (spring) periods. Bone properties that are affected by body mass (e.g. bone geometrical properties) tended to be larger in male compared to female bears. There were no differences (p>0.226) in bone structure, mineral content, or mechanical properties between fall and spring bears. Bone geometrical properties differed by less than 5% and bone mechanical properties differed by less than 10% between fall and spring bears. Porosity (fall: 5.5±2.2%; spring: 4.8±1.6%) and ash fraction (fall: 0.694±0.011; spring: 0.696±0.010) also showed no change (p>0.304) between seasons. Statistical power was high (>72%) for these analyses. Furthermore, bone geometrical properties and ash fraction (a measure of mineral content) increased with age and porosity decreased with age. These results support the idea that bears possess a biological mechanism to prevent disuse and age-related osteoporoses.     

The tensile strength of black bear (Ursus americanus) cortical bone is not compromised with aging despite annual periods of hibernation

Black bears (Ursus americanus) may not develop disuse osteoporosis during long periods of disuse (i.e. hibernation) because they may be able to maintain bone formation. Previously, we found that cortical bone bending strength was not compromised with age in black bears' tibias, despite annual periods of disuse. Here we showed that cortical bone tensile strength (166-198MPa) also does not decrease with age (2-14 years) in black bear tibias. There were also no significant age-related changes in cortical bone porosity in black bear tibias. It is likely that the ability of black bears to maintain bone formation during hibernation keeps bone porosity low (2.3-8.6%) with aging, notwithstanding annual periods of disuse. This low porosity likely preserves ultimate stress with aging. Female bears give birth and nurse during hibernation; however, we found no significant differences between male and female tensile material properties, mineral content, or porosity. Our findings support the idea that black bears, which hibernate 5-7 months annually, have evolved biological mechanisms to mitigate the adverse effects of disuse on bone porosity and strength.     

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α₂-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α₂-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.     

Minimal seasonal alterations in the skeletal muscle of captive brown bears

Abstract Previous studies on wild black bears (Ursus americanus) have shown that skeletal muscle morphology, composition, and overall force-generating capacity do not differ drastically between seasons despite prolonged inactivity during hibernation. However, the amount and characteristics of the seasonal variations were not consistent in these studies. The goals of this study were to compare the amount of muscle atrophy in captive brown bears (Ursus arctos) with that observed in wild black bears and measure seasonal differences in twitch characteristics. Samples from the biceps femoris muscle were collected during the summer and winter. Protein concentration, fiber-type composition, and fiber cross-sectional area were measured along with twitch characteristics. The protein concentration of the winter samples was 8.2% lower than that of the summer samples; fiber cross-sectional area and the relative proportion of fast and slow fibers remained unchanged between seasons. Myosin heavy chain isoforms I, IIa, and IIx were identified by immunoblotting and electrophoresis, and the proportions did not change between seasons. The half-rise time in the twitch contractions increased in winter relative to summer samples, which is unexpected under disuse conditions. These results agreed with a study that showed minimal skeletal muscle atrophy between seasons in wild black bears.     

Room without a view—Den excavation in relation to body size in brown bears

Hibernation is an adaptive strategy to survive harsh winter conditions and food shortage. The use of well‐insulated winter dens helps animals minimize energy loss during hibernation. Brown bears (Ursus arctos) commonly use excavated dens for hibernation. Physical attributes of excavated dens are expected to impact the bear's heat retention and energy conservation. The objective of this study was to examine the determinants of cavity size of excavated dens and the impact of physical attributes of excavated dens on energy conservation in hibernating bears, hypothesizing that bears excavate dens in a way to minimize heat loss and optimize energy conservation during hibernation. We predicted that den cavity size would be determined by the bear's body size and that older bears would excavate better‐fitting cavities to minimize heat loss, due to their previous experience. We further predicted that physical attributes of excavated dens would affect the bears’ posthibernation body condition. Our results revealed that bears excavated a den cavity in relation to their body size, regardless of sex, and that older bears tended to excavate better‐fitting den cavities compared to young bears, as we expected. Older bears excavated better‐fitting den cavities, suggesting a potentially experience‐based shift with age in den‐excavation behavior and an optimum cavity size relative to a bear's body size. Our key finding is that insulation of excavated dens provided by wall/rood thickness and bedding materials had a significant positive effect on bears’ posthibernation body condition. We believe that our study provides new insight into how not only the quality of denning habitat, but also the quality of dens may affect hibernating animals, by presenting a potential adaptive aspect of den preparation (age effect on efficiency in den excavation) and effect of den attributes on the posthibernation body condition of brown bears.     

Annual changes in serum sex steroids in male and female black (Ursus americanus) and polar (Ursus maritimus) bears

The adaptation of black and polar bears to their environments is proportional to the severity of climate and food restriction. Both black and polar bears mate during the spring, despite differences in their recent metabolic state. Reproductive activity in black bears follows 4 mo of torpor, whereas reproduction in polar bears occurs prior to torpor. The goals of this study were to measure the annual changes in serum sex steroids in male and female black and polar bears, and to determine if changes in serum levels of these steroids were associated with metabolic condition or photoperiod. Serum testosterone (T) concentrations were elevated during spring in black and polar bears. Moreover, this increase in serum T in polar bears during spring was correlated with age and testis size. Serum progesterone (P4) concentrations increased in pregnant polar bears in fall coincident with the time of expected implantation. No increases in serum P4 were observed in nonpregnant black and polar bears. Serum estradiol (E2) was elevated in nonpregnant and pregnant polar bears 2 mo prior to the time of expected implantation. We found that serum sex steroids measured in black and polar bears change independent of torpor. Therefore, our results suggest that photoperiod may be a more important regulator of serum steroid levels and reproduction than metabolic condition.     

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.     

Detection of pregnancy in a hibernator based on activity data

Pregnancy and parturition are often difficult to detect in wild-living animals, especially in species that give birth to altricial young in burrows or caves. Current methods to detect pregnancy and birth in hibernating animals often entail disturbances that can affect the animals' reproductive success. We developed a new method to confirm pregnancy and parturition in hibernating brown bears (Ursus arctos) using activity data recorded in dual-axis motion sensors mounted on GPS–GSM neck collars on 30 brown bears during 52 hibernation seasons in Sweden. We adjusted recorded activity to the individual basal activity level for each bear. Pregnant females showed characteristic activity patterns during the period of pregnancy, with significantly higher daily activity levels and frequency of active periods during pregnancy than nonpregnant bears. Pregnant females were active on average 2.20 times more often during the pregnancy period than during the postpartum period, compared with 0.97 times for nonpregnant females. A pregnancy index was defined as the average of the proportion of mean daily activity levels and the proportion of activity events during the pregnancy period compared with the postpartum period. It averaged 2.61?±?1.73 (SD) for pregnant females and 0.94?±?0.24 for nonpregnant females. Using this method, we evaluated a group of adult females that had an unverified reproductive status but were classified as not pregnant because no cubs had been observed after den emergence. The results suggested that 35 % of those females had, in fact, been pregnant.     

Annual skeletal changes in the little brown bat, Myotis lucifugus lucifugus, with particular reference to pregnancy and lactation

Studies of bone from summer-active little brown bats, Myotis lucifugus lucifugus, have demonstrated sex differences in the renewal of skeletal mineral reserves following spring-arousal from hibernation. Patterns of bone remodeling in both sexes of bats indicate that new bone formation does not occur during hibernation: All new bone formation occurs during the summer-active season. Results show that a short period of time elapses after hibernation before the initial demands of a large fetus and rapidly growing neonate are expressed on maternal skeletal reserves. Bone loss in summer-active females was associated with pregnancy and lactation, whereas summer-active males did not show evidence of bone loss but, instead, uninterrupted bone accretion throughout the summer-active season. Osteoclasts and bone-forming osteoblasts, absent during the hibernation period, reappeared on bone surfaces following spring-arousal from hibernation. There was no apparent increase in osteoclast numbers or activity during lactation but resorption cavities were found in deep cortical lamellae distant from bone surfaces. The increase in bone resorption in lactating bats appeared to be by osteocytic osteolysis, suggesting that it might be a significant mechanism of bone/calcium regulation in this hibernating mammal throughout the year.     

Cardiac function adaptations in hibernating grizzly bears (Ursus arctos horribilis)

Research on the cardiovascular physiology of hibernating mammals may provide insight into evolutionary adaptations; however, anesthesia used to handle wild animals may affect the cardiovascular parameters of interest. To overcome these potential biases, we investigated the functional cardiac phenotype of the hibernating grizzly bear (Ursus arctos horribilis) during the active, transitional and hibernating phases over a 4 year period in conscious rather than anesthetized bears. The bears were captive born and serially studied from the age of 5 months to 4 years. Heart rate was significantly different from active (82.6 ± 7.7 beats/min) to hibernating states (17.8 ± 2.8 beats/min). There was no difference from the active to the hibernating state in diastolic and stroke volume parameters or in left atrial area. Left ventricular volume:mass was significantly increased during hibernation indicating decreased ventricular mass. Ejection fraction of the left ventricle was not different between active and hibernating states. In contrast, total left atrial emptying fraction was significantly reduced during hibernation (17.8 ± 2.8%) as compared to the active state (40.8 ± 1.9%). Reduced atrial chamber function was also supported by reduced atrial contraction blood flow velocities and atrial contraction ejection fraction during hibernation; 7.1 ± 2.8% as compared to 20.7 ± 3% during the active state. Changes in the diastolic cardiac filling cycle, especially atrial chamber contribution to ventricular filling, appear to be the most prominent macroscopic functional change during hibernation. Thus, we propose that these changes in atrial chamber function constitute a major adaptation during hibernation which allows the myocardium to conserve energy, avoid chamber dilation and remain healthy during a period of extremely low heart rates. These findings will aid in rational approaches to identifying underlying molecular mechanisms.     

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation

Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.     

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus)

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.