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.     

Induction of summer hibernation in the 13-lined ground squirrel shown by comparative serum transfusions from arctic mammals.

A “trigger” substance was again indicated to be present in sera of hibernating animals. Sera from the hibernating 13-lined ground squirrel, hibernating woodchuck, hibernating Arctic ground squirrel, and hibernating Arctic marmot were all capable of inducing the 13-lined ground squirrel to hibernate in the summer, a season when that species would normally be active. The hibernation trigger is thus not species specific. It is effective whether drawn from these two Arctic species of hibernators or drawn from these two species of hibernators from the midwestern states. The normothermic Arctic marmot appears to have an “anti-trigger” substance in its serum in the summer, which impedes fall hibernation in the transfused 13-lined ground squirrel. This is similar to the anti-trigger observed in the summer serum of active 13-lined ground squirrels and active woodchucks. With respect to hypothermia, it was induced in Artic marmots and in Arctic foxes at Point Barrow, Alaska, in summer. Though in such cases body temperatures fell significantly (as in hibernation), no trigger was recovered from their hypothermic sera that could be shown to be capable of inducing summer hibernation in the ground squirrel. Neither was anti-trigger found in the serum of hypothermic experimentals. These latter experiments thus suggest that the release of trigger into the blood during hibernation is dependent on a mechanism more complex than simply lowering body temperature.     

Resistance of Erythrocytes of Hibernating Mammals to Loss of Potassium during Hibernation and during Cold Storage

In two species of hibernators, hamsters and ground squirrels, erythrocytes were collected by heart puncture and the K content of the cells of hibernating individuals was compared with that of awake individuals. The K concentration of hamsters did not decline significantly during each bout of hibernation (maximum period of 5 days) but in long-term bouts in ground squirrels (i.e. more than 5 days) the K concentration of cells dropped significantly. When ground squirrels were allowed to rewarm the K content of cells rose toward normal values within a few hours. Erythrocytes of both hamsters and ground squirrels lose K more slowly than those of guinea pigs (nonhibernators) when stored in vitro for up to 10 days at 5°C. In ground squirrels the rate of loss of K during storage is the same as in vivo during hibernation, and stored cells taken from hibernating ground squirrels also lose K at the same rate. The rate of loss of K from guinea pig cells corresponded with that predicted from passive diffusion unopposed by transport. The actual rate of loss of K from ground squirrel cells was slower than such a predicted rate but corresponded with it when glucose was omitted from the storage medium or ouabain was added to it. Despite the slight loss of K that may occur in hibernation, therefore, the cells of hibernators are more cold adapted than those of a nonhibernating mammal, and this adaptation depends in part upon active transport.     

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.     

The "hibernation induction trigger": specificity and validity of bioassay using the 13-lined ground squirrel

Even though the existence of the blood-borne "hibernation induction trigger" has been reported in the 13-lined ground squirrel, transfusion of plasma from hibernating rodents with other hibernating species as the recipients failed to induce the occurrence of summer hibernation. In order to verify whether the response to the "trigger" substance is species specific, the present study was carried out to compare the effect of plasma from hibernating Richardson's ground squirrels on the incidence of summer hibernation in both juvenile Richardson's and adult 13-lined ground squirrels. In two series of experiments, 13-lined ground squirrels entered hibernation quite readily independent of the treatment. The rate of occurrence of hibernation ranged from 78% after sham injection to 86% after warm saline, fresh summer active plasma, and fresh hibernating plasma, respectively. There were no differences in the number of hibernation bouts and the number of days in hibernation after each treatment. In contrast, none of the juvenile Richardson's ground squirrels entered hibernation after any of the treatments up to the end of the 8-week observation period. These results not only argue against the existence of blood-borne "trigger" substance, at least in the Richardson's ground squirrel, but also caution against the use of the 13-lined ground squirrel as a standard test animal for the bioassay of the "trigger" substance.     

Myosin light chains of skeletal and cardiac muscles of ground squirrel Citillus undulatus in different periods of hibernation

The isoform composition of myosin light chains and the extent of their phosphorylation in skeletal and cardiac muscles of ground squirrel Citellus undulatus in different periods of hibernation were studied. Regulatory myosin light chains of skeletal muscles of hibernating ground squirrels were completely dephosphorylated, while 25% of these light chains in active animals were phosphorylated. During hibernation, a shift of isoform composition of essential and regulatory skeletal muscle myosin light chains toward slower isoforms was observed, which is evidenced by the data obtained on m. psoas and on the totality of all skeletal muscles. In the atrial myocardium of hibernating ground squirrels, ventricular myosin light chains 1 (up to 60%) were registered. In contrast, during arousal of ground squirrels, in ventricular myocardium the appearance of atrial myosin light chains 1 (up to 30%) was revealed. A possible role of posttranslation changes in myosin light chains and their isoform shifts in the hibernation scenario is discussed.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.     

Seasonal Changes in Microsomal Fraction Enriched with Na,K-ATPase from Kidneys of the Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.     

Thermoregulatory changes anticipate hibernation onset by 45 days: data from free-living arctic ground squirrels

Hibernation is a strategy of reducing energy expenditure, body temperature (T(b)) and activity used by endotherms to escape unpredictable or seasonally reduced food availability. Despite extensive research on thermoregulatory adjustments during hibernation, less is known about transitions in thermoregulatory state, particularly under natural conditions. Laboratory studies on hibernating ground squirrels have demonstrated that thermoregulatory adjustments may occur over short intervals when animals undergo several brief, preliminary torpor bouts prior to entering multiday torpor. These short torpor bouts have been suggested to reflect a resetting of hypothalamic regions that control T(b) or to precondition animals before they undergo deep, multiday torpor. Here, we examined continuous records of T(b) in 240 arctic ground squirrels (Urocitellus parryii) prior to hibernation in the wild and in captivity. In free-living squirrels, T(b) began to decline 45 days prior to hibernation, and average T(b) had decreased 4.28 °C at the onset of torpor. Further, we found that 75 % of free-living squirrels and 35 % of captive squirrels entered bouts of multiday torpor with a single T(b) decline and without previously showing short preliminary bouts. This study provides evidence that adjustments in the thermoregulatory component of hibernation begin far earlier than previously demonstrated. The gradual reduction in T(b) is likely a component of the suite of metabolic and behavioral adjustments, controlled by an endogenous, circannual rhythm, that vary seasonally in hibernating ground squirrels.     

Effect of ionizing radiation on the synthetic activity of blood system cells in ground squirrels in different physiological states of animals

The functional (synthetic) activity of blood lymphocytes and bone marrow haemopoietic cells in ground squirrels during the annual cycle as well as in hibernating and awaken animals in winter have been studied by fluorescent microspectrometry. The effect of ionizing radiation on animals in different functional states of the hibernation-arousal bout was investigated too. It was shown that the synthetic activity (parameter alpha) in blood lymphocytes was minimal in hibernating state in winter and maximal in active euthermic spring animals, then slightly decreased in June and more considerably decreased in the prehibernating autumn period. In awake animals in winter, the values of parameter alpha reached the same values as in summer. The changes of parameter alpha in bone marrow haemopoietic cells were essentially the same: the minimal values were observed in the prehibernation autumn period and in awake animals in winter the alpha values were slightly higher than in active euthermic animals in summer. The maximal synthetic activity in bone marrow haemopoietic cells in active euthermic spring animals is due mainly to cells in G1-G2 phases of the cell cycle. The decrease of the synthetic activity in summer is a result of the cell transition from G2 to mitosis and transition of a part of cells to G0 When investigating the hibernation-arousal bout in ground squirrels in winter, during arousal, we found two stages considerably differing in both the values of parameter alpha in bone marrow haemopoietic cells and the number of blood cells. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of deep hibernation did not differ significantly from the state of non-irradiated hibernating animals. The negative effect of radiation appeared upon the arousal of these animals but it was expressed to a lesser degree in comparison with the animals irradiated in the active state. It was found that the acute irradiation of animals during arousal from hibernation in the second stage caused the most pronounced functional inactivation and cell death. The physiological state of ground squirrels subjected to ionizing irradiation at different phases of the hibernation-arousal bout plays a determining role in the changes of the qualitative and quantitative characteristics of blood system cells. Thus, the hypometabolic state of ground squirrels in hibernation is a factor of protection from the action of ionizing radiation on the organism and the immune system.     

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.     

The effect of hibernation on lipids of the liver mitochondrial fraction in the Yakut ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

We demonstrated that the level of phospholipids in the liver mitochondrial fraction is increased by 60% during the winter hibernation season in the Yakut ground squirrel S. undulatus; the phospholipid composition in sleeping animals is characterized by an increase in phosphatidylethanolamine compared with summer animals. A sharp increase in the level of cholesterol, as well as fatty acid, monoglycerides, and diglycerides was found in the mitochondrial fraction of hibernating ground squirrels in relation to summer ground squirrels. Functional changes during hibernation concern the level of phosphatidylserine (the growth in sleeping animals compared with active animals). Seasonal modification of the lipid composition of the liver mitochondria (particularly, an increase in the level of cholesterol) can play a role in the resistance of mitochondria to the seasonal increase in the level of fatty acids in the liver. Lipids of the liver mitochondrial fraction are involved in the ground squirrel adaptation to the hibernation season.     

中国达乌尔黄鼠的夏季诱发冬眠——对有否冬眠触发物质的探讨

用中国北方草原地区的季节性冬眠动物达乌尔黄鼠,经1981和1984两年的工作,重复了Dawe(1969)注射冷藏的冬眠动物血清,诱发夏季活泼黄鼠冬眠的原始实验。实验成功地实现了在非冬眠季节诱发达乌尔黄鼠冬眠,发现禁食在人工诱发冬眠中起重要作用,却不能证实血源性冬眠触发物(HIT)的存在。     

Influence of energy stores on activation of reproductive function in male golden-mantled ground squirrels

Summary Male golden-mantled ground squirrels were captured in the field in late summer and placed on either unlimited or restricted (80% normal) food rations in the laboratory until each animal began to hibernate. At entrance into hibernation mean body mass was 274.5 g for the unlimited group and 224.5 g for the restricted group. Only six of 21 males subsequently underwent reproductive maturation during winter. Each of these six received the unlimited ration prior to hibernation, and these six included the five heaviest animals in the experiment. In addition to activating their reproductive systems, these heavier squirrels began hibernation later and ended hibernation earlier than the lighter squirrels. Squirrels that remained reproductively quiescent averaged 25.5% body mass as fat in early spring and thus were not severely limited in energy stores during winter.In another experiment a group of 13 male and female squirrels were housed together in a common outdoor enclosure in order to examine the possible relationship between reproductive condition and social standing of males. Although there appeared to be a social hierarchy among males, each male fattened substantially in fall and became reproductively active during winter.These results suggest that the level of energy stores accumulated as fat prior to hibernation in fall affects the potential of male ground squirrels to breed in spring. This dependence of reproductive development on energy stores may reflect the high energetic costs associated with breeding in males when they emerge from hibernation in spring when food availability is low.     

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h = 1.70 and S _0.5 = 6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K⁺ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h = 1.16) and GLK affinity for substrate was reduced (S _0.5 = 23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h = 1.60, S _0.5 = 9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h = 1.20, S _0.5 = 45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.     

Blood Plasma Phospholipids and Cholesterol during Hibernation of the Long-Tailed Ground Squirrel

Seasonal changes in the levels of phospholipids, diglycerides, cholesterol, and total protein in the blood plasma were investigated during hibernation of the long-tailed ground squirrel Spermophilus undulatus. During the winter period, the levels of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin phospholipids (per 1 mg of plasma protein) were increased in both torpid and active ground squirrels by 70–80, 50, 600–700, 70, and 150–200%, respectively; the level of phosphatidylserine did not change in comparison to the summer period. The plasma phospholipid composition differed between hibernating and active summer animals: in winter, the phosphatidylcholine mol % decreased by 20%, phosphatidylinositol and phosphatidylethanolamine increased by 3–4 times, and the phosphatidylserine mol % decreased by 50%, while sphingomyelin and lysophosphatidylcholine did not change in comparison to summer animals. In hibernating ground squirrels, the plasma cholesterol levels increased by two times, the diglyceride content diminished by 60%, and the level of protein (in milligrams per 1 mL plasma) increased by 20%. The simultaneous increase in the levels of cholesterol and total phospholipids, as well as the pronounced specific changes in the levels of individual phospholipids in the blood plasma of hibernating ground squirrels, indicate the involvement of plasma lipoprotein lipids in the molecular mechanisms of adaptation to natural hypobiosis in mammals and a possible role of these mechanisms in systemic reactions to damaging factors.     

Seasonal variations in the rate and capacity of cardiac SR calcium accumulation in a hibernating species.

The rate of calcium uptake and the level of calcium accumulation was measured in cardiac muscle SR from hibernating and nonhibernating Richardson's ground squirrels. In whole heart homogenates, the rate of calcium uptake was higher (P less than 0.05) in hibernating animals than it was in active animals. Further purification of homogenates into sacroplasmic reticulum (SR) preparations showed that the hibernating animals had the highest rate of calcium uptake and the greatest level of calcium accumulation. These results could not be explained by variations in non-SR membrane contaminants nor by changes in the maximal activity or total amount of a SR marker enzyme, the Ca(2+)-ATPase. The addition of ryanodine to the calcium uptake medium increased the level of calcium accumulation in all groups by a similar amount. It is concluded that the high rate of calcium uptake by isolated cardiac SR vesicles from hibernating ground squirrels reflects the activity of the organelle in vivo, and that the ability of the ryanodine-insensitive population of SR vesicles to accumulate calcium is affected by hibernation.