Adaptation of intestinal enzymes to seasonal and dietary changes in a hibernator: the European hamster (Cricetus cricetus)

Summary Effects of diet, hibernation and seasonal variations on hydrolase activities were determined in mucosa and purified brush border membranes of the small intestine of European hamsters. Wild hamsters captured in April and fed for several weeks with an equilibrated laboratory chow (20% protein, 50% carbohydrates) exhibited a rise in disaccharidase activities (sucrase, isomaltase, lactase) but no changes in aminopeptidase N activity. During deep hibernation, in contrast to sucrase and isomaltase activities which showed only minor changes, lactase activity was significantly enhanced along the jejunoileum, and aminopeptidase N activity was maximum in the ileum. After a short period (48 h) of wakefulness and feeding following 10 days of starvation during the hibernation period, the activities of the disaccharidases and of aminopeptidase N returned to values measured in active animals. In contrast to the nutritional state, which has an important impact on the activities of intestinal enzymes, season has little effect on the intestine of the active animal under a controlled environment. The pattern of enzyme activities which occurs along the small intestine in the hibernating animal may be a prerequisite for optimum digestion during the short phases of waking during the hibernation period of the European hamster.     

Response of Gut Microbiota to Fasting and Hibernation in Syrian Hamsters

Although hibernating mammals wake occasionally to eat during torpor, this period represents a state of fasting. Fasting is known to alter the gut microbiota in nonhibernating mammals; therefore, hibernation may also affect the gut microbiota. However, there are few reports of gut microbiota in hibernating mammals. The present study aimed to compare the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses. Hamsters were allocated to either torpid, fed active, or fasted active groups. Hibernation was successfully induced by maintaining darkness at 4°C. Flow cytometry analysis of cecal bacteria showed that 96-h fasting reduced the total gut bacteria. This period of fasting also reduced the concentrations of short chain fatty acids in the cecal contents. In contrast, total bacterial numbers and concentrations of short chain fatty acids were unaffected by hibernation. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments indicated that fasting and hibernation modulated the cecal microbiota. Analysis of 16S rRNA clone library and species-specific real-time quantitative PCR showed that the class Clostridia predominated in both active and torpid hamsters and that populations of Akkermansia muciniphila, a mucin degrader, were increased by fasting but not by hibernation. From these results, we conclude that the gut microbiota responds differently to fasting and hibernation in Syrian hamsters.Some mammalian species have evolved with the physiological phenomenon of hibernation to survive unfavorable winter environments (9). Hibernation is realized by entering torpor in order to eliminate the need to maintain a constant, high body temperature. During torpor, typical hibernating mammals, such as hamsters and ground squirrels, lower their body temperature to only a few degrees above ambient temperatures to reduce energy expenditure. Torpor is interrupted by periods of intense metabolic activity. During these interbout arousals, physiological parameters are restored rapidly to near-normal levels. Thus, hibernators alternate between hypothermic and euthermic states during hibernation.Some hibernating mammals awake to forage during torpor, while food-storing hibernators such as hamsters eat cached food during interbout arousals. However, hibernation essentially involves periods of fasting. Fasting is known to affect the gut microbiota in nonhibernating mammals such as mice (12); therefore, it is possible that hibernation also influences the gut microbiota. Given that the gut microbiota plays important roles in mammalian tissue development and homeostasis (28), it was of interest to investigate the changes in the gut microbiota that may take place during hibernation. To date, this issue has received little attention; to our knowledge, there are only two reports on the gut microbiota in hibernating mammals. Schmidt et al. showed that although the total counts of coliforms, streptococci, and psychrophilic organisms in the feces of arctic ground squirrels held in a cold room at 3°C remained constant the composition changed, with a decrease in coliform count and a 1,000-fold increase in the number of aerobic psychrophilic gram-negative bacteria (31). Barnes and Burton reported that although there was some reduction in total numbers of viable bacteria in the cecum during hibernation, composition of the microbiota remained stable (6). In terms of amphibians, Banas et al. and Gossling et al. reported a reduction and compositional changes of the gut microbiota in hibernating leopard frogs (4, 5, 18, 19).Only 20 to 40% of bacterial species from the mammalian intestinal tract can be cultured and identified using classical culture methods (22, 34, 36). In contrast, culture-independent methods based on the amplification of bacterial 16S rRNA genes by PCR have revealed a great diversity of microbiota in environmental samples (3, 37). The present study compared the gut microbiota in hibernating torpid Syrian hamsters with that in active counterparts by using culture-independent analyses.     

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.     

Study of the testis in the active and hibernating hedgehog

The structure and ultrastructure of the hedgehog's testis has been studied during active and hibernating states. The maximum activity of the hedgehog's testis manifest itself in spring and summer. In autumn they enter in a seasonal resting, in which, the interstitial cells atrophy and it can observe primary spermatocytes resting next to spermatogonia and supporting cells in the seminiferous tubules. These have a minimal diameter and contain only primary spermatocytes in hibernating period also the seminiferous epithelium that compound the walls of the tubules are about 72 microns thick in the active period and about 47 microns in hibernation. The Sertoli cells shows a small diameter in hibernation than in the active season. The nucleus has one or two nucleoli with distinguish themselves from those observed in the active season because of their closer union with the inner nuclear envelope. In autumn (resting period) the interstitial tissue regress and the Leydig cells are aggregated in blocks with little cytoplasm, minimum size nuclei and 1 or 2 nucleolus closely to the inner nuclear membrane. Like then in the ovary, the hedgehog's testis follows a seasonal cycle determined by the temperature and environmental changes and it have a power of physiological adaptation inherent of a typical hibernating animal.     

Diet quality does not affect resting metabolic rate or body temperatures selected by an herbivorous lizard

Diet quality can influence many aspects of digestion, but the links between diet quality and resting metabolism are poorly understood. In nature, it might be beneficial to reduce energy expenditure when only poor quality diets are available. Alternatively, animals might increase the processing capacity of the gut to more thoroughly extract energy. If maintaining the processing capacity of the gut is energetically expensive, then increasing gut size or function might result in higher resting metabolism. In ectotherms, most digestive functions are temperature dependent, thus another strategy to maintain energy balance might be to alter selected body temperatures. We tested whether differing concentrations of dietary fiber affected the resting metabolic rate or body temperatures selected by chuckwallas (Sauromalus obesus) – lizards that naturally experience marked variation in dietary fiber. Resting metabolic rates measured at two temperatures and over three time intervals did not differ between groups of lizards force-fed low- (30% neutral-detergent fiber; NDF) and high-fiber (45% NDF) diets, nor did these diet differences influence body temperatures selected in a thermal gradient. We conclude that ecologically relevant differences in diet quality may have negligible effects on resting metabolic rates and body temperatures selected by chuckwallas. Accepted: 5 January 1998     

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.     

Comparative physiological and biochemical aspects of hypothermia as a model for hibernation

It is obvious that research is far from the last chapter in developing a model for natural hibernation. The relationships and comparative mechanisms for thermogenesis and survival in hibernation and experimental hypothermia are still unclear. Yet, two primary areas appear to be most promising, namely, the control of thermogenesis via the glucocorticoids and the specific role of the central nervous system (CNS) in survival of hypothermic subjects and arousal of hibernating subjects. Although there have been several approaches to understanding the role of the CNS in terms of circulation, integrity of the blood-brain barrier (BBB) system, and CNS sites of activity, it may appear that more questions have been raised than have been answered. However, a more optimistic view can also be taken. The development of a laboratory model, using experimental hypothermia for natural hibernation, is progressing. This view is justified in terms of results from the use of glucocorticoids in metabolic regulation of available carbohydrates, i.e., available glucose in hypothermia, and the continued promising parallel studies of physiological and biochemical integrity of areas of the CNS in hypothermic and hibernating subjects.     

Hibernation confers resistance to intestinal ischemia-reperfusion injury

The damaging effects of intestinal ischemia-reperfusion (I/R) on the gut and remote organs can be attenuated by subjecting the intestine to a prior, less severe I/R insult, a process known as preconditioning. Because intestines of hibernating ground squirrels experience repeated cycles of hypoperfusion and reperfusion, we examined whether hibernation serves as a model for natural preconditioning against I/R-induced injury. We induced intestinal I/R in either the entire gut or in isolated intestinal loops using rats, summer ground squirrels, and hibernating squirrels during natural interbout arousals (IBA; body temperature 37-39 degrees C). In both models, I/R induced less mucosal damage in IBA squirrels than in summer squirrels or rats. Superior mesenteric artery I/R increased MPO activity in the gut mucosa and lung of rats and summer squirrels and the liver of rats but had no effect in IBA squirrels. I/R in isolated loops increased luminal albumin levels, suggesting increased gut permeability in rats and summer squirrels but not IBA squirrels. The results suggest that the hibernation phenotype is associated with natural protection against intestinal I/R injury.     

Comparison of Brain Transcriptome of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) in Active and Torpid Episodes

Hibernation is an energy-saving strategy which is widely adopted by heterothermic mammals to survive in the harsh environment. The greater horseshoe bat (Rhinolophus ferrumequinum) can hibernate for a long period in the hibernation season. However, the global gene expression changes between hibernation and non-hibernation season in the greater horseshoe bat remain largely unknown. We herein reported a comprehensive survey of differential gene expression in the brain between winter hibernating and summer active greater horseshoe bats using next-generation sequencing technology. A total of 90,314,174 reads were generated and we identified 1,573 differentially expressed genes between active and torpid states. Interestingly, we found that differentially expressed genes are over-represented in some GO categories (such as metabolic suppression, cellular stress responses and oxidative stress), which suggests neuroprotective strategies might play an important role in hibernation control mechanisms. Our results determined to what extent the brain tissue of the greater horseshoe bats differ in gene expression between summer active and winter hibernating states and provided comprehensive insights into the adaptive mechanisms of bat hibernation.     

How do food passage rate and assimilation differ between herbivorous lizards and nonruminant mammals?

Summary What digestive adaptations permit herbivorous nonruminant mammals to sustain much higher metabolic rates than herbivorous lizards, despite gross similarity in digestive anatomy and physiology? We approached this question by comparing four herbivorous species eating the same diet of alfalfa pellets: two lizards (chuckwalla and desert iugana) and two mammals (desert woodrat and laboratory mouse). The mammals had longer small and large intestines, greater intestinal surface area, much higher (by an order of magnitude) food intake normalized to metabolic live mass, and much faster food passage times (a few hours instead of a few days). Among both reptiles and mammals, passage times increase with body size and are longer for herbivores than for carnivores. The herbivorous lizards, despite these much slower passage times, had slightly lower apparent digestive efficiencies than the mammals. At least for chuckwallas, this difference from mammals was not due to differences in body temperature regime. Comparisons of chuckwallas and woodrats in their assimilation of various dietary components showed that the woodrat's main advantage lay in greater assimilation of the dietary fiber fraction. Woodrats achieved greater fiber digestion despite shorter residence time, but possibly because of a larger fermentation chamber, coprophagy, and/or different conditions for microbial fermentation. We conclude with a comparative overview of digestive function in herbivorous lizards and mammals, and with a list of four major unsolved questions.     

Ecology of natural hibernation in the marsupial mountain pygmy-possum (Burramys parvus)

The hibernating marsupial mountain pygmy-possum (Burramys parvus, 40 g) has to raise its slow-growing offspring during a short alpine summer. Only females provide parental care, while after mating males emigrate to marginal habitats often at lower altitudes which can sustain only low possum densities. We predicted that the hibernation strategies in mountain pygmy-possums are distinct from those of similar-sized placental hibernators, because of the developmental constraints in marsupials and because hibernation differs between the sexes. Using temperature-sensitive radio transmitters, we studied the hibernation patterns of free-living male and female mountain pygmy-possums living in a north- and a south-facing boulder field (Kosciusko National Park) for two consecutive winters. Individual possums commenced hibernation several months before the snow season. As in other hibernators, torpor in the mountain pygmy-possum was interrupted by periodic arousals which occurred most often during the late afternoon. Torpor bouts initially lasted a few days when the hibernacula temperature (T _hib) ranged from 4 to 7°C. As the hibernation season progressed, torpor bouts became longer and possum body temperatures (T _b) approached 2°C. The T _bs of females were significantly lower and torpor bouts were longer in the second half of the hibernation season than in males. Between torpor bouts, both sexes were often active and left hibernacula for periods of up to 5 days. Especially during the first months of the hibernation season, possums also frequently changed hibernacula sites probably in an attempt to select a site with a more suitable microclimate. Emergence from hibernation was closely coupled with the disappearance of snow from the possum habitat (September 1995, October 1996) and the limited fat stores probably dictate an opportunistic spring emergence. However, in 1995, spring was early and males emerged significantly earlier than females. In 1996, when snow melt was delayed, this difference vanished. Testes are regressed in males during hibernation and the time needed for testes growth and spermatogenesis favours an earlier emergence for males which was probably achieved by their preference for the more sun exposed north-facing boulder field. A sexual dimorphism in hibernation strategies and spring emergence therefore enables mountain pygmy-possums to cope with their harsh alpine environment. Received: 22 May 1997 / Accepted: 21 August 1997     

Influence of photoperiod and gonadal steroids on hibernation in the European hamster

Torpor was monitored daily in adult male and female European hamsters (Cricetus cricetus) induced to hibernate by exposure to a cold environment (6 degrees C). The effect of photoperiodic manipulations or administration of exogenous gonadal steroids was examined in gonadectomized or intact hamsters. 1. Gonadal regression occurred in all short day, but only in some long day, cold-exposed hamsters. Entry into hibernation was not observed until reproductive regression had occurred. Thus, gonadal atrophy appears to be a necessary precondition for hibernation. 2. Castrated hamsters in the short day cold condition showed a significantly greater incidence of torpor than those in the long day cold condition. Hence, photoperiod affected torpor independently of its effect on the gonadal cycle. 3. Testosterone, when administered via silastic capsules at near physiological levels, completely inhibited torpor in gonadectomized male and female hamsters hibernating in the short day cold condition. 4. In ovariectomized females, torpor was unaffected by progesterone treatment, but partially inhibited by estradiol. A greater inhibition of torpor was observed when estradiol-primed females were administered both estradiol and progesterone simultaneously. Thus, the effect of both hormones may be functionally comparable to that of the single testicular hormone. 5. Estradiol inhibited torpor to a greater extent in intact and ovariectomized female hamsters hibernating in long days than those in short days, suggesting an effect of photoperiod on responsiveness to estradiol. These results indicate an inverse relationship between the gonadal and hibernation cycles, and a probable role for gonadal steroids to influence the timing of the hibernation season. However, non-gonadal factors must also be involved in controlling hibernation, since photoperiod affected the incidence of torpor in gonadectomized animals and because hamsters were able to terminate hibernation in the absence of gonadal hormones.     

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.     

Proteomic analysis of the winter-protected phenotype of hibernating ground squirrel intestine

The intestine of hibernating ground squirrels is protected against damage by ischemia-reperfusion (I/R) injury. This resistance does not depend on the low body temperature of torpor; rather, it is exhibited during natural interbout arousals that periodically return hibernating animals to euthermia. Here we use fluorescence two-dimensional difference gel electrophoresis (DIGE) to identify protein spot differences in intestines of 13-lined ground squirrels in the sensitive and protected phases of the circannual hibernation cycle, comparing sham-treated control animals with those exposed to I/R. Protein spot differences distinguished the sham-treated summer and hibernating samples, as well as the response to I/R between summer and hibernating intestines. The majority of protein changes among these groups were attributed to a seasonal difference between summer and winter hibernators. Many of the protein spots that differed were unambiguously identified by high-pressure liquid chromatography followed by tandem mass spectrometry of their constituent peptides. Western blot analysis confirmed significant upregulation for three of the proteins, albumin, apolipoprotein A-I, and ubiquitin hydrolase L1, that were identified in the DIGE analysis as increased in sham-treated hibernating squirrels compared with sham-treated summer squirrels. This study identifies several candidate proteins that may contribute to hibernation-induced protection of the gut during natural torpor-arousal cycles and experimental I/R injury. It also reveals the importance of enterocyte maturation in defining the hibernating gut proteome and the role of changing cell populations for the differences between sham and I/R-treated summer animals.     

Seasonal rhythms in the functioning of the hypophyseo-thyroid system of the suslik Citellus parryi

In experiments on the Arctic ground squirrel Citellus parryi, radioimmune assay of the content of thyrotropin of the hypophysis and thyroxine and triiodothyronine of the thyroid in the peripheral blood has been made at monthly intervals from July until May. It was found that during hibernation period, thyroxine and triiodothyronine concentrations in the blood of sleeping animals are high as compared with those during pre-hibernation period in autumn and active period in May. Thyrotropin content of the blood increases from October to May, being the lowest however during the season of the deepest hibernation from December to February. It is suggested that activation of the hypophysial--thyroid system after resting period in summer and autumn begins in October. During deep sleeping, it is depressed, being recovered in March. High levels of the thyroid hormones during hibernation period may be explained by metabolic strategy of the organism in hibernating animals which is directed to optimization of energy supply of hibernation.     

冬眠和非冬眠状态达乌尔黄鼠肾单位及相关功能因子的比较

冬眠是动物应对冬季低温和食物匮乏的一种生存策略。达乌尔黄鼠(Spermophilus dauricus)是典型的贮脂类冬眠动物。为研究冬眠动物肾脏的适应机制,本实验采用组织学、血液生化分析及酶联免疫方法检测了夏季活动期(7月)、冬眠期(12月)和早春出眠后(3月)达乌尔黄鼠肾单位形态学及血清肌酐、尿素和抗利尿激素(ADH)的变化,并用qPCR方法检测了肾脏水通道蛋白基因(AQP1、AQP2和AQP3)、ADH受体(V2R)及内皮型一氧化氮合酶基因(eNOS)的表达。结果发现,冬眠期和早春出眠期的达乌尔黄鼠肾小球密度、近曲小管和远曲小管的相对管径、皮质部近曲小管数与远曲小管数比值均低于夏季活动期;冬眠期血清肌酐和尿素浓度高于夏季活动期和早春出眠期,ADH浓度及其受体V2R基因表达低于夏季活动期;冬眠期AQP1基因表达高于早春出眠期,AQP3基因表达低于夏季活动期,AQP2基因表达无显著差异;冬眠期eNOS基因表达低于早春出眠期。这些结果表明冬眠的达乌尔黄鼠表现出较低的肾功能;不同时期的水通道蛋白,eNOS及ADH表现出适应性的功能调节。该实验结果丰富了对冬眠动物肾脏适应机制的认识。     

Timing of torpor bouts during hibernation in European hamsters (Cricetus cricetus L.)

Body temperature (T _b) of seven European hamsters maintained at constant ambient temperature (T _a = 8 °C) and constant photoperiod (LD 8:16) was recorded throughout the hibernating season using intraperitoneal temperature-sensitive HF transmitters. The animals spent about 30% of the hibernation season in hypothermia and 70% in inter-bout normothermy. Three types of hypothermia, namely deep hibernation bouts (DHBs), short hibernation bouts (SHBs), and short and shallow hibernation bouts (SSHBs), were distinguished by differences in bout duration and minimal body temperature (T _m). A gradual development of SSHBs from the diel minimum of T _b during normothermy could be seen in individual hamsters, suggesting a stepwise decrease of the homeostatic setpoint of T _b regulation during the early hibernation season. Entry into hibernation followed a 24-h rhythm occurring at preferred times of the day in all three types of hypothermia. DHBs and SHBs were initiated approximately 4 h before SSHBs, indicating a general difference in the physiological initiation of SSHBs on the one hand and DHBs and SHBs on the other. Arousals from SHBs and SSHBs also followed a 24-h rhythm, whereas spontaneous arousals from DHBs were widely scattered across day and night. Statistical analyses of bout length and the interval between arousals revealed evidence for a free-running circadian rhythm underlying the timing of arousals. The results clearly demonstrate that entries into hypothermia are linked to the light/dark-cycle. However, the role of the circadian system in the timing of arousals from DHBs remains unclear. Accepted: 11 December 1996     

Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel.

Hibernating animals transiently reduce renal function during their hypothermic periods (torpor), while completely restoring it during their periodical rewarming to euthermia (arousal). Moreover, structural integrity of the kidney is preserved throughout the hibernation. Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) is a crucial vasodilatory mediator and a protective factor in the kidney. We investigated renal NOS expression in hibernating European ground squirrels after 1 day and 7 days of torpor (torpor short, TS, and torpor long, TL, respectively), at 1.5 and at 10 h of rewarming (arousal short, AS, and arousal long, AL, respectively), and in continuously euthermic animals after hibernation (EU). For that purpose, we performed NOS activity assay, immunohistochemistry and real-time PCR analysis. Immunohistochemistry revealed a decreased glomerular eNOS expression in hibernating animals (TS, TL, AS, and AL) compared to non-hibernating animals (EU, p < 0.05), whereas no difference was found in the expression of interstitial eNOS. Expression of iNOS and nNOS did not differ between all groups. The reduced glomerular eNOS was associated with a significantly lower eNOS mRNA levels and NOS activity of whole kidney during torpor and arousal (TS, TL, AS, and AL) compared to EU. In all methods used, torpid and aroused squirrels did not differ. These results demonstrate differential regulation of eNOS in glomeruli and interstitium of hibernating animals, which is unaffected during arousal. The differential regulation of eNOS may serve to reduce ultrafiltration without jeopardizing tubular structures during hibernation.     

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.