Torpid Ground Squirrels Research Articles

Torpid 13-lined ground squirrel liver mitochondria resist anoxia-reoxygenation despite high levels of protein damage.

Hibernation confers resistance to ischemia-reperfusion injury in tissue, but the underlying mechanisms remain unclear. Suppression of mitochondrial respiration during torpor may contribute to this tolerance. To explore this concept, we subjected isolated liver mitochondria from torpid, interbout euthermic (IBE) and summer 13-lined ground squirrels (Ictidomys tridecemlineatus) to 5 min of anoxia, followed by reoxygenation (A/R). We also included rat liver mitochondria as a non-hibernating comparison group. Maximum respiration rates of mitochondria from torpid ground squirrels were not affected by A/R, but in IBE and summer, these rates decreased by 50% following A/R and in rats they decreased by 80%. Comparing net ROS production rates among groups, revealed seasonal differences; mitochondria from IBE and torpor produced 75% less ROS than summer ground squirrels and rats. Measurements of oxidative damage to these mitochondria, both freshly isolated, as well as pre- and post-A/R, demonstrated elevated damage to protein, but not lipids, in all groups. Hibernation likely generates oxidative stress, as freshly isolated mitochondria had greater protein damage in torpor and IBE than in summer and rats. When comparing markers of damage pre- and post-A/R, we found that when RET was active, rat macromolecules were more damaged than when RET is inhibited, but in TLGS markers of damage were similar. This result suggests that suppression of RET during hibernation, both in torpor and IBE, lessens oxidative stress produced during arousal. Taken together our study suggests that ischemia-reperfusion tolerance at the mitochondrial level is associated with metabolically suppressed oxidative phosphorylation during hibernation.

Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin–junctophilin axis

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC-RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC-RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a "reverse model of heart failure" at the molecular level, suggesting a strategy for treating heart diseases.

Characterizing the regulation of pyruvate kinase in response to hibernation in ground squirrel liver (Urocitellus richardsonii).

The Richardson's ground squirrel (Urocitellus richardsonii) undergoes numerous changes to its core physiological and metabolic processes over the months it spends hibernating during the winter. Winter torpor is characterized by an overall reduction in metabolic rate, a lowering of core body temperature, and a switch to preferential consumption of lipids instead of carbohydrates. The alterations in central metabolic pathways are often accomplished by the regulation of key enzymes within the glycolytic pathway. The regulation of one such enzyme, pyruvate kinase (PK), was characterized in the present study in the liver of torpid ground squirrels. PK was purified from liver tissue of euthermic and hibernating U. richardsonii and subsequently assayed to determine the kinetic parameters of the enzyme at 22° and 5°C. Additional studies assessed the relative degree of post-translational modifications in PK from control and hibernating ground squirrels. The results from this study demonstrated significantly lowered maximal activity in the hibernating form of the enzyme and decreased sensitivity to the activator FBP when compared to the control. Immunoblotting demonstrated increased relative serine and threonine phosphorylation (~3 fold) in the hibernating PK. Taken together these results suggest that phosphorylation of liver PK is an important step in inhibiting glycolytic activity in the liver of the Richardson's ground squirrel during torpor.

Are long chain acyl CoAs responsible for suppression of mitochondrial metabolism in hibernating 13-lined ground squirrels?

Hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus) is associated with a substantial suppression of whole-animal metabolism. We compared the metabolism of liver mitochondria isolated from torpid ground squirrels with those from interbout euthermic (IBE; recently aroused from torpor) and summer euthermic conspecifics. Succinate-fuelled state 3 respiration, calculated relative to mitochondrial protein, was suppressed in torpor by 48% and 44% compared with IBE and summer, respectively. This suppression remains when respiration is expressed relative to cytochrome c oxidase activity. We hypothesized that this suppression was caused by inhibition of succinate transport at the dicarboxylate transporter (DCT) by long-chain fatty acyl CoAs that may accumulate during torpor. We predicted, therefore, that exogenous palmitoyl CoA would inhibit respiration in IBE more than in torpor. Palmitoyl CoA inhibited respiration ~70%, in both torpor and IBE. The addition of carnitine, predicted to reverse palmitoyl CoA suppression by facilitating its transport into the mitochondrial matrix, did not rescue the respiration rates in IBE or torpor. Liver mitochondrial activities of carnitine palmitoyl transferase did not differ among IBE, torpor and summer animals. Although palmitoyl CoA inhibits succinate-fuelled respiration, this suppression is likely not related exclusively to inhibition of the DCT, and may involve additional mitochondrial transporters such as the adenine-nucleotide transporter.

Effects of hibernation on regulation of mammalian protein phosphatase type-2-A

Hibernation is used by many small mammals for winter survival. Animals undergo cycles of deep torpor characterized by strong reductions in metabolic rate, body temperature, and all physiological activities. One prominent biochemical mechanism involved in regulating coordinated transitions to and from the hypometabolic state is reversible protein phosphorylation, a dynamic covalent modification maintained by the actions of protein kinases and protein phosphatases. The present study characterizes protein phosphatase type 2A (PP2A) from liver and skeletal muscle of Richardson’s ground squirrels, Urocitellus richardsonii, to determine if the properties of this enzyme are modified between euthermic and hibernating states to provide differential control of the enzyme or its target proteins. Kinetic analysis showed a significant 1.5-fold increase in the Km for the synthetic phosphorylated peptide substrate (RRApTVA) for PP2A from skeletal muscle (but not liver) of torpid ground squirrels, as compared with euthermic controls, when measured at both 25°C and 5°C. This correlated with enhanced phosphorylation of PP2Aα Tyr-307 (a 2.3-fold increase) during hibernation in skeletal muscle (but not liver), a modification known to inhibit PP2A action. Hence, both kinetic and phosphorylation data suggest a suppression of PP2A function in the torpid state in skeletal muscle. However, total PP2A protein levels (assessed by immunoblotting) and maximal activities did not change in either tissue between euthermic and hibernating states. Liver PP2A from hibernating animals showed a broader pH profile with high activity retained at lower pH values (6.0–6.5) than the euthermic enzyme which might aid enzyme function in vivo under conditions of respiratory acidosis during torpor.

Seasonal changes in the isoform composition of the myosin heavy chains in skeletal muscles of hibernating ground squirrels Spermophilus undulatus

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca(2+)-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isorfom decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca(2+)-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions.

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation.

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.

The Suprachiasmatic Nucleus Is Essential for Circadian Body Temperature Rhythms in Hibernating Ground Squirrels

Body temperature (T(b)) was recorded at 10 min intervals over 2.5 years in female golden-mantled ground squirrels that sustained complete ablation of the suprachiasmatic nucleus (SCNx). Animals housed at an ambient temperature (T(a)) of 6.5 degrees C were housed in a 12 hr light/dark cycle for 19 months followed by 11 months in constant light. The circadian rhythm of T(b) was permanently eliminated in euthermic and torpid SCNx squirrels, but not in those with partial destruction of the SCN or in neurologically intact control animals. Among control animals, some low-amplitude T(b) rhythms during torpor were driven by small (<0.1 degrees C) diurnal changes in T(a). During torpor bouts in which T(b) rhythms were unaffected by T(a), T(b) rhythm period ranged from 23.7 to 28.5 hr. Both SCNx and control squirrels were more likely to enter torpor at night and to arouse during the day in the presence of the light/dark cycle, whereas entry into and arousal from torpor occurred at random clock times in both SCNx and control animals housed in constant light. Absence of circadian rhythms 2.5 years after SCN ablation indicates that extra-SCN pacemakers are unable to mediate circadian organization in euthermic or torpid ground squirrels. The presence of diurnal rhythms of entry into and arousal from torpor in SCNx animals held under a light/dark cycle, and their absence in constant light, suggest that light can reach the retina of hibernating ground squirrels maintained in the laboratory and affect hibernation via an SCN-independent mechanism.

MRNA stability and polysome loss in hibernating Arctic ground squirrels (Spermophilus parryii).

All small mammalian hibernators periodically rewarm from torpor to high, euthermic body temperatures for brief intervals throughout the hibernating season. The functional significance of these arousal episodes is unknown, but one suggestion is that rewarming may be related to replacement of gene products lost during torpor due to degradation of mRNA. To assess the stability of mRNA as a function of the hibernation state, we examined the poly(A) tail lengths of liver mRNA from arctic ground squirrels sacrificed during four hibernation states (early and late during a torpor bout and early and late following arousal from torpor) and from active ground squirrels sacrificed in the summer. Poly(A) tail lengths were not altered during torpor, suggesting either that mRNA is stabilized or that transcription continues during torpor. In mRNA isolated from torpid ground squirrels, we observed a pattern of 12 poly(A) residues at greater densities approximately every 27 nucleotides along the poly(A) tail, which is a pattern consistent with binding of poly(A)-binding protein. The intensity of this pattern was significantly reduced following arousal from torpor and undetectable in mRNA obtained from summer ground squirrels. Analyses of polysome profiles revealed a significant reduction in polyribosomes in torpid animals, indicating that translation is depressed during torpor.

Acridine orange as an indicator of the cytoplasmic ribosome state

In hippocampal neurons of ground squirrels and neuroblastoma culture the ribosome state was analyzed by staining with acridine orange (AO), labeling with radioactive amino acids, and electron microscopy. Electron microscopy indicated that the extent to which ribosomes associated in polysomes varied from 25% in brain cells of torpid ground squirrels up to 93% in growing neuroblastoma cells. In control rat neurons, it was 75%. The fluorescence of the AO-rRNA complex in ribosomes changed with the polysome/monosome ratio. The red fluorescence of AO-single-stranded rRNA complex as well as ribosome association in polysomes decreased greatly as a function of the shift from polysomes to monosomes. The green fluorescence of AO intercalated in the double-stranded rRNA changed insignificantly. As a result, the ratio of red to green fluorescence intensity, Kalpha, changed more than 3-fold with the changing polysome/monosome ratio. Protein labeling also showed strong positive correlation with Kalpha. Thus, rRNA showed different accessibility for AO binding in active and inactive ribosomes. A possible mechanism of partial rRNA shielding with proteins is proposed. AO fluorescence in the cytoplasm, i.e., AO binding to rRNA in ribosomes, is presumed to reflect adequately the profound changes in the state of cell protein synthesizing system that can be regulated by both functional activity and stress factors.

Uridine uptake and RNA synthesis in the brain of torpid and awakened ground squirrels

1. 1. Uptake of [ 3H]uridine into the nucleotide precursor pool after intraventricular injection occurs with the same intensity in the brain of torpid and normothermic awakened ground squirrels. This indicates that the membrane uridine transporters and uridine kinases operate in the hibernator' brain in a hypothermia-tolerant way. 2. 2. Utilization of the [ 3H]uridine pool for synthesis of the rapidly labelled RNA in the brain of torpid ground squirrels falls more than eight times against RNA labelling in the brain of the active animals between bouts of hibernation. 3. 3. Two hours from the beginning of the artificially provoked awakening, RNA uridine incorporation in the brain of ground squirrels has risen 6.5 times. 4. 4. Drastic changes in [ 3H]uridine RNA labelling under the stable uridine uptake exclude the precursors and energy supply as the main factors determining changes in intensity of the brain RNA synthesis in the different stages of hibernation.

Temperature dependence of in vitro androgen production in testes from hibernating ground squirrels, Spermophilus lateralis

The effect of temperature on the in vitro androgen secretion of testes from hibernating ground squirrels was measured in response to stimulation by luteinizing hormone (LH). We wished to determine whether hibernating ground squirrels can maintain responsiveness of gonads while at the low body temperatures of torpor. In gonads incubated at 32 °C, secretion of testosterone increased in a dose-dependent manner in response to ovine-LH or ground squirrel pituitary homogenate. This responsiveness was reduced at 20 and 9 °C and absent at 5 °C, the temperature that most closely approximates the body temperature of torpid ground squirrels. This temperature sensitivity was similar to that in the nonhibernating laboratory mouse. Superfusion of ground squirrel testes revealed a lag of testosterone secretion in response to LH and, additionally, an ability of testes to secrete testosterone after being only briefly exposed to ovine-LH while at 5 °C. These results provide evidence against a hypothesis of temperature compensation that would allow continued testis function during torpor, and support a previous study which indicated that gonadal growth is restricted to intervals of normothermy during and after the hibernation season.

Thyroxine metabolism in active and torpid ground squirrels, Spermophilus richardsoni

Sereral aspects of thyroxine (T 4) metabolism were examined in active and torpid ground squirrels, Spermophilus richardsoni. A [ 125I]T 4 turnover study showed thyroxine half-time was increased while the total distribution volume and metabolic clearance rate were decreased in hibernators compared with active animals. However, T 4 degradation rates were similar in hibernators and nonhibernators due to increased serum titres of total and, more importantly, free T 4 (FT 4) in torpid animals. Percentage FT 4 was estimated by equilibrium dialysis, and serum total T 4 concentration, by radioimmunoassay. The estimated average FT 4 values were 1.5 ng/100 ml in hibernators and 0.7 ng/100 ml in active animals (when serum was dialysed at the respective body temperatures). Serum levels of 3,5,3′-triiodo- l-thyronine (T 3) and 3,3′,5′-triiodo- l-thyronine (rT 3) were measured by radioimmunoassay. The titres of both iodothyronines were increased in dormant animals, but the ratio of rT 3 to T 3 in hibernators was less than that in active ground squirrels. These results provide further evidence for continued thyroid function during dormancy in a mammalian hibernator.

Serum T 4 and T 3 in active and torpid ground squirrels, Spermophilus richardsoni

Serum titres of l-thyroxine (T 4) and 3,5,3′-triiodo- l-thyronine (T 3) were measured by radioimmunoassay at monthly intervals over the annual cycle of Richardson's ground squirrel, Spermophilus richardsoni. Animals were sampled in the field between March and October and from laboratory colonies of hibernating and nonhibernating ground squirrels between October and February. In nonhibernating animals, T 4 concentrations ranged from mean values of 10–30 ng/ml and T 3 titres varied from 1 to 2 ng/ml over the year. In hibernators, killed on the second day of a dormancy bout, hormone levels increased to 45–75 ng of T 4/ml and 5–6 ng of T 3/ml. Within the dormancy phase, T 4 concentrations fell by 50% during hibernation bouts and T 3 titres decreased sharply within a few hours of arousal. Levels of both iodothyronines increased before or on reentry into torpor. The results indicate continued thryoid function during dormancy in S. richardsoni.

Mitochondrial α-glycerophosphate dehydrogenase activity in active and torpid ground squirrels, Spermophilus richardsoni

1. 1. Mitochondrial α-glycerophosphate dehydrogenase (GPD) activity was assayed in liver homogenates from active and torpid ground squirrels. 2. 2. Arrhenius plots of GPD activity were linear in non-hibernating animals and discontinuous in hibernators. Compared with non-hibernators, the energy of activation in hibernators was reduced between 37 and 25°C, but increased between 25 and 6°C. 3. 3. A dose-response relation between GPD activity and injected thyroxine was determined in active animals. No correlation was found between enzyme activity at 37 or 6°C and circulating titres of thyroid hormones, in ground squirrels sampled during the preparative and hibernating phases.

HIBERNATION OF THE THIRTEEN-LINED GROUND SQUIRREL,CITELLUS TRIDECEMLINEATUS(MITCHILL). III. THE RISE IN RESPIRATION, HEART BEAT AND TEMPERATURE IN WAKING FROM HIBERNATION

The increase in respiration, heart beat and temperature as shown in graphs is very gradual at first in waking in a cold room of 0° to 8° C., whereas in a warm room of near 30° C. the process is more rapid from the first. In both cases there is a very rapid rise of all near or at the time of the opening of the eyes when the respiration rate commonly ranged between 100 and 200 a minute, and the heart beat rate usually ranged between 300 and 400 a minute, and the temperature between 20° and 34° C. The anterior part of the animal wakes more rapidly than the posterior regions, and œsophageal and food pouch temperatures indicate the increase in metabolism more accurately than deep rectal or anal temperatures. There is a general correlation between the curves of rise of respiration, heart beat and temperature. In the production of waking heart beat appears to be somewhat more effective than respiration. Waking may be induced by stimulation but its rate is not proportional to the duration or intensity of the stimulation applied, except in the case of a surrounding high temperature. Transference to a room of about 0° C. appears to stimulate waking in some but not in all torpid ground squirrels. This temperature does not prevent hibernation.