The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

Adaptive behavior of titin isoforms from skeletal and cardiac muscles of ground squirrels (Citellus undulatus) during hibernation

By the use of modified SDS electrophoresis in agarose-strengthened 2% polyacrylamide gels, the adaptive behavior of titin isoforms in skeletal and cardiac muscles of ground squirrels (Citellus undulatus) during hibernation was studied. The presence of two titin isoforms (short and long) with molecular weights approximately 3700 and approximately 3800 kDa in m. soleus, approximately 3400 and approximately 3600 kDa in m. psoas, approximately 3000 and approximately 3400 kDa in the left ventricle of myocardium was found. It was found that the content of the short titin isoform in the above muscles of hibernating and arousing ground squirrels is considerably lower than that of the long titin isoform. The preservation of the long titin isoform in skeletal and cardiac muscles of hibernating and arousing ground squirrels can be regarded as an evolutionarily determined adaptive mechanism favoring the survival of animal under extreme conditions without pathological consequences.     

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [32P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [H3]-labeled GTP. Type II T4 5'-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T4 5'-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T4 5'-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.     

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.     

Pharmacological characterization of alpha1- and beta-adrenergic synergism of 5'DII activity in rat brown adipocytes

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.     

Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.     

Induction of summer hibernation in the 13-lined ground squirrel, Citellus tridecemlineatus

The induction of summer hibernation in the 13-lined ground squirrel (Citellus tridecemlineatus) by intravenous injection of plasma obtained from winter hibernating ground squirrels was confirmed. Hibernation was also induced by injection of urine from arousing winter ground squirrels. Results support the “trigger” theory of hibernation proposed by Dawe and Spurrier (3) and also suggest that tissues are set free from “trigger” influence during winter arousal by the excretion of “trigger.”     

Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.     

Markers of tissue remodeling and inflammation in the white and brown adipose tissues of a model hibernator

The thirteen-lined ground squirrel is a model fat-storing hibernator that nearly doubles its weight in the fall to fuel metabolism with triglycerides throughout the winter months. Hibernator brown and white adipose tissue (BAT, WAT) are important to study in terms of their inflammatory profile and tissue remodeling mechanisms since controlled and natural regulation of these processes could inform new pharmacological interventions that limit oxidative stress and inflammation in the adipose tissues of humans suffering from obesity, promote non-shivering thermogenesis-mediated weight loss, or prevent tissue damage in transplantable organs emerging from cold-storage. Thus, markers of inflammation like cytokines and soluble receptors and tissue remodeling proteins such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were investigated in normothermic, torpid, and arousing ground squirrels. Multiplex protein assays and western blotting revealed fewer changes in WAT compared to BAT. Pro-inflammatory IL-1α levels increased during torpor and soluble epidermal growth factor receptor protein levels increased during arousal in BAT. Given their known roles in other model systems, these proteins could regulate processes like adipogenesis, lipid catabolism, or cell motility. Decreased TIMP2 levels combined with maintained MMP2 or MMP3 protein levels suggested that BAT may avoid tissue remodeling until arousal. No changes in WAT inflammatory cytokines or soluble receptors as well as decreased MMP2 levels during torpor and arousal suggested inflammation and modification to the extracellular matrix is likely suppressed in WAT. This study emphasizes the fat-but-fit nature of the hibernating ground squirrel and the ability of its fat stores to suppress inflammation.     

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [³²P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [³H]-labeled GTP. Type II T₄ 5′-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T₄ 5′-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T₄ 5′-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.     

Hibernation induces glutathione redox imbalance in ground squirrel intestine

Glutathione (GSH) is the major thiol-disulfide redox buffer in cells and is a critical component of antioxidant defense. Here we examined GSH redox balance in the intestinal mucosa during the annual cycle of 13-lined ground squirrels (Spermophilus tridecemlineatus). The ratio of reduced GSH to its oxidized form (glutathione disulfide, GSSG), which is an index of oxidative stress, was five-fold lower in hibernating compared with summer-active squirrels, an effect due primarily to elevated GSSG concentration in hibernators. During hibernation the total pool of GSH equivalents was lowest in squirrels undergoing arousal and highest in squirrels during interbout arousals. Hibernation decreased intestinal GSSG reductase activity by approximately 50%, but had no effect on activities of glutathione peroxidase or glucose-6-phosphate dehydrogenase. Within the hibernation season, expression of the stress protein HSP70 in intestinal mucosa was highest in squirrels entering torpor and early in a torpor bout, and lowest in squirrels arousing from torpor and during interbout euthermia. The results suggest that hibernation in ground squirrels is associated with a shift in intestinal GSH redox balance to a more oxidized state. Higher levels of HSP70 during the early phases of torpor may reflect induction of the stress response due to aberrations in protein folding or may be a mechanism to increase enterocyte tolerance to subsequent stress imposed by extended torpor or the arousal process.     

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.     

Mitochondrial metabolism in hibernation: metabolic suppression, temperature effects, and substrate preferences

We compared liver and skeletal muscle mitochondrial function among activity states to characterize regulated reversible metabolic suppression in the mammalian hibernator Spermophilus tridecemlineatus. At 37 degrees C, succinate oxidation was 70% lower in the liver mitochondria from torpid animals than in those from summer-active animals or in animals arousing from torpor. Respiration was very sensitive to temperature (Q(10) 5.8-9.8), and when measured at 25 degrees or 5 degrees C there was no difference among the three states. Liver mitochondria from summer-active animals oxidized pyruvate and beta -hydroxybutyrate at higher rates than those from torpid animals, and flux through complex 4 of the electron transport chain was about three- and fivefold higher than flux through complexes 2-4 and complexes 1-4, respectively. In the hibernating and arousing animals there was no difference in flux through complexes 2-4 and complex 4, suggesting a downregulation of cytochrome c oxidase in liver mitochondria during the hibernation season. Muscle mitochondrial respiration did not differ between the torpid and summer-active states in any of the parameters measured. The data support a regulated, reversible decrease of liver (but not muscle) mitochondrial oxidative phosphorylation in hibernating ground squirrels.     

Summer hibernation of infant (six week old) 13-lined ground squirrels, Citellus tridecemlineatus

In an endeavor to more clearly define the physiological action of hibernation trigger (previously identified by the authors) an experiment was devised using infant ground squirrels. In one case, pregnant ground squirrel mothers were transfused intravenously with cold dialysate of serum of hibernating woodchuck, and in another case, infant (1 week to 3 week) squirrels were transfused intraperitoneally with the same material. When isolated and placed in a cold room (15 °C) at 6 weeks post partum, both the infant born of the transfused mothers, as well as the infant which has been transfused directly, hibernated in the summer. This was in contrast to controls of several kind, i.e., animals not injected, or receiving active or arousing woodchuck serum or heat-treated hibernation serum. Some details for a theory, which had been advanced in a previous article (4), were essentially substantiated by this experiment. It is our belief that this represents the first time mammalian hibernation has been observed in infant hibernators.     

Uncoupling protein1 mRNA, mitochondrial GTP-binding, and T4 5'-deiodinase of brown adipose tissue in euthermic Daurian ground squirrel during cold exposure

Regulation of thermogenic activity and uncoupling protein1 (UCP1) expression in brown adipose tissue (BAT) were studied in euthermic Daurian ground squirrel after acute and chronic cold exposure at 4 degrees C. The UCP1 concentration was indirectly determined by titration with its specific ligand [3H]-labeled GTP, and Ucp1 mRNA was detected by using a [32P]-labeled antisense oligonucleotide probe. Both acute and chronic cold exposure stimulated up-regulation of Ucp1 mRNA. Although UCP1 concentration is not significantly increased after 24 h of cold exposure, it is markedly elevated by 75% in squirrels after 4-week cold adaptation compared with controls raised at 22 degrees C. Changes in T4 5'-deiodinase activity were closely associated with variations of Ucp1 mRNA level. Ucp1 gene expression is significantly affected by cold exposure in BAT from euthermic Daurian ground squirrels. In addition, the activation of T4 5'-deiodinase may be an important regulatory factor in cold-induced Ucp1 expression.