Phosphorylation of myosin light chains and C-protein in the myocardium of hibernating ground squirrel Citellus undulatus

A comparative study was made of the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of the hibernant ground squirrel Citellus undulatus during the periods of hibernation and activity. During hibernation, the light chains were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averaged 40–45%. The extent of cardiac C-protein phosphorylation in hibernation was about twice higher than in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.     

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.     

Phosphorylation of sarcomeric cytoskeletal proteins--an adaptive factor for inhibiting the contractile activity of muscle during hibernation

The influence of phosphorylation in vitro of the sarcomere cytoskeletal proteins titin and X-protein of skeletal muscles as well as C-protein of cardiac muscle of ground squirrel Citellus undulatus on the actin-activated ATPase activity of myosin and its Ca2+ sensitivity was studied. It was shown that phosphorylation lowers the activating effect of titin and C-protein and increases the inhibitory effect of X-protein on the enzymatic properties of actomyosin. The phosphorylation of the proteins has the most pronounced influence on Ca2+ sensitivity of actomyosin: it drops to a greater extent in the presence of phosphorylated C-protein and titin and is completely inhibited by phosphorylated X-protein. The inhibitory influence of phosphorylation in vitro of sarcomere cytoskeletal proteins on the above functional properties of the actomyosin system as well as the increase in the extent of phosphorylation of titin in vivo upon hibernation allow one to conclude that this posttranslation modification contributes to adaptive mechanisms of suppression of the contractile ability of muscles in this period.     

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.     

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.     

Distribution of superoxide dismutase in the ground squirrel (Citellus citellus): effect of the hibernation and arousal

The activity of superoxide dismutase (SOD) was studied in the liver, kidney, interscapular brown adipose tissue (IBAT), lung, heart and spleen of the active and hibernating ground squirrel (Citellus citellus). One group was examined immediately after the arousal from the hibernation. A considerable activity of this enzyme was found in homogenates of all tissues studied except the lung. This activity was lower in the liver and lung of the ground squirrel than in the rat (P less than 0.01). In the other tissues studied the enzyme activity was about the same level in both animals. In the ground squirrel hibernation didn't produce the significant change in SOD activity, as compared with the active state, except in the spleen. Tested immediately after the arousal, SOD activity was significantly higher in all tissues studied except in the IBAT, as compared with the hibernating ones (P less than 0.01).     

Regulation of Akt during torpor in the hibernating ground squirrel, Ictidomys tridecemlineatus

The 13-lined ground squirrel (Ictidomys tridecemlineatus) is capable of entering into extended periods of torpor during winter hibernation. The state of torpor represents a hypometabolic shift wherein the rate of oxygen consuming processes are strongly repressed in an effort to maintain cellular homeostasis as the availability of food energy becomes limited. We are interested in studying hibernation/torpor because of the robust state of tolerance to constrained oxygen delivery, oligemia, and hypothermia achieved by the tissues of hibernating mammals. The role of the serine/threonine kinase Akt (also known as PKB) has been examined in torpor in previous studies. However, this is the first study that examines the level of Akt phosphorylation in the liver during the two transition phases of the hibernation cycle: entrance into torpor, and the subsequent arousal from torpor. Our results indicate that Akt is activated in the squirrel liver by phosphorylation of two key residues (Thr³⁰⁸ and Ser⁴⁷³) during entrance into torpor and arousal from torpor. Moreover, we observed increased phosphorylation of key substrates of Akt during the two transition stages of torpor. Finally, this study reports the novel finding that PRAS40, a component of the TORC1 multi-protein complex and a potentially important modulator of metabolism, is regulated during torpor.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

The Van't Hoff rule in studying various phases of hibernation of the squirrel Citellus undulatus Pallas. Physico-chemical route to cold adaptation

Taking into account the van't Hoff's law the rates of relative erythrocyte hemolysis in ground squirrel Citellus undulatus Pallas during different phases of hibernation were first studied by the method of acid erythrograms with some modifications. The temperature of the erythrogram registration (8 degrees C) models the body temperature in hibernation, and temperature of 35 degrees C, corresponds to the body temperature of the awakened ground squirrel. The positions of the erythrogram maxima for the ground squirrel during short-term arousal and during hibernation coincide for each temperature studied: 8 or 35 degrees C. Therefore, the increase in HCl concentration at 8 degrees C reflects an increase in the stability of erythrocyte membranes or a decrease in the rate of relative hemolysis for the ground squirrel during hibernation. Thus, the adaptive response of erythrocytes in hibernating ground squirrel is revealed. The correlation of the rates of physicochemical processes in vitro with the rates of physiological ones in vivo is shown using the ratio of the rates mentioned above for the short-term arousal and for hibernation. First the physicochemical way of cold adaptation due to the van't Hoff's law is proved.     

Ultrastructure of cells from the liver-endocrine pancreas system in hibernating animals

In hibernated ground squirrel (Citellus erythrogenys) during various periods of hibernation morphological changes in organs participating in insurance of energoplastic homeostasis (liver, endocrine part of the pancreas) have been followed. At the beginning of hibernation certain signs of functional strain in the liver are observed-shortening and swelling of the endoplasmic reticulum (ER) canaliculi, decreases of glycogen resources and activation of the lysosomal apparatus, as well as certain features demonstrating its decreased activity--shortening cisterns in the Golgi complex, poorly manifested granular ER, tightening of mitochondrial matrix. At a deep hibernation, the signs of strain disappear, ultrastructure of hepatocytes corresponds to a new lower level of functioning in comparison to that in the active state and at the beginning of hibernation. During these periods the structure of endocrine cells in the pancreas demonstrates certain changes in the character of functioning of the cells. Ultrastructural transformations in the liver and endocrine part of the pancreas in the ground squirrel are mutually correlated.     

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.     

Changes of characteristics of preoptic neurons and NA metabolism in hypothalamus of ground squirrel (Citelleus Dautieus) in different seasons and hibernating phases

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The evaluation of the sizes of erythrocytes of the hibernating ground squirrel Citellus undulatus Pallas

A topographical image of individual erythrocytes of the ground squirrel Citellus undulatus Pallas, in unfixed unstained smears was first obtained by scanning probe microscopy for two states of the animal: hibernation and the active state. The scannig of single discocytes, i.e. erythrocytes having a typical discal form, was fulfilled. For the active male, the diameter of the discocyte was found to be approximately 6500 nm. For the hibernating female, the diameter is approximately 6000 nm. According to the data of light microscopy, the discocyte diameters are: 6610 +/- 100 nm for the active state of animal and 6430 +/- 160 nm for the hibernating state.     

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.     

Changes of characteristics of preoptic neurons and NA metabolism in hypothalamus of ground squirrel (Citelleus Dautieus) in different seasons and hibernating phases

The unit firing activities of neurons in the preoptic area (POA) of ground squirrel hypothalamic tissue slices were recorded and the metabolism of NA in hypothalamus was measured with high performance liquid chromatography (HPLC). Thermosensitivity, proportions, the critical temperature (Tc) and the lowest temperature (TL) of firing activity of the above-mentioned neurons, and NA metabolism in hypothalamus were compared in different seasons and hibernating phases. In comparison with that in summer euthermar, it was shown that (i) the percentage and thermosensitivity of the POA neurons varied respectively in the hibernating phases; (ii) TL and Tc of the POA neurons in winter, both euthermar and hibernation, were markedly decreased; (iii) the POA neurons in hibernation became much more sensitive to NA, and the response of cold-sensitive neurons to NA changed from inhibiting pattern in summer to exciting one in hibernation; (iv) the contents and metabolism of NA in hypothalamus decreased significantly in the entering phase and deep hibernation phase, while the metabolism of NA increased remarkably in the arousal phase. These changes might explain the regulatory mechanism how ground squirrel actively decreases body temperature (Tb) in entering into hibernation and quickly recovers body temperature in arousal phase. Project supported by the National Natural Science Foundation of China (Grant Nos. 39230060 and 39570100)     

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus)

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for β-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.     

Seasonal variations of DNA synthesis in intestinal epithelial cells of hibernating animals--I. DNA synthesis in intestinal epithelial cells of ground squirrel (Citellus undulatus) during deep hibernation

The conditions for obtaining crypt cells from ground squirrel small intestine were chosen which allow flow-through cytofluorometric analysis of the DNA synthesis of this tissue. DNA synthesis was found to be greatly reduced in the intestinal crypt cells of ground squirrel during deep hibernation in torpid animals, in animals during spontaneous arousals and in animals prevented from hibernation. The conclusion is made about endogenous control of the DNA synthesis in the cells of true hibernators.     

Gradual reappearance of post-hibernation circadian rhythmicity correlates with numbers of vasopressin-containing neurons in the suprachiasmatic nuclei of European ground squirrels

European ground squirrels (Spermophilus citellus) in outside enclosures show suppressed circadian rhythmicity in body temperature patterns during the first days of euthermia after hibernation. This may reflect either gradual reappearance of circadian rhythmicity following suppressed functioning of the circadian system during hibernation, or it may reflect transient days during re-entrainment of the circadian system which, during hibernation, has drifted out of phase with the environmental light-dark cycle. Here we report that animals kept under continuous dim light conditions also showed absence of circadian rhythmicity in activity and body temperature in the first 5-15 days after hibernation. After post-hibernation arrhythmicity, spontaneous circadian rhythms re-appeared gradually and increased daily body temperature range. Numbers of arginine-vasopressin immunoreactive neurons in the suprachiasmatic nuclei correlated positively with individual circadian rhythmicity and increased gradually over time after hibernation. Furthermore, circadian rhythmicity was enhanced rather than suppressed after exposure to a light-dark cycle but not after a single 1-h light pulse (1,700 lux). The results support the view that the functioning of the circadian system in the European ground squirrel is suppressed during hibernation at low temperatures and that it requires several days of euthermia to resume its summer function.     

The intestinal epithelial cells of ground squirrel (Citellus undulatus) accumulate at G2 phase of the cell cycle throughout a bout of hibernation

1. The mitotic index was found to be greatly reduced in the intestinal crypt cells of ground squirrel during bout of hibernation. The percentage of mitosis rose abruptly at least 2 hr after arousal. 2. An increase in the number of G2 cells was found in the intestinal tract of ground squirrel during bouts of hibernation. 3. The conclusion can be drawn that the cells are progressing steadily through the cell cycle. The cells accumulate at G2 in hibernation. 4. It was assumed that the block in G2 prevents the cells from possible damage in mitosis under hypothermia accompanying hibernation and, therefore, it represents an adaptive reaction.