Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Regulation of the α-ketoglutarate dehydrogenasecomplex during hibernation in a small mammal,the Richardson's ground squirrel (Urocitellus richardsonii)

The citric acid cycle (CAC) is a central metabolic pathway that links carbohydrate, lipid, and amino acid metabolism in the mitochondria and, hence, is a crucial target for metabolic regulation. The α-ketoglutarate dehydrogenase complex (KGDC) is the rate-limiting step of the CAC, the three enzymes of the complex catalyzing the transformation of α-ketoglutarate to succinyl-CoA with the release of CO₂ and reduction of NAD to NADH. During hibernation, the metabolic rate of small mammals is suppressed, in part due to reduced body temperature but also active controls that suppress aerobic metabolism. The present study examined KGDC regulation during hibernation in skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii). The KGDC was partially purified from skeletal muscle of euthermic and hibernating ground squirrels and kinetic properties were evaluated at 5°, 22°, and 37 °C. KGDC from hibernator muscle at all temperatures compared with euthermic controls exhibited a decreased affinity for CoA as well as reduced activation by Ca²⁺ ions at 5 °C from both euthermic and hibernating conditions. Co-immunoprecipitation was employed to isolate the E1, E2 and E3 enzymes of the complex (OGDH, DLST, DLD) to allow immunoblot analysis of post-translational modifications (PTMs) of each enzyme. The results showed elevated phospho-tyrosine content on all three enzymes during hibernation as well as increased ADP-ribosylation and succinylation of hibernator OGDH. Taken together these results show that the KGDC is regulated by posttranslational modifications and temperature effects to reorganize enzyme activity and mitochondrial function to aid suppression of mitochondrial activity during hibernation.     

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson’s ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4 ± 1.27 pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3–5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S_0.5 and n_H values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10 °C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22 °C were directly compared with the hibernator enzyme at 10 °C. Arrhenius plots showed significantly different activation energies of 40.8 ± 0.7 and 54.3 ± 2.7 kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor.     

Evidence for a posttranslational covalent modification of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa (Jaculus orientalis)

The specific activity of d-glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) (GPDH, EC 1.2.1.12) found in liver of induced hibernating jerboa (Jaculus orientalis) was 2–3-fold lower than in the euthermic animal. However, the comparative analysis of the soluble protein fraction of these tissues by SDS-PAGE and Western blotting showed no significant changes in the intensity of the 36 kDa protein band of the GPDH subunit. After using the same purification procedure, the GPDH from liver of hibernating jerboa exhibited lower values for both apparent optimal temperature and specific activity than the enzyme from the euthermic animal. Similar non-linear Arrhenius plots were obtained, but the E_a values calculated for the GPDH from hibernating tissue were higher. Although in both purified enzyme preparations four isoelectric GPDH isoforms were resolved by chromatofocusing, those of hibernating liver exhibited more acidic pI values (pI 7.3–6.1) than the hepatic isoforms of euthermic animals (pI 8.7–8.1). However, all liver GPDH isoforms exhibited similar native and subunit molecular masses and cross-reacted with an antibody raised against muscle GPDH. The comparison of the kinetic parameters of both purified preparations and the main isoforms isolated from euthermic and hibernating tissues showed the decreased catalytic efficiency of hibernating enzyme being exclusively due to a lower V_max for both substrates G3P and NAD⁺. Phosphodiesterase treatment of cell-free extracts increased GPDH activity in the case of hibernating liver only. The pI of the main isoform purified from this tissue, about 6.9, changed after this treatment to an alkaline value (pI 8.44) similar to those of the euthermic GPDH isoforms. Differential ultraviolet absorption spectra of these isoforms indicated that a substance absorbing at 260 nm, that was released by the phosphodiesterase digestion, was present in the enzyme of hibernating tissue. Incubation of purified GPDH with the NO-releasing agent sodium nitroprussite produced under conditions that promote mono-ADP-ribosylation a dramatic decrease of activity (up to 60%) of both euthermic and phosphodiesterase-treated hibernating preparations but only a marginal inhibition of the hibernating enzyme. These data suggest that liver GPDH of hibernating jerboa exhibits a posttranslational covalent modification, being probably a mono-ADP-ribosylation. The resulting inhibition of enzyme activity could contribute to the wide depression of the glycolytic metabolic flow associated with mammalian hibernation.     

Mechanisms of glycolytic control during hibernation in the ground squirrel Spermophilus lateralis

Summary The mechanisms of glycolytic rate control during hibernation in the ground squirrel Spermophilus lateralis were investigated in four tissues: heart, liver, kidney, and leg muscle. Overall glycogen phosphorylase activity decreased significantly in liver and kidney to give 50% or 75% of the activity found in the corresponding euthermic organs, respectively. The concentration of fructose-2,6-bisphosphate (F-2,6-P₂) decreased significantly in heart and leg muscle during hibernation to 50% and 80% of euthermic tissue concentrations, respectively, but remained constant in liver and kidney. The overall activity of pyruvate dehydrogenase (PDH) in heart and kidney from hibernators was only 4% of the corresponding euthermic values. Measurements of phosphofructokinase (PFK) and pyruvate kinase (PK) kinetic parameters in euthermic and hibernating animals showed that heart and skeletal muscle had typical rabbit skeletal M-type PFK and M₁-type PK. Liver and kidney PFK were similar to the L-type enzyme from rabbit liver, whereas liver and kidney PK were similar to the M₂ isozyme found primarily in rabbit kidney. The kinetic parameters of PFK and PK from euthermic vs hibernating animals were not statistically different. These data indicate that tissue-specific phosphorylation of glycogen phosphorylase and PDH, as well as changes in the concentration of F-2,6-P₂ may be part of a general mechanism to coordinate glycolytic rate reduction in hibernating S. lateralis.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenonine triphoshate - EDTA ethylenediaminetetra-acetic acid - EGTA ethylene glycol tetra-acetic acid - F-6-P fructose 6-phosphate - F-1,6-P₂ fructose 1,6-bisphosphate - F-2,6-P₂ fructose-2,6-bisphosphate - K _a activation coefficient - I₅₀ concentration of inhibitor which reduces control activity by 50% - PDH pyruvate dehydrogenase - PEP phosphoenolpyruvate - PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson’s ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4 ± 1.27 pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3–5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S_0.5 and n_H values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10 °C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22 °C were directly compared with the hibernator enzyme at 10 °C. Arrhenius plots showed significantly different activation energies of 40.8 ± 0.7 and 54.3 ± 2.7 kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor.     

Sexual differences in over-winter torpor patterns of Richardson's ground squirrels in natural hibernacula

Summary Over-winter torpor patterns of Richardson's ground squirrels hibernating in southern Alberta were monitored with temperature-sensitive radiocollars to determine if these patterns differed between males and females in a manner related to the greater costs of mating effort by males than females. The hibernation season (from immergence to emergence) was composed of three periods: post-immergence euthermy, heterothermy, and pre-emergence euthermy. The hibernation season was shorter for juveniles than adults both among males (< 150 versus 234 days) and females (185 versus 231 days), a reflection of the later immergence into hibernation by juveniles. However, regardless of the absolute duration of hibernation, heterothermy accounted for a smaller proportion of the hibernation season of males (93±5%) than females (98±1%) and, within the heterothermal period, males had shorter torpor bouts and longer inter-torpor arousals. Overall, males spent a smaller proportion of the hibernation season in torpor (85±6%) than females (92±1%). This sexual difference was largely attributable to the longer duration of preemergence euthermy for males than females. Males terminated torpor in January and February, when hibernacula were at their coldest, then remained euthermic for 8.8 days (range 0.5–25.0 days) before emergence. In contrast, females terminated torpor in March, when hibernaculum temperatures were increasing, then remained euthermic for only 1.1 days (range 0.5–2.0 days) before emergence. Males lost less mass per euthermic day during hibernation than females (7.0 versus 9.3 g/day). Males and females hibernated at similar depths (56 cm), but males had larger chambers than females (18 versus 16 cm³/g). Many males, but no females, cached seeds in the hibernaculum. Males met the costs of thermogenesis and euthermy from a combination of fat reserves and food caches, whereas females relied solely on fat. Access to food caches permitted males to terminate torpor several weeks in advance of emergence, during which time they recouped mass and developed sperm in preparation for the forthcoming mating season.     

The influence of hibernation patterns on the critical enzymes of lipogenesis and lipolysis in prairie dogs

White-tailed prairie dogs (Cynomys leucurus) are spontaneous hibernators that enter torpor each fall, whereas black-tailed prairie dogs (C. ludovicianus) hibernate facultatively only when food- or water-stressed during the winter. The body masses of both species greatly increase during the fall feeding period, with most of this gain in the form of depot fat. Body fat is utilized during winter fasting and/or hibernation. We measured the activities of fatty acid synthase (FAS), ATP-citrate lyase (ACL), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), and hormone-sensitive lipase (HSL) in the tissues of both C.leucurus (hibernating and euthermic) and C. ludovicianus (euthermic only) under controlled conditions. The activities of FAS, ACL, and G6PDH in the liver all decreased during hibernation. The activities of ME and G6PDH in white adipose tissue (WAT) were also reduced during hibernation. Euthermic C. leucurus and euthermic C. ludovicianus differed only in brown adipose (BAT) ACL and WAT G6PDH activities. No significant differences in HSL activities were found between these two species or between euthermic and hibernating animals. These results suggest that this seasonal body fat cycle is due, at least in part, to seasonal variations in the activities of FAS, ME, ACL, and G6PDH that affect the rate of fatty acid synthesis. This study also demonstrates that spontaneous hibernators do not have a greater capacity to synthesize fatty acids during the fall than facultative hibernators, as previously suggested.     

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.     

Hibernating patterns of free-ranging Columbian ground squirrels

Summary The patterns of torpor and euthermy during hibernation was documented for 28 free-ranging Columbian ground squirrels (Spermophilus columbianus) fitted with temperature-sensitive radio transmitter collars. Adult males began hibernation earlier, were euthermic for a greater proportion of the hibernating season and emerged earlier than other age and sex classes. The patterns of hibernation of adult females did not differ significantly from those of juveniles. Emergence from the hibernaculum was preceded by a long (3–12 d) euthermic interval in adult males but not in adult females or juveniles. Changes in soil temperature did not appear to initiate emergence. The greater time spent euthermic by adult males is interpreted as a significantly greater energy cost of hibernation for adult males than for other age and sex classes. The benefits offsetting these costs may be increased reproductive potential in spring and avoidance of predation in late summer.     

Spontaneous and induced summer hibernation in 13-lined ground squirrels

Among several mammalian hibernators, an endogenous circannual sequence of physiological events is believed to mediate the timing of torpor. Dawe and Spurrier (3) reported that a bloodborne substance (hibernation induction trigger) is important in initiating the torpor phase of those events in the 13-lined ground squirrel. We have reported the induction of summer hibernation among 13-lined ground squirrels using dialysates of serum from hibernating golden hamsters (a nonseasonal hibernator). While those animals receiving saline injections hibernated in 36.3 ± 2.9 days, an earlier induction (22 ± 8.8 days) occurred among those receiving the hibernation serum dialysate (P = 0.05). It was also observed that naive animals departed from a strict circannual rhythm and displayed a high incidence of hibernation, although not significant when compared to the experimental saline controls. The spontaneity of torpor in summer among the naive sample may in part be a characteristic of wild-caught animals employed in the bioassay. Nevertheless, the induction of hibernation among those animals receiving the hibernation serum preparations is supportive of the studies of Dawe and Spurrier (2, 3). That a “trigger” material apparently is present in the hamster, a phylogenetically distinct nonseasonal hibernator, suggests that a characteristic of rodent hibernators is the presence of a material which is associated with the initiation of torpor.     

Energy expenditure increases during the active season in the small,free-living hibernator Muscardinus avellanarius

Little is known about strategies employed by small mammals to reduce energy expenditure during the summer. To understand whether ambient conditions impact euthermic energy demands in a small free-living hibernator, we measured metabolic rate of hazel dormice (Muscardinus avellanarius) in the field. Furthermore, we aimed to reveal which variables influence torpor use. Our results show that hazel dormice altered euthermic energy expenditure during summer but not as expected as a response to environmental conditions. Euthermic resting metabolic rate was lowest directly after emergence from hibernation and increased by about 95% until the end of August. A considerable part of this increase was presumably caused by the changing influence of gender and rain on energy demands during different months, variation in food quality and quantity, and reversible size changes of organs that had been atrophied during hibernation. Torpor use in hazel dormice occurred more frequently when it was colder, earlier during the day, and in lighter individuals. Torpor was used routinely in males and non-reproductive females. We show that torpor is used more frequently than previously suggested by studies that only used visual proof of torpor use by surveying nest boxes.     

Body temperature patterns and use of torpor in an alpine glirid species,woolly dormouse

Woolly dormice, Dryomys laniger Felten and Storch (Senckenbergiana Biol 49(6):429–435, 1968), are a small (20–30 g), omnivorous (mainly insectivorous), nocturnal glirid species endemic to Turkey. Although woolly dormice have been assumed to hibernate during winter, no information exists on body temperature patterns and use of torpor in the species. In the present study, we aimed to determine body temperature patterns and use of torpor in woolly dormice under controlled laboratory conditions. Accordingly, body temperature (Tb) of woolly dormice was recorded using surgically implanted Thermochron iButtons, small and inexpensive temperature-sensitive data loggers. Woolly dormice exhibited robust, unimodal daily Tb rhythmicity during the euthermic stage before the beginning of hibernation. They displayed short torpor before they began hibernation, although the tendency to enter short torpor was different among individuals. Woolly dormice began hibernation within 1–3 days after exposure to cold and darkness, i.e., on October 22–24, and ended hibernation in the first half of April. Hibernation consisted of a sequence of multiday torpor bouts, interrupted by euthermic intervals. Thus, the patterns of hibernation in woolly dormice were similar to those observed in classical hibernating mammals.     

Metabolic Hormone FGF21 Is Induced in Ground Squirrels during Hibernation but Its Overexpression Is Not Sufficient to Cause Torpor

Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes that are lethal to humans. Near freezing body temperatures, heart rates of 3–10 beats per minute, absence of food consumption, and depressed metabolism are characteristic of hibernation torpor bouts that are periodically interrupted by brief interbout arousals (IBAs). The molecular basis of torpor induction is unknown, however starved mice overexpressing the metabolic hormone fibroblast growth factor 21 (FGF21) promote fat utilization, reduce body temperature, and readily enter torpor–all hallmarks of mammalian hibernation. In this study we cloned FGF21 from the naturally hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and found that levels of FGF21 mRNA in liver and FGF21 protein in serum are elevated during hibernation torpor bouts and significantly elevated during IBAs compared to summer active animals. The effects of artificially elevating circulating FGF21 concentrations 50 to 100-fold via adenoviral-mediated overexpression were examined at three different times of the year. This is the first time that a transgenic approach has been used in a natural hibernator to examine mechanistic aspects of hibernation. Surgically implanted transmitters measured various metrics of the hibernation phenotype over a 7-day period including changes in motor activity, heart rate and core body temperature. In April fed-state animals, FGF21 overexpression decreased blood insulin and free fatty acid concentrations, effects similar to those seen in obese mice. However, elevated FGF21 concentrations did not cause torpor in these fed-state animals nor did they cause torpor or affect metabolic parameters in fasted-state animals in March/April, August or October. We conclude that FGF21 is strongly regulated during torpor and IBA but that its overexpression is not sufficient to cause torpor in naturally hibernating ground squirrels.