Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.     

Hepatic changes in the freeze-tolerant turtle Chrysemys picta marginata in response to freezing and thawing

Select hepatic changes in the freeze-tolerant hatchling turtle, Chrysemys picta marginata, were studied in response to freezing at -2.5 degrees C and thawing. Upon freezing, a small, selective increase in the liver weight with no increase in body weight was seen suggestive of an hepatic capacitance response. In all turtles studies, lobular differences in the hepatic content of glycogen were evident: the smaller lobe contained twice as much glycogen as the larger lobe. The response to freezing and thawing was comparable. Total hepatic glycogen levels of turtles were reduced approximately 60 per cent from control levels in the frozen state and recovered to >80 per cent of control levels in the thawed state. Compared to the control state, turtle blood glucose levels were: unchanged after 12 h in the cool state; reduced 28 per cent after 24 h and increased two-fold after 48 h in the frozen state; and increased 4.5-fold in the thawed state. Thus, changes in hepatic glycogen metabolism occur without large changes in blood glucose levels. In turtle liver plasma membranes, the hepatic alpha(1)-adrenergic receptor was barely detectable and did not change. The beta(2)-adrenergic receptor was expressed at high levels and, compared to control levels, was: unchanged after 12 h in the cool state; reduced 20 per cent after 24 h and 40 per cent after 48 h in the frozen state. On thawing, this receptor was 50 per cent of control levels. While catecholamines working through the beta(2)-adrenergic receptor may effect early hepatic glycogen breakdown in response to freezing, other factors must be involved to complete the process. The plasma membrane-bound enzyme gamma-glutamyltranspeptidase displayed a different pattern of changes indicative of selective modulation: it was increased 2.7-fold over control levels in the cool state; unchanged in the frozen state; and increased 1.8-fold in the thawed state. The activity of the kidney enzyme was decreased in the cool state and slightly increased in the frozen and thawed states emphasizing the tissue-specific nature of the changes in the activity of gamma-glutamyltranspeptidase in response to freezing and thawing. The similarities and differences of the hepatic changes in response to freezing and thawing in the freeze-tolerant hatchling turtle to those we have previously reported for the freeze-tolerant frog are discussed.     

Postfreeze reduction of locomotor endurance in the freeze-tolerant wood frog,Rana sylvatica

Considerable study has focused on the physiological adaptations for freeze tolerance in the wood frog, Rana sylvatica, a northern species that overwinters within the frost zone, but little attention has been paid to the associated costs to organismal performance. Here we report that freezing causes transient impairment of locomotor endurance and adverse changes in exercise physiology that persist for at least 96 h. Wood frogs frozen at -2 degrees C for 36 h exhibited normal behaviors and hydro-osmotic status and near-normal metabolite (glycogen, glucose, and lactate) levels within 24 h after thawing began. However, when exercised to exhaustion on a treadmill, these frogs showed a 40% reduction in endurance as compared to sham-treated (unfrozen) controls, a reduction that persisted for at least 96 h. Previously frozen frogs exhibited higher rates of lactate accumulation during exercise than controls, suggesting that prior freezing forces greater reliance on the glycolytic pathways of energy production to support exercise. Given that this species breeds in late winter, when subzero temperatures are common, freezing may result in reduced fitness by hampering their ability to reach the pond, avoid predators, and successfully obtain mates.     

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation

The wood frog, Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state. MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 degrees C acclimated), 24 h frozen at -2.5 degrees C, 4 or 8 h thawed at 5 degrees C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K(m) and V(max) values compared with controls; e.g., K(m) Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V(max) decreased by 40%. After thawing, K(m) was restored and V(max) rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V(max) implicating reversible phosphorylation in MCP regulation during freeze-thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze-thaw.     

Freeze-thaw effects on metabolic enzymes in wood frog organs.

To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.     

Freeze-Induced Alterations of Translatable mRNA Populations in Wood Frog Organs

To investigate the roles that gene expression and new protein synthesis play in freezing survival by the wood frog, Rana sylvatica, we compared the in vitro translation products made from mRNA isolated from six tissues (liver, brain, heart, muscle, kidney, gut) of control (5 degrees C), frozen (24 h at -2.5 degrees C), and thawed (24 h at 5 degrees C after 24 h frozen) frogs. [(35)S]Methionine-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and located by fluorography. Results indicated specific changes in the translatable populations of mRNA in tissues of freezing-exposed frogs that were largely reversed upon thawing. Differential protein expression was greatest in the comparison of liver from control versus frozen frogs with proteins ranging from 45 to 14.8 kDa identified as enhanced or unique to the frozen state. One unique protein appeared in skeletal muscle (116 kDa) of freeze-exposed frogs while another (52.5 kDa) was enhanced. Analysis of brain and heart each revealed the presence of one protein unique to the frozen state in each (58.9 and 5.9 kDa, respectively) whereas no change in the pattern of in vitro translation products was seen in gut (stomach + intestine combined) or kidney between the three experimental states. These freeze-induced alterations in the populations of translatable mRNA suggest that changes in the complement of specific proteins underlie various adaptive responses that contribute to the freezing survival of this amphibian.     

New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps

New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25-2.0 g) of skeletal muscle. Thebeta(2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalbeta(2)-adrenergic receptor was expressed in frogs and mammals at similar levels of expression (336-454 fmol. x mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol. x mg(-1)), oxalate stimulation was variable: low in frogs ( approximately 1.05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4-9 nmol. x mg(-1)) and much higher in mammals (322-383 nmol. x mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (<70-fold) than in mammals (1.6-2-fold). It is concluded that thebeta(2)-adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

In vitro formation of glycogenolytic enzyme complexes with the sarcoplasmic reticulum in the skeletal muscles of skates and the frog

Experimental evidence is presented concerning the existence of complexes of glycogenolytic enzymes with sarcoplasmic reticulum (SR) in skeletal muscles of the skates Dasyatis pastinaca and Raja clavata and frog Rana temporaria. At various stages of preparation of kinase of glycogen phosphorylase (KGP) from ectothermic animals, in contrast to rabbit, association of KGP with the SR and glycogen granules persisted in calcium-free medium. Complex of KGP with glycogen phosphorylase and ATPase could be fractionated only during chromatographic procedure on Sepharose 4B, chromatographic pictures being distinctly different from those obtained for rabbit. It may be suggested that activation of KGP by Ca2+ in a multienzyme SR--glycogenolytic complex plays an important role in regulation of glycogenolysis in muscle tissue of skates, since hormonal stimulation of glycogen phosphorylation had not yet been described for these fishes.     

Pressure shift freezing of pork muscle: effect on color, drip loss, texture, and protein stability

Cylindrical specimens (50 mm diameter and 160 mm length) of fresh pork muscle (boneless rib portions) packed in plastic bags were frozen by pressure shift freezing (PSF) at 100, 150, and 200 MPa, air blast freezing (ABF), and liquid immersion freezing (LIF). Temperature and phase transformations of the muscle tissue were monitored during the freezing process at three locations: center, midway between the center and the surface, and near the surface. Pork muscle quality changes [color, drip loss (both thawing and cooking), texture (shear force), and protein stability (DSC thermal profiles)] were evaluated after thawing the frozen samples at room temperature (20 degrees C). Employing pressures above 150 MPa caused very significant (P < 0.01) color changes in pork muscle during the PSF process. The PSF process reduced thawing drip loss of pork muscle but did not cause obvious changes in total drip loss following thawing and subsequent cooking. PSF at 150 and 200 MPa resulted in considerable denaturation of myofibrillar proteins of pork muscle. The PSF process also caused an increase in the pork muscle toughness as compared with that of unfrozen, ABF, and LIF samples.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Freeze tolerance in the gray treefrog: cryoprotectant mobilization and organ dehydration.

Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.     

Regulation of hexokinase by reversible phosphorylation in skeletal muscle of a freeze-tolerant frog

Hexokinase (HK) was isolated from hind leg skeletal muscle of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K(m) and V(max)) of HK showed significant increases in K(m) glucose (from 144 ± 4.4 to 248 ± 1 2.0 μM) and K(m) ATP (from 248 ± 8.5 to 330 ± 20.9 μM), as well as a decrease in V(max) (from 86.1 ± 0.40 to 52 ± 0.49 mUmg(-1) of protein) in frogs following freezing exposure, indicating lower affinity for HK substrates and lower enzyme activity in this state. Subsequent analyses indicated that differential phosphorylation of HK between the two states was responsible for the altered kinetic properties. HK was analyzed by SDS-PAGE; phosphoprotein staining revealed a 33% decrease in phosphate content of HK from frozen frogs but immunoblotting showed no change in total HK protein content. Muscle extracts from control and frozen frogs were incubated with ions and second messengers to stimulate the actions of protein kinases and protein phosphatases, with results indicating that HK can be phosphorylated by protein kinases A and C, and AMP-activated protein kinase, and can be dephosphorylated by protein phosphatases 1, 2A and 2C. The data indicate that in control frogs, HK is in a higher phosphate form and displays a high substrate affinity and high activity, whereas in frozen frogs HK is less phosphorylated, with lower substrate affinity and lower activity. Studies also showed that HK affinity for ATP decreases further in response to low temperature, but that high cryoprotective glucose concentrations can prevent these changes in affinity. Finally, the activity and structure of HK from frozen frogs is more sensitive to non-compatible osmolytes than the enzyme in control frogs.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.