Freeze-tolerant Frog Research Articles

Novel observations of "freeze resistance" and dynamic blue and green dorsal coloration in frozen and thawing Dryophytes chrysoscelis.

Freeze tolerant animals survive the winter by tolerating the freezing and thawing of up to 70% of body water and the respective cessation and resumption of essential functions including circulation and respiration during each freeze-thaw cycle. Cope's gray treefrog Dryophytes chrysoscelis is a freeze tolerant anuran that uses a system of cryoprotectants to prevent intracellular freezing and mitigate osmotic stress during freezing and thawing episodes. Morphological features were documented in D. chrysoscelis using a repeated freeze-thaw protocol. Dorsal skin in frozen frogs was distinctly blue and green before reverting to brown during thawing. The dorsal color change in frozen frogs does not function similarly to other known color change events in amphibians. The return to brown skin color in thawing animals coincides with recovery of vital functions in freeze tolerant frogs, suggesting that dorsal color change is an indicator of postfreeze recovery in D. chrysoscelis. We also provide evidence of "freeze resistance" in D. chrysoscelis. Two individuals did not freeze following three successive bouts of ice inoculation at -2.5°C and maintained brown dorsal color despite ice crystallization on the dorsum and contact with frozen substrate. Both frogs had similar plasma osmolality, circulating cryoprotectants, and incidence of cryoinjury compared to frogs that were frozen and thawed once or three times. Freeze resistance may be explained by physical changes in the skin including lipid accumulation and dehydration. This integrative study presents novel attributes of organismal freeze tolerance in D. chrysoscelis.

Hypoxia inducible factor-1α responds to freezing, anoxia and dehydration stresses in a freeze-tolerant frog

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state.

Muscles in Winter: The Epigenetics of Metabolic Arrest.

The winter months are challenging for many animal species, which often enter a state of dormancy or hypometabolism to “wait out” the cold weather, food scarcity, reduced daylight, and restricted mobility that can characterize the season. To survive, many species use metabolic rate depression (MRD) to suppress nonessential metabolic processes, conserving energy and limiting tissue atrophy particularly of skeletal and cardiac muscles. Mammalian hibernation is the best recognized example of winter MRD, but some turtle species spend the winter unable to breathe air and use MRD to survive with little or no oxygen (hypoxia/anoxia), and various frogs endure the freezing of about two-thirds of their total body water as extracellular ice. These winter survival strategies are highly effective, but create physiological and metabolic challenges that require specific biochemical adaptive strategies. Gene-related processes as well as epigenetic processes can lower the risk of atrophy during prolonged inactivity and limited nutrient stores, and DNA modifications, mRNA storage, and microRNA action are enacted to maintain and preserve muscle. This review article focuses on epigenetic controls on muscle metabolism that regulate MRD to avoid muscle atrophy and support winter survival in model species of hibernating mammals, anoxia-tolerant turtles and freeze-tolerant frogs. Such research may lead to human applications including muscle-wasting disorders such as sarcopenia, or other conditions of limited mobility.

Response of Anatolian mountain frogs (Rana macrocnemis and Rana holtzi) to freezing, anoxia, and dehydration: Glucose as a cryoprotectant

Cryoprotectants play an essential role in the survival of some amphibians in response to different stress conditions such as freezing, anoxia, and dehydration. Glucose is one of the cryoprotectants important for freeze-tolerant frogs. The aim of the present study was to investigate the survival strategies of Anatolian mountain frogs (Rana macrocnemis and Rana holtzi), which are terrestrial hibernators, by examining the changes in glucose and water content in some tissues at subzero temperatures. In the current study, animals were exposed to freezing (−2.5 °C), anoxia, and dehydration treatments. During these treatments, all frogs survived. The glucose levels in the plasma, liver, and skeletal muscle and the water content of the tissues were measured during the freezing, anoxia, and dehydration. Changes in body weight were also recorded in both species. During the freezing, a 3.3-fold increase was seen in the blood glucose level of R. macrocnemis (1.35 ± 0.25 to 4.45 ± 0.51 μmol mL−1), whereas the blood glucose level of R. holtzi exhibited a 4.5-fold increase (1.90 ± 0.25 to 8.67 ± 2.22 μmol mL−1). In the liver, a 6.7-fold increase was seen in the glucose level of R. macrocnemis (5.66 ± 0.15 to 38.27 ± 8.53 μmol g−1) and the increase in R. holtzi was approximately 6.0-fold (2.25 ± 0.46 to 13.36 ± 1.32 μmol g−1) during freezing. The liver glucose levels of both species also increased significantly in response to the anoxia and dehydration. In both species, the glucose levels of the skeletal muscle were found to be higher in dehydration than with freezing and anoxia. In conclusion, our results suggest that glucose may be identified as an important cryoprotectant that plays an important role in the survival of Anatolian mountain frogs during extreme conditions.

The complete mitochondrial genome of Dryophytes versicolor: Phylogenetic relationship among Hylidae and mitochondrial protein-coding gene expression in response to freezing and anoxia.

Dryophytes versicolor is one of the most extreme freeze-tolerant frogs from eastern North America. In this study, the mitochondrial genome of D. versicolor was sequenced to analyze the phylogenetic relationships among Hylidae and investigate mitochondrial gene expression in response to freezing and anoxia. The total length of the D. versicolor mitogenome is the longest known to date among the available family members of Hylidae. Both maximum likelihood (ML) and Bayesian inference (BI) analyses strongly supported D. versicolor as a sister clade to (D. japonica + D. ussuriensis) + (D. suweonensis + D. immaculata (KP212702)), and indicated that Dryophytes is monophyletic. Using the mitochondrial genome, gene expression analysis was performed using RT-qPCR in skeletal muscle samples, and determined that relative levels of D. versicolor COX2 increased by 2.40 ± 0.23 fold in response to anoxia, but did not change with exposure to freezing. In addition, ND3 transcript levels decreased in response to anoxia but remained constant during freezing. By contrast, COX1 transcript levels decreased with exposure to freezing, but did not change under anoxic conditions. These results suggest that modulations of protein-coding mitochondrial genes of D. versicolor may play a role in the molecular response to freezing and anoxia tolerance.

Glucose and urea metabolic enzymes are differentially phosphorylated during freezing, anoxia, and dehydration exposures in a freeze tolerant frog

Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress.

Freeze Tolerance and Cryoprotection of Erythrocytes from Warm and Cold Acclimated D. chrysoscelis

Dryophytes chrysoscelis (Cope's gray treefrog) is a freeze tolerant frog that survives the freezing of extracellular fluids throughout winter. Prior to freezing, D. chrysoscelis goes through a cold acclimation period during which the temperature, hours of daylight, and food intake decrease. Cold‐acclimated frogs have elevated plasma levels of glycerol and urea, solutes that function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing. To minimize damage, urea and glycerol must be taken up by erythrocytes. Erythrocytes from cold‐acclimated frogs have enhanced abundance and membrane localization of an aquaglyceroporin, HC‐3, through which the intra‐ and extracellular flux of glycerol and urea occur. It is hypothesized that erythrocytes frozen in solution containing glycerol/urea would have greater post‐freeze (PF) viability than cells frozen without glycerol or urea. It is further hypothesized that cells frozen with naturally accumulating, and HC‐3 permeating, solutes (glycerol and urea) would have enhanced PF viability compared to cells frozen with solutes that do not accumulate during cold acclimation (glucose, NaCl, sorbitol). In this study, erythrocytes were obtained from warm‐acclimated (22°C) or cold‐acclimated (4°C) frogs, suspended in phosphate buffered saline (PBS) ± solutes, then incubated at 0 or −8°C for 30 minutes. A separate aliquot of cells was frozen in liquid nitrogen for a 100% hemolysis control. Hemolysis was determined by a colorimetric assay. The percentage of free hemoglobin from the total hemoglobin in the erythrocyte sample was calculated and was normalized to the 100% hemolysis control. Viability of the liquid nitrogen sample was taken as 0%. Viability of cells from warm‐acclimated frogs was reduced by 85% when cells were incubated at −8°C in PBS as oppose to 0°C (p<0.001). Cell viability was reduced approximately 50%, when cells from cold‐acclimated frogs were incubated at −8°C compared to 0°C (p<0.001). Erythrocytes from cold‐acclimated frogs have a 2.6‐fold increase in PF viability compared to cells from warm‐acclimated frogs. Cells from warm‐acclimated frogs had a 2.8‐fold increase in PF viability with the addition of urea in the PBS (p<0.005). Cells from cold‐acclimated frogs had a 0.6‐fold increase in PF viability with the addition of urea or glycerol to the PBS (p<0.001 or p<0.005, respectively). The addition of glucose, NaCl, or sorbitol to PBS did not significantly alter PF viability of cells from warm‐ or cold‐acclimated frogs (p>0.05). Enhanced PF viability of cells from cold‐acclimated frogs, compared to cells from warm‐acclimated frogs suggests cold acclimation aids in the freeze tolerance of D. chrysoscelis by mechanisms other than glycerol/urea accumulation. The results of the cryoprotection assay support the involvement of glycerol and urea in the complex cryoprotectant system of D. chrysoscelis.Support or Funding InformationThis research was supported by NSF IOS‐1121457 (CMK and DLG), Schuellein Chair in the Biological Sciences to CMK, and Schuellein Graduate Research Fellowship to LG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The cryoprotectant system of Cope\u2019s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing

Cope’s gray treefrog (Dryophytes chrysoscelis) is one of few freeze-tolerant frogs that mobilize glycerol as a cryoprotectant, yet cold and freezing-induced accumulation of this and other osmolytes has received little attention in this species. This study investigated the development of freeze tolerance in D. chrysoscelis, analyzing the response of the cryoprotectant system to cold acclimation, freezing, and thawing. Glycerol production was low and unresponsive to acclimation temperature, or duration of acclimation to 5 °C, except for one cold-acclimated frog that presented elevated glycerol in plasma, liver, and skeletal muscle. Curiously, glycerol concentration in skeletal muscle was higher than that of plasma and liver, in both warm- and cold-acclimated frogs, suggesting glycerol synthesis in muscle. Urea concentration in plasma doubled in response to cold acclimation but did not change during freezing. Freezing induced hepatic glycogen catabolism and an increase in glycerol and glucose in several tissues, although the mobilization dynamics differed between these cryoprotectants, possibly as a result of different transport mechanisms. Although hepatic glucose mobilization was of considerable magnitude, glucose accumulation in peripheral tissues was low and was surpassed by that of glycerol and urea. The muscle production of glycerol and the cold-induced accumulation of urea imply a role for skeletal muscle metabolism in the mobilization of cryoprotective solutes in D. chrysoscelis. The cryoprotectant system of D. chrysoscelis is complex, highly variable, and unique, with glycerol, glucose, and likely urea serving as cryoprotectants.

12. From ice in the veins, through unfrozen fish and frozen frogs to isochoric preservation, ad Astra

Star Wars V: The Empire Strikes Back – One of the most dramatic scenes in the movie is when the Dark Lord successfully entombs Han Solo in carbonite in a state of suspended animation for transportation to Tatooine to receive a bounty from Jabba. Recently records show that the film has made so far $819,808,300. I doubt that the entire scientific community, together, through the last century, has received from society, 1% of this amount to carry out research on cryobiology. Imagine the state of science in cryobiology if society would have invested 10% of the amount, this one movie grossed, in research. This 6 min presentation will give a brief review of the research in my lab on cryobiology and evidence on why cryobiology could work. Cells survive freezing to cryogenic temperature while organs do not. To understand why, we build a directional solidification stage that can mimic the process of freezing in bulk tissue under a microscope (Rubinsky and Ikeda, 1985). The research has shown, that because of geometrical constrains unique to tissue and absent in single cells, freezing causes damage to the extracellular matrix of the tissue (Rubinsky and Pegg, 1988). Avoiding this mechanism of damage should be an important target in designing cryopreservation protocols. A way to avoid this damage is by finding ways to reduce temperature without formation of ice or by forcing the ice to form at sites where it will not cause mechanical damage. This is what nature does. Arctic and Antarctic fish produce chemicals that allow them to live without ice in their system at temperatures below the normal freezing temperature of physiological saline (Devries, 1971). Using a combination of antifreeze proteins for their cell membrane protection properties (Rubinsky, Arav, et al., 1991) and freezing depression properties (Rubinsky and Devries, 1989) and glycerol for the freezing depression properties we successfully cryopreserved a liver at subzero temperatures (Ishine, Rubinsky, et al., 2000) and transplanted the liver in an animal that lived normally, following the transplantation. Rana Sylvatica frogs survive freezing by modulating the freezing process with glucose, in such a way that the tissue extracellular matrix is not impaired (Storey, Bischof, et al., 1992) and thawing is achieved in an optimal manner for a living animal (Rubinsky, Wong, et al., 1994). Combining the knowledge from Antarctic fish and freeze tolerant frogs, we successfully preserved aa heart for 24 h and transplanted the organ (Amir, Rubinsky, et al., 2004). One way to reach high subzero temperatures without freezing a cooled organ is by isochoric subcooling (Rubinsky, Perez, et al., 2005). Unpublished results on long-term preservation of hearts with this method will be shown.

21. A lesson from Nature for controlling ice during organ cryopreservation by vitrification

Banking of organs using current tissue banking practices employing conventional cryopreservation by freezing is not feasible due to the well known damage caused by intra and extra-cellular ice formation. An alternative cryopreservation approach is known as vitrification. There has been considerable success in ice-free vitrification of small tissue samples but better ice control is required for samples > 10 mL in volume. Development of novel cryoprotectants and methods that will facilitate clinically effective banking by vitrification of large vascularized tissues and organs is needed. There are lessons to be learned for organ vitrification from Nature. Freeze avoiding insects such as Cujucus clavipes beetle larvae from the interior of Alaska vitrify and survive temperatures that routinely reach −60 °C with some individuals surviving exposure to −100 °C. At the onset of winter these insects combine changes in behavior with increases in cryoprotectant content (such as glycerol, proline, trehalose), dehydration, production of antifreeze compounds (proteins and glycolipids), and diapause. The cryoprotectant concentration can achieve 4.2 M, approximately half the concentration required for ice-free cryopreservation of tissue and organ therapy products. Recombinant copies of insect-derived antifreeze proteins are available, so they can be manufactured, and they are up to 10 times more effective in ice modulation (thermal hysteresis) than the well known fish-derived antifreeze proteins that have proven ineffective or detrimental for cell cryopreservation. Recently we have discovered that certain insect-derived antifreeze proteins significantly increase cell viability during cryopreservation by freezing. Similarly, in unpublished studies we have found that an insect-derived antifreeze glycolipid, with thermal hysteresis activity similar to that of hyperactive insect antifreeze proteins, can significantly increase cell viability post-cryopreservation using freezing methods. These glycolipids have been found in several species including freeze tolerant and avoiding insects, freeze tolerant frogs, and freeze tolerant plants. Unlike the antifreeze proteins, which are diverse in structure across species, these glycolipids are nearly identical. While we anticipate that eventually it will be possible to synthesize the saccharide component (s1-4 linked xylose-mannose), at present the antifreeze glycolipid is harvested from natural sources, especially plants. Translation of this lesson from Nature requires a better understanding of how animals prepare themselves for diapause and hibernation so that we can reproduce these phenomena in organ donors in vivo, under physiological conditions post-mortem prior to organ procurement, and/or organs ex-vivo before cryopreservation in cryoprotectant formulations that incorporate antifreeze compounds. It is anticipated that utilization of antifreeze proteins and glycolipids will enable utilization of less cytotoxic cryoprotectant formulations with enhanced ice control during cooling and warming of larger biological structures than currently possible. In the big picture it is likely that they will be combined with other strategies presented in this session on controlling ice formation including improved cryoprotectant addition and removal methods, pressure assisted cooling, stress relief by annealing, synthetic antifreeze compounds, and nanoheater assisted rewarming. Once we have ice formation under control we must be concerned with ischemic reperfusion injury, the final hurdle upon in vivo transplantation in patients.

A02 Session 1: Translational Hibernation : Stress response and adaptation: A new molecular toolkit for the 21st century

Molecular studies of cold and frozen systems “be they naturally cold-hardy animals, cryopreserved cells/tissues/organs, or tissues ablated by cryosurgery” all have one thing in common. This is a need to identify and quantify biochemical markers of stress, injury and survival. Physical assessment methods (cell rupture, enzyme leakage, etc.) have long been used as have “bulk” protective strategies (e.g. cryoprotectant infusion). However, in-depth knowledge of the key biochemical parameters of cell systems under freezing stress, the negative versus positive responses that can constitute molecular markers of stress versus endurance, and the features that differentiate natural versus man-made freezing survival still leave much to be determined. Assessing complex cell functions has traditionally been difficult and time consuming absorbing many grad student careers for energy-intensive methods like gene screening, PCR, immunoblotting and enzyme assay. However, new technologies have recently emerged that are re-focusing research efforts and allowing huge amounts of data to be gathered from very small cell/tissue samples. Among these, accelerated rates of genome sequencing are giving us access to genomic tools to find inherent differences in genes between species and tools for screening a stress-responsive transcriptome (e.g. RNA seq.; the RT2 profiler array technology) or proteome are becoming easier and cheaper. This is providing ways to identify signatures of cells/organisms under stress. A relatively new analytical tool, multiplex suspension array technology (Luminex), is also radically changing biomedical analytical capabilities for both research and diagnostic purposes by providing a way to quantify panels of multiple analytes simultaneously in single small samples in a very short time. For example, this approach can allow multiple members of integrated signal transduction pathways to be quantified in a single sample. Critical assessments of metabolic status can also be derived from analyzing the patterns of posttranslational modification of macromolecules including the phosphoproteome or the epigenome. Quick ‘snapshots’ of transcriptional and translational control using state-of-the-art methodologies can identify derangements as varied as changes in cell cycle control, the status of apoptosis and autophagy, or microRNA involvement in stress and adaptation. Most of these technologies are now available in kit formats (that can often be customized) to screen for large numbers of targets simultaneously at an ever-decreasing cost to researchers. To illustrate the power of these new methodologies, a variety of examples of their use by my lab in probing the molecular adaptations for low temperature function in hibernating mammals, freeze tolerant frogs, and freeze/anoxia tolerant intertidal snails will be provided. For comparative and cryo-biochemistry the future is bright as we now have the capacity to apply the latest molecular technologies to analyze stress and adaptation.

Expression of freeze‐responsive proteins, Fr10 and Li16, from freeze‐tolerant frogs enhances freezing survival of BmN insect cells

To date, two novel freeze-responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze-intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no-transfection sample. Li16 and Fr10 showed 1.8 ± 0.1- and 1.7 ± 0.2-fold, respectively, greater survival after freezing at -6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.

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.

The emerging roles of microRNAs in the molecular responses of metabolic rate depression

Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, estivation, anoxia and diapause. Studies of the molecular mechanisms that regulate reversible transitions to and from hypometabolic states have identified principles of regulatory control. These control mechanisms are conserved among biologically diverse organisms and include the coordinated reduction of specific groups of key regulatory enzymes or proteins in the cell, a process likely driven by microRNA target repression/degradation. The present review focuses on a growing area of research in hypometabolism and mechanisms involving the rapid and reversible control of translation facilitated by microRNAs. The analysis draws primarily from current research on three animal models: hibernating mammals, anoxic turtles and freeze-tolerant frogs (with selected examples from multiple other sources). Here, we demonstrate a link between metabolic rate depression, a well-documented response to periods of environmental stress, and microRNA expression. Microarray-based expression profiles and PCR-driven studies have revealed that specific microRNAs are induced in response to environmental stress. Selected members of this group decrease pro-apoptotic signaling, reduce muscle wasting and reduce protein translation, whereas other members contribute to cell cycle arrest and mitogen-activated protein kinase signaling. Many of the same microRNAs are frequently deregulated in numerous disease pathologies and, hence, the hypometabolism model could provide a novel approach for the treatment of stroke and heart attack in humans.

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver 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 G6PDH showed a significant increase in K(m) G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K(m) NADP(+) (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K(m) G6P of 94.1 ± 1.1 μM and K(m) NADP(+) of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K(m) G6P was 172 ± 4.3 μM, K(m) NADP(+) was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.

Overwintering Physiology and Hibernacula Microclimates of Blanchard's Cricket Frogs at Their Northwestern Range Boundary

Blanchard's Cricket Frogs (Acris crepitans blanchardi) in the central portion of their range show minimal capacities for freezing tolerance and survive overwinter by using terrestrial hibernacula where they avoid freezing. However, frogs may exhibit greater freeze-tolerance capacity at high latitude range limits, where winter climate is more severe. We studied freezing tolerance, glucose mobilization during freezing, and hibernacula microclimates of cricket frogs in southeastern South Dakota, at the northwestern limit of their range. Cricket frogs from South Dakota generally survived freezing exposure at −1.5 to −2.5°C for 6-h periods (80% survival), but uniformly died when exposed to these same temperatures for 24-h freezing bouts. Hepatic glucose levels and phosphorylase a activities increased significantly during freezing, but hepatic glucose levels during freezing remained low, only reaching levels approximating those prior to freezing in freeze-tolerant species. Moreover, muscle glucose and hepatic glycogen levels did not vary with freezing, suggesting little mobilization of glucose from hepatic glycogen stores during freezing, contrasting with patterns in freeze-tolerant frogs. Temperatures in soil cracks and burrows potentially used for hibernacula were variable, with some sites remaining above the freezing point of the body fluids throughout the winter, some sites dropping below the freezing point for only short periods, and some sites dropping below the freezing point for extended periods. These data suggest that cricket frogs in South Dakota, as in other portions of their range, survive overwinter by locating hibernacula that prevent freezing, although their toleration of short freezing bouts may expand the range of suitable hibernacula. These data also suggest that overwinter mortality may be high at the northern range boundary and might limit cricket frogs from expanding their range northward.

Annual variation in glycerol mobilization and effect of freeze rigor on post-thaw locomotion in the freeze-tolerant frog Hyla versicolor

This study documents post-thaw recovery of jump distance and cryoprotectant mobilization in the freeze-tolerant frog Hyla versicolor over two successive years. Cold acclimated frogs had plasma glycerol levels near 1.0 mM in 2004 but it was nearly 70x higher during 2005. Freezing of frogs induced nearly identical levels of plasma glycerol (ca. 177 mM) during 2004 and 2005. Plasma glucose was only mobilized upon somatic freezing, with averages ranging between 21 and 36 mM. Control jump distance showed no difference between the two years of the study. The post-thaw jump response was identical during the first 2 years despite large differences in glycerol mobilization between these 2 years. Recovery proceeded much faster in 2005 when frogs mobilized glycerol prior to freeze exposure. Frogs were more impaired in their locomotion performance during the initial stages of recovery period when they were frozen at a lower temperature (-3 vs. -1.5 degrees C) but they eventually recovered. Moderate lengthening of the freeze duration (3 vs. 7 days) with the 2004 collection group did not affect recovery of jump distance when frogs were frozen at -1.5 degrees C. Hence, postfreeze impairment of locomotion is dependent of the intensity of the freeze temperature but it is a reversible process that is mitigated when glycerol is more freely distributed to body tissues.

158. Creatine kinase regulation by reversible phosphorylationin a freeze tolerant frog

158. Creatine kinase regulation by reversible phosphorylationin a freeze tolerant frog

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-2α 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.

Nonmetabolizable Glucose Compounds Impart Cryotolerance to Primary Rat Hepatocytes

We herein report a novel method for the cryopreservation of hepatocytes using a non-metabolizable glucose derivative in an attempt to mimic the natural cryoprotective adaptations observed in freeze-tolerant frogs. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity. The 3OMG-loaded hepatocytes were then frozen in a controlled rate freezer down to -80 degrees C and stored in liquid nitrogen at -196 degrees C. Hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (<0.2 M) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes. This is the first study to demonstrate the use of the non-metabolizable glucose derivative 3OMG in hepatocyte cryopreservation.