Organic solutes in freezing tolerance

Comp. Biochem. Physiol. Vol. 117A, No. 3, pp. 319–326, 1997 Copyright  1997 Elsevier Science Inc. ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00270-8 Organic Solutes in Freezing Tolerance Kenneth B. Storey Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Canada K1S 5B5 ABSTRACT. The accumulation of high levels of low-molecular-weight solutes (polyhydric alcohols, saccharides) provides cryoprotection to freeze-tolerant animals by minimizing, via colligative effects, the percentage of body water converted to extracellular ice and the extent of cell volume reduction. Many freeze-tolerant insects accumulate high levels of polyols during autumn cold hardening, whereas freeze-tolerant frogs respond to ice formation in peripheral tissues by synthesizing large amounts of glucose in the liver and rapidly distributing the sugar throughout the body. Seasonal patterns of enzymatic change occur in cold-hardy insects; activities associated with cryoprotectant synthesis rise in the fall, whereas enzymes associated with polyol degradation dominate in the spring. Enzyme profiles also revealed the route of glycerol degradation via polyol dehydrogenase and the novel enzyme, glyceraldehyde kinase. Proton magnetic resonance imaging of freezing and thawing in whole frogs showed a new adaptive effect of the very high glucose levels in core organs; during thawing, organs such as liver and heart melted first, allowing recovery of their vital functions to begin while the rest of the frog thawed. New studies have examined signal transduction in the stimulation of glucose production by wood frog liver, revealing the key role of β-adrenergic receptors and cAMP-mediated activation of glycogenolysis for cryoprotectant synthesis. The seasonal elevation of plasma membrane glucose transporters was also shown to be key to cryoprotectant distribution during freezing. Other new work has shown that frog freeze tolerance probably grew out of preexisting mechanisms of amphibian dehydration tolerance and that both freeze-tolerant and -intolerant frogs show a hyperglycemic response to desiccation at 5°C. comp biochem physiol 117A;3:319–326, 1997.  1997 Elsevier Science Inc. KEY WORDS. Anuran winter hibernation, cell volume regulation, cryoprotectants, Euosta solidaginis, glycerol biosynthesis, insect cold hardiness, Rana sylvatica INTRODUCTION For numerous types of animals, winter survival at subzero temperature depends on freeze tolerance, the ability to endure the conversion of up to 65% of total body water into extracellular ice and to survive for days or weeks without movement, breathing and blood circulation. Freeze tolerance is well developed in many species of insects, various other terrestrial and intertidal marine invertebrates and selected species of terrestrially hibernating amphibians and reptiles (1,9,31,33,37). One of the most important biochemical adaptations supporting freeze tolerance is the use of low-molecular-weight organic solutes as cryoprotectants. These are of two general types. Colligative cryoprotectants are accumulated in high concentrations (often 0.2–2 M) and their action in raising the osmolality of body fluids reduces the percentage of total body water that can accumulate as extracellular ice and prevents intracellular volume Address reprint requests to: K. B. Storey, Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Canada K1S 5B5. Tel. (613) 520-3678; Fax (613) 520-4389; e-mail [email protected]. Received 7 September 1995; accepted 20 October 1995. from falling below a critical minimum. In most cases, the upper limit for survival is about 65% of total body water converted to extracellular ice. However, the subzero temperature at which this limit is reached can vary widely, largely as a function of the osmolality of body fluids and keyed to the natural freezing temperatures that are encountered by each species (31). The second type of cryoprotectant is membrane protectants that are accumulated in lower amounts (generally ,0.2 M) and have a specific function in stabilizing membrane bilayer structure to prevent an irreversible transition to the gel state when the plasma membrane is compressed during cell volume reduction. Trehalose and proline are the compounds that are most often associated with membrane protection, and they interact directly with the polar head groups of membrane lipids to stabilize the bilayer structure. Their actions have been extensively described in anhydrobiotic systems (8) and have been confirmed for freezing preservation in studies with isolated membranes (26). Notably, both trehalose and proline levels are elevated in freeze-tolerant insects during the winter, and proline is typically one of the major intracellular free amino acids in euryhaline marine invertebrates and one whose concentration can be changed rapidly in response to os- 320 motic stress. Other osmolytes have also been cited as membrane stabilizers, including several of the common intracellular osmolytes accumulated by marine invertebrates (e.g., amino acids) (20). These compounds, as well as the high intracellular free amino acid pool that serves to maintain the cells of marine invertebrates isosmotic with seawater, appear to be sufficient to protect intertidal species during freezing. Thus, none of the common carbohydrate cryoprotectants have been detected in these species nor does cold acclimation or freezing exposure further elevate the osmolality of body fluids as occurs in terrestrial freeze-tolerant species. The focus of this article is on the colligative cryoprotectants and, specifically, on recent advances in our understanding of the regulation of their synthesis, clearance and transport in both freeze-tolerant insects and frogs. Furthermore, recent evidence that the cryoprotectant response in amphibians probably grew out of pre-existing mechanisms of cell volume regulation and desiccation tolerance is presented. The natural colligative cryoprotectants accumulated by terrestrial animals are almost exclusively carbohydrates that are synthesized from the large reserves of glycogen that are laid down (in fat body of insects, liver of frogs) during late summer and early autumn feeding. Among insects, glycerol is by far the most common cryoprotectant and is produced by both freeze-tolerant and freeze-avoiding species. Other polyhydric alcohols also occur in some species (e.g., sorbitol, ribitol, erythritol, threitol, ethylene glycol), and disaccharides (trehalose, sucrose) are also used in some cases (31). Various species accumulate two or more protectants; glycerol and sorbitol are a common pairing, and the independent time courses over which each is synthesized and catabolized in species such as larvae of the gall fly (Eurosta solidaginis) and eggs of the silkmoth (Bombyx mori) indicate differential roles for the two compounds. The popularity of glycerol as a natural cryoprotectant probably derives from a number of factors, including: high solubility, nontoxicity and compatibility with biological macromolecules, biosynthetic and catabolic pathways that are central and constitutive, an optimal output of osmotically active particles (1 hexose unit from glycogen is converted to 2 glycerol, as opposed to 1 sorbitol or 0.5 trehalose) and optimal conversion efficiency (no loss of CO2 as in the biosynthesis of C2, C4 or C5 polyols). Of the freeze-tolerant amphibians, however, only the gray tree frog Hyla versicolor and the Siberian salamander Hynobius keyserlingi accumulate glycerol (33). Other frogs use glucose instead (33), and the choice of this sugar appears to suit the circumstances of freezing in frogs. Thus, whereas insects accumulate polyols over the long term (days/weeks) as part of temperaturemodulated autumn cold hardening or as a hard-wired function of entry into diapause, frogs produce glucose only as an immediate response to the initiation of ice formation in body extremities. The biosynthetic pathway for glucose in liver is short, involves high activity enzymes, is ATP-inde- K. B. Storey pendent (important as ischemia develops during freezing), can be quickly activated under catecholamine control and plasma membrane glucose transporters are constitutive in all vertebrate organs and can be modified to accommodate high rates of glucose transport. The actions of colligative cryoprotectants have been the subject of much study by cryomedical researchers (for review, see (2,10)]. For a freeze-tolerant animal, the most important action of these and other solutes is the colligative effect of dissolved osmolytes on the freezing behavior of a solution. At any given subzero temperature, ice will form in a solution (removing pure water into the crystalline form) until the osmolality of the remaining unfrozen fraction rises to a value whose melting point is equivalent to the present temperature (11). Therefore, the greater the initial osmolality of the solution, the lower the percentage of total water that has to freeze before the osmolality of the remaining solution rises to this maximum. For an animal freezing with high cryoprotectant concentrations in its body fluids (compared with one without cryoprotectants), the consequences are, therefore, a lower percentage of total body water converted to extracellular ice, a lesser reduction of cell volume due to water exiting to extracellular ice masses and, when freezing is initiated in a supercooled animal, a lower initial ice surge (ice produced instantly upon nucleation) and a lesser osmotic stress on cells. Similar to the effect of osmolytes on the equilibrium between water in solution and water in ice, osmolytes also reduce water loss from solution in other situations, including reducing evaporative water loss from cells or water outflow when cells are placed in hyperosmotic solutions. Indeed, from the cell’s point of view, the removal of intracellular water and the osmotic stress that it creates, whether it be by extracellular ice formation, desiccation or hypersaline stress, is undoubtedly all the same. Not surprisingly, therefore, animals have evolved the same responses of elevating the intracellular concentrations of selected low-molecular-weight organic solutes under all three forms of water stress and, indeed, a small group of osmolytes (e.g., glycerol, urea, neutral free amino acids, taurine) reappear frequently in nature in a variety of systems that experience water stress. Furthermore, as is discussed later, new studies are revealing strong links between desiccation tolerance and freeze tolerance in frogs and are suggesting that various adaptations for freezing survival are triggered by cell volume change. Low-molecular-weight organic osmolytes also have other effects on biological fluids and the macromolecules in them. Carbohydrate cryoprotectants are typically compatible solutes that do not perturb macromolecular conformation even at very high concentrations and, furthermore, they are generally excellent stabilizing agents that can protect proteins and other macromolecules from the denaturing effects of extreme heat, cold and desiccation. However, within the biological range of freezing temperatures for most organisms, it is doubtful whether either low temperature or cell dehy- Cryoprotectants in Freezing Tolerance dration stresses are great enough to cause macromolecular disruption, but clearly cryoprotectants can take on this function under extreme conditions as has been illustrated with anhydrobiotic animals and isolated protein systems (8). Another effect of elevated organic osmolyte concentrations is to lower the freezing and supercooling points of a solution, and these properties are exploited by freezeavoiding organisms to prevent the freezing of body fluids within the range of normally experienced winter temperatures (37). Freeze-tolerant animals also have this function available to them, and, indeed, freezing point suppression is an unavoidable property of elevated solute concentrations. However, freeze-tolerant animals generally avoid extensive supercooling and instead induce freezing at or near the freezing point of body fluids via the action of endogenous ice nucleators (often specifically synthesized ice nucleating proteins) or via transcutaneous inoculation by contact with environmental ice crystals. Thus, although there are advantages of a supercooled state (as opposed to a frozen state), such as the ability to maintain mobility and aerobic metabolism, animals also run the risk of instantaneous and lethal freezing should nucleation occur in a deep supercooled state. Freeze-tolerant animals avoid the risk of having to deal with high rates of ice formation and major osmotic shock by arranging for freezing to begin at or just below the freezing point of body fluids where the rate of freezing can be slow and controlled. For example, when wood frogs are frozen at 22.5°C, it takes nearly an hour postnucleation before freezing can be confirmed by an icy stiffness of the skin, several hours before animals are unable to extend their limbs and about 24 hr to reach maximal ice content. Within this slow and controlled freeze, the frogs have ample time to initiate various protective adjustments, including the synthesis and release of high concentrations of the cryoprotectant glucose from the liver and metabolic adjustments to deal with the ischemic state imposed by freezing. RECENT ADVANCES IN INSECT CRYOPROTECTANT METABOLISM Many aspects of cryoprotectant metabolism in cold-hardy insects have been addressed by numerous authors [for recent review, see (32,37)]. Generally, cryoprotectant synthesis begins in the fall, typically triggered by thermoperiod (extended exposures over several days to temperatures at or below a trigger value) but facilitated by previous glycogen accumulation in fat body and elevation of the activities of requisite enzymes, frequently regulated by either developmental or photoperiod cues. Recent studies in our laboratory have monitored the coordinated changes in enzyme activity profiles that take place over the winter months in both a freeze-tolerant (Eurosta solidaginis) and freezeavoiding (Epiblema scudderiana) goldenrod gall former that affect not only the potential for cryoprotectant synthesis vs degradation but also other aspects of metabolism (14–16). 321 Larvae of both species experience diapause from late autumn through to about February, and this is followed by a quiescent period until temperatures rewarm in the spring. Correlated with diapause, a coordinated suppression of the activities of key enzymes associated with mitochondrial oxidative metabolism was noted during the midwinter months in both species; citrate synthase, NAD-isocitrate dehydrogenase and glutamate dehydrogenase activities were all reduced by about 50% (14). This is consistent with the suppressed oxidative metabolism that is a general characteristic of dormancy. However, despite the diapause state and the low environmental temperature, active enzymatic reorganization continued and, after an autumn peak, activities of enzymes associated with polyol synthesis generally decreased, whereas those involved in polyol catabolism increased to reach their maxima by early spring (15,16). These studies of the seasonal patterns of enzymes associated with polyol synthesis vs degradation revealed that adjustments in the activities of enzymes are key to directing either the synthesis or degradation of cryoprotectants. Both species studied accumulated glycerol as a cryoprotectant, E. scudderiana larvae to about 2 M levels and E. solidaginis larvae to about 250 mM, but the freeze-tolerant insect also built up about 150 mM sorbitol (15,16). The enzymatic routes of sorbitol synthesis and degradation have been well characterized previously in both E. solidaginis and other species. Synthesis arises from glucose-6-P, relying on glucose6-phosphatase and NADPH-dependent polyol dehydrogenase, whereas degradation takes another route involving NAD-dependent sorbitol dehydrogenase to produce fructose and then hexokinase (32). Indeed, the induction of sorbitol dehydrogenase to begin catabolism of the polyol is a characteristic feature of the termination of diapause in B. mori eggs (36). Seasonal changes in these enzymes in E. solidaginis strongly supported this division of synthetic vs catabolic pathways (15); glycogen phosphorylase, glucose6-phosphatase, and polyol dehydrogenase (measured with glucose as the substrate) activities all rose together during the autumn and then fell over the winter months, whereas sorbitol dehydrogenase and hexokinase/glucokinase activities were very low in the autumn, peaked in late winter when sorbitol is catabolized and then fell again by late spring (Fig. 1). By contrast, the enzymatic routes of glycerol synthesis and degradation have previously been in question for two routes for the interconversion of triose phosphates (from glycolysis) and glycerol were possible. One route derives from dihydroxyacetone phosphate and uses the reversible NAD(H)-linked glycerol-3-phosphate dehydrogenase coupled with glycerol-3-phosphatase in the synthetic direction and ATP-dependent glycerol kinase in the degradative direction. These pathways occur widely in animal cells and are involved in triglyceride metabolism and gluconeogenesis from glycerol. The second pathway derives from glyceraldehyde-3-phosphate (GAP) and involves glyceraldehyde-3- 322 K. B. Storey FIG. 1. Seasonal changes in the levels of cryoprotectants (glycerol and sorbitol) and in the activities of enzymes associated with their synthesis and catabolism in larvae of the freeze tolerant gall fly Eurosta solidaginis. Data are mean 6 SEM, n 5 4– 6 with polyol concentrations in mmol/g wet mass and enzyme activities in mmol substrate utilized/min/g wet mass at 25°C. Abbreviations: GAPase, glyceraldehyde-3-phosphatase; PDHald, polyol dehydrogenase using glyceraldehyde as the substrate: GAK, glyceraldehyde kinase; G6Pase, glucose-6-phosphatase; PDHgluc, polyol dehydrogenase using glucose as the substrate; SDH, sorbitol dehydrogenase; HK 1 GK, hexokinase 1 glucokinase. Data were analyzed with a one-way ANOVA followed by the Student-Neuman-Keuls test. s, Significantly different from the September value, P , 0.05; * significantly different from the value for the previous sampling date, P , 0.05. Modified from Joanisse and Storey (15). phosphatase in the biosynthetic direction, ATP-dependent glyceraldehyde kinase in the degradative direction, with NADP(H)-linked polyol dehydrogenase used in both cases to interconvert glyceraldehyde and glycerol. Substantially higher activities of glycerol-3-phosphate dehydrogenase, a high enzyme affinity for substrate, and the build up of glycerol-3-phosphate at the cessation of glycerol synthesis in insects all gave initial support for the first route of glycerol metabolism (32), but accumulated evidence now strongly supports the route involving glyceraldehyde as the intermediate. Thus, it is well established that the relative flux of carbohydrate through the pentose phosphate cycle, compared with glycolysis, increases during cold-induced polyol synthesis in insects (35). This generates reducing power in the form of NADPH. Although transhydrogenases undoubtedly exist to allow reducing equivalents to be transferred to NADH for use by glycerol-3-phosphate dehydrogenase, the direct use of NADPH by polyol dehydrogenase seems more logical. Furthermore, the activity of polyol dehydrogenase is elevated during autumn cold hardening. In the freeze-avoiding E. scudderiana, the activity of glyceraldehyde-utilizing polyol dehydrogenase increased over 7-fold from September to November, whereas in the freeze-tolerant E. solidaginis, activities of polyol dehydrogenase using both glyceraldehyde (producing glycerol) and glucose (producing sorbitol) rose in concert in the autumn (Fig. 1). What was more convincing, however, was the pattern of enzymatic changes associated with polyol degradation. In Cryoprotectants in Freezing Tolerance general, in animal tissues, glycerol enters intermediary metabolism by way of ATP-dependent glycerol kinase, which phosphorylates the polyol to produce glycerol-3-phosphate. The absence of glycerol kinase in cold-hardy insects has been noted previously but, in autumn-sampled animals, this seemed to be a logical way to avoid glycerol catabolism and the waste of ATP that it would involve, under metabolic conditions where a high glycerol pool needed to be maintained. What our recent studies have shown, however, is that glycerol kinase activity could not be detected in either cold-hardy species at any time, even during spring dehardening (15,16). Instead, the activity of a new enzyme, ATPdependent glyceraldehyde kinase, appeared during late winter (Fig. 1), paralleling the late winter appearance of sorbitol dehydrogenase and indicating that pathways for the degradation of both polyols were put in place concurrently as spring approached. To our knowledge, no previous studies have looked for a glyceraldehyde kinase activity in coldhardy insects, but its presence and elevation in late winter clearly indicate that the route of glycerol degradation in both freeze-tolerant and freeze-avoiding insects is via glycerol → glyceraldehyde → GAP. By using this route, reducing equivalents are recovered as NADPH that could be directly used for biosynthesis along with the triose phosphates; for example, both might be used for the biosynthesis of triglycerides. Previous studies have shown that typically only a small percentage of glycerol carbon is reconverted to its precursor, glycogen, in cold-hardy insects and this seemed puzzling, especially in light of the nearly stoichiometric return of sorbitol to the polysaccharide reserve (32). But even though GAP is a good gluconeogenic substrate and the gluconeogenic potential of the larvae clearly rises in the spring (as evidenced by the ratio of phosphofructokinase to fructose-1,6-bisphosphatase) (15,16), GAP reconversion to glycogen does not consume the NADPH that is produced in a 1 : 1 molar ratio with the triose phosphate. But by channeling the carbon into another biosynthesic fate, such as lipid biosynthesis, both the carbon and the reducing equivalents generated during glycerol clearance can be consumed. RECENT ADVANCES IN VERTEBRATE CRYOPROTECTANT METABOLISM The terrestrially hibernating wood frog uses glucose as its cryoprotectant. Glucose is synthesized from huge reserves of glycogen stored in the liver. A significant increase in glucose output from liver can be detected in as little as 2–5 min after freezing begins at peripheral sites on the skin, and this implicated the involvement of nervous or hormonal mediation in linking peripheral stimulus with the liver response of glycogenolysis (33). This is supported by several lines of evidence; thus, freezing of isolated hepatocytes in vitro did not elevate their glucose content, freezing-induced cryoprotectant production in whole animals is suppressed 323 by injections of the β-adrenergic blocker propranolol, β2adrenergic receptors dominate (as opposed to α-adrenergic receptors) in hepatic plasma membranes during the early hours of freezing exposure, and glucose output from both liver in vivo and hepatocytes in vitro is stimulated with glucagon or catecholamines (13,21,28,30). As the result of high rates of glycogenolysis, wood frogs catabolize a huge reserve of liver glycogen (as much as 600–700 µmol/g wet weight in glucose equivalents or about 18% of liver fresh weight) and export glucose to other organs to raise their sugar content (32). Maximal levels of glucose rise to about 200–300 mM in core organs with progressively lower concentrations in more peripheral sites (e.g., glucose in skeletal muscle and skin may rise to only 25–50 mM). This gradient of organ glucose concentrations is the result of the progress of the freezing front that moves from the periphery inward and halts circulation as ice forms in extracellular and vascular spaces, thereby concentrating glucose delivery more and more to the core organs. A key advantage of the uneven distribution of cryoprotectant within the frog’s body was recently revealed by proton magnetic resonance imaging (25). MRI showed, not unexpectedly, that whole frogs freeze in a directional manner with the freezing front moving inward from peripheral sites and penetrating through the core until liver and heart are the last organs to freeze. Parenthetically, MRI also showed that freezing begins first in large fluid species (e.g., the abdominal cavity, ventricles of the brain) before penetrating into tissues themselves. However, thawing was a surprise. Frogs melted from the inside out; liver and heart melted before the extraorgan ice surrounding them and brain melted before the ice in ventricles. This unusual pattern of thawing is due to the colligative action of high glucose levels in core organs that lowers the melting point of these tissues. The adaptive significance of this for the frog is that core organs can begin to recover (readjusting metabolic and volume status) well before peripheral organs and, hence, vital functions such as heart beat and breathing can be re-established as soon as possible. Indeed, the reappearance of heart beat is the first visible vital sign during thawing and occurs well before any contractile response can be elicited from skeletal muscles. MRI also revealed another interesting feature of natural freezing in wood frogs: core organs shrink in size during freezing due to the loss of water to abdominal ice masses (25). This is also visible during dissection and sampling of frozen frogs, and the decrease in organ water content during freezing has been quantified (7). For example, frogs frozen slowly at 22.5°C showed reductions of organ water contents of 8.7, 12.7, 19.5 and 24.2% for brain, skeletal muscle, liver and heart, respectively. The extraorgan sequestration of ice that is the cause of this shrinkage appears to be a useful survival strategy because it reduces the amount of ice that will then form in extracellular vascular spaces within organs. Damage due to ice expansion in the vascular spaces of complex tissues and organs and the subsequent loss of 324 vascular integrity after thawing is a critical problem in cryomedicine (24). Freeze-tolerant frogs appear to avoid this by moving a substantial percentage of organ water out into extraorgan spaces where it can freeze in bulk ice masses and thereby minimize the ice buildup within delicate vascular spaces. Furthermore, this action further elevates the concentration of remaining solutes within the organ and, thereby, may enhance the cryoprotection of the cells. The obvious importance of water and solute movements across plasma membranes for freeze-tolerant frogs led us to analyze the transport of the cryoprotectant, glucose. The lipid bilayer of the plasma membrane allows few compounds to cross it by simple diffusion. For most compounds, entry into or exit from cells is largely gated by transport proteins that span the membrane and provide facilitated transport for solutes moving in the direction of an osmotic gradient and active transport to move solutes against their concentration gradient. During extracellular freezing, the osmotic and ionic imbalance set up by the exclusion of solutes from the rapidly growing ice crystals requires a rapid redistribution of water, ions and organic solutes across cell membranes; reverse movements accompany thawing. Cryomedical studies have shown that compounds such as glycerol and dimethylsulfoxide can rapidly equilibrate across the plasma membrane of mammalian cells, making these useful cryoprotectants. By contrast, glucose is regarded as a much poorer protectant because it cannot enter cells rapidly. In mammalian systems, glucose movement across cell membranes is carrier-mediated by glucose transporter proteins (22); hence, we proposed that one of the adaptive mechanisms needed to support freezing survival by wood frogs must be to optimize the transport of glucose across plasma membranes. Not surprisingly, then, we found that the numbers of glucose transporters in R. sylvatica plasma membranes are specifically increased to deal with the demands of rapid cryoprotectant movement (17,18). Membrane vesicles prepared from liver of autumn-collected wood frogs had an 8.2-fold greater rate of carrier-mediated glucose transport and a 4.7-fold higher number of glucose transporters (quantified by the amount of cytochalasin B binding) than did liver membrane vesicles from the freezing-intolerant, aquatic-hibernating leopard frog (R. pipiens). Glucose transport rate by wood frog skeletal muscle vesicles was also 8fold higher than in leopard frog vesicles, showing that transporter systems of both the cryoprotectant-producing organ and a receiving organ are modified in concert. Furthermore, the capacity for rapid cryoprotectant transport was modified seasonally in wood frog liver; frogs collected in September (then held at 5°C for 3 weeks) showed a 6-fold higher rate of carrier-mediated glucose transport by liver vesicles and an 8.5-fold higher number of transporter sites than did frogs collected in June. The importance of elevated glucose transport capacity for freeze tolerance by frogs raises the question of whether other specific transporters are involved in cell volume regulation K. B. Storey during freezing. For example, are adaptations made to facilitate rapid and large fluxes of water across membranes during freezing and thawing? Water moves across cell membranes both by simple diffusion and by channel-mediated facilitated transport, the latter being particularly important in epithelia with very high rates of transmembrane water movement such as erythrocytes and renal proximal tubules (6). Channel-mediated water movement differs from simple diffusion in being sensitive to inhibitors (mercuric chloride) and having a low Arrhenius activation energy (whereas diffusion is sensitive to the effects of low temperature on lipid packing in membranes). Aquaporins, or water channel proteins, have been identified in both animals and plants, and these 28- to 30-kDa proteins belong to a highly conserved and ancient family of channel proteins. There is not yet any information on aquaporins in freeze-tolerant animals, but a logical hypothesis would be that these species would elevate aquaporin numbers in membranes to facilitate water movements during freezing and thawing. It is also interesting to note that of about 20 related membrane proteins that have been identified in the aquaporin family, one is the glycerol facilitator of Escherichia coli (23). It may be, therefore, that glycerol movements across membranes in freeze-tolerant animals are also facilitated by specific transporters or, alternatively, that glycerol (whose hydrogen and hydroxyl groups are oriented very similarly to those in water) may pass through water channels. This latter suggestion could be one reason why glycerol is used widely in nature for cell volume regulation, not just in freeze-tolerant animals but also in other natural situations of water stress (e.g., hyperosmotic stress in algae, desiccation resistance in Artemia), and would also support the status of glycerol as the cryoprotectant of choice in many cryomedical applications. By contrast, most mammalian cells are impermeable to sorbitol, yet this is one of the major polyols accumulated by freezetolerant insects. This strongly implicates the presence of a sorbitol transporter in insect plasma membranes, perhaps similar to the sorbitol permease that has been identified in mammalian erythrocytes and renal papillary epithelial cells (19). The probable development of freeze tolerance as an extension of pre-existing mechanisms of dealing with water stress in animals is further supported by some recent experiments on the metabolic responses to dehydration in frogs. Because ice forms extracellularly, the major stress perceived by cells during freezing is a sharp volume reduction. Hence, it seemed logical to predict that protective metabolic responses to freezing might be triggered or regulated by dehydration or changes in cell volume. Indeed, this seems to be the case for the cryoprotectant synthesis response of frogs. Two freeze-tolerant species, the wood frog R. sylvatica and the spring peeper P. crucifer, were subjected to controlled whole body dehydration stress at 5°C at a rate of 0.5–1% of total body water lost per hr (achieved by holding frogs in closed containers with desiccant in the bottom) (3,4). Both species tolerated the loss of 50–60% of total body wa- Cryoprotectants in Freezing Tolerance FIG. 2. Effect of dehydration and rehydration on glucose lev- els in liver, heart and brain of the freeze-tolerant frogs Rana sylvatica and Pseudacris crucifer and the freeze-intolerant Rana pipiens. All frogs were autumn-collected, acclimated at 5°C and then dehydrated at 5°C at a rate of 0.5–1% of total body water lost per hour in closed containers over a layer of silica gel desiccant. For rehydration, 50% dehydrated frogs were placed in containers with 1–2 cm of distilled water and sampled after 24 hr at 5°C. h, controls at 5°C; , dehydrated to 25% of total body water lost; , dehydrated to 50% of total body water lost; ■, 50% dehydrated then fully rehydrated. Data compiled from Churchill and Storey (3–5). ter (just as they tolerate the conversion of about the same percentage of body water into ice), which puts them among the best of the desiccation-tolerant anurans (27). Our critical discovery, however, was that both species responded to dehydration with a rapid glycogenolysis in liver and glucose output to other organs. Glucose rose progressively in all six organs tested of autumn-collected wood frogs when animals were dehydrated (see Fig. 2 for liver, heart and brain); the maximal increase ranged from 9-fold in gut to 313-fold in liver of frogs that had lost 50% of total body water. Final liver glucose was 1280 nmol/mg or 127 µmol/g wet weight, a value not much less than the 200–300 µmol/g wet weight typically stimulated by freezing exposure (31). A similar response was seen during dehydration of autumn-collected P. crucifer with glucose rising by 120-fold to 2690 6 400 nmol/ mg protein or 220 µmol/g wet weight in liver of 50% dehydrated frogs (Fig. 2), a value again virtually identical to the effect of freezing on liver glucose in this species. Glucose in other organs of P. crucifer rose by 3- to 60-fold. Glucose levels in both species fell again when animals were rehydrated (Fig. 2), as also occurs when frogs are thawed, and the glucose output response to dehydration was much greater in autumn-collected vs spring-collected frogs, as is also the case for freezing-induced cryoprotectant synthesis (31,33). Parallel dehydration experiments with the leopard 325 frog R. pipiens showed that the liver glycogenolysis and glucose output responses to whole body dehydration were also shared by a freeze-intolerant species (5). Glucose rose progressively with dehydration in R. pipiens liver, increasing by 24-fold overall to a final value of about 300 nmol/mg or 20 µmol/g wet weight in frogs that had lost 50% of total body water (Fig. 2); concomitantly, glucose content also rose in most other organs. The magnitude of the glucose output response to dehydration is clearly much lower in R. pipiens than in the two freeze-tolerant species, but the metabolic response of liver glycogenolysis to cellular water loss is clearly in place in the freeze-intolerant species. This suggests that freeze-tolerant frogs have probably elaborated on an underlying glycogenolytic response to dehydration to achieve the rapid cryoprotectant output response to freezing. What is also intriguing about this glucose output response is that it does not appear to be a direct response to water loss by liver cells themselves. In all three species undergoing dehydration, water was lost first from extraorgan pools and the water content of core organs was defended until a high percentage of total body water was lost (3–5). Indeed, R. sylvatica excelled at this, and even when 50–60% of total body water was lost, liver water content remained unchanged (3). Rather, it appears that water loss (due to dehydration or freezing) is probably detected by peripheral target cells, perhaps in the skin, and is transmitted by nervous or hormonal signals to the liver that responds metabolically with glucose output. As discussed earlier, the signal involved may be catecholamine-based and directed through changes in cAMP levels (33). Indeed, recent research with other systems has shown that cell volume change can trigger a wide variety of cellular responses, including changes to intermediary metabolism and to gene expression, and that various extracellular stimuli, by stimulating cell swelling or cell shrinkage, induce a pattern of coordinated cellular responses (12). Insulin, for example, stimulates cell swelling in rat liver, whereas glucagon and catecholamines (both glycogenolytic hormones) induce shrinkage (12). Thus, glycogenolysis has an ancient link to cell volume reduction, and this may have formed the basis of the cryoprotectant (glucose) output response to freezing by frog liver. One of the key areas for future research in natural freeze tolerance will be to fully explore the range of cryoprotective responses stimulated and coordinated by changes in cell volume. Thanks to J.M. Storey for critical commentary and editing of the manuscript. 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