INTRODUCTION

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THIS PAPER FOCUSES ON the processes of metabolic rate reduction that allow the common frog Rana temporaria to survive natural periods of overwintering submergence. Hibernating underwater affords the amphibian protection from the environmental stresses of freezing and desiccation. Several aquatic amphibian species in the northern hemisphere remain submerged for up to 9 mo of the year, as a consequence of the ice cover in the ponds and lakes in which they hibernate, becoming covered with ice (4, 11, 34). Because R. temporaria is the most northerly distributed ectothermic tetrapod in Europe (65° north; see Ref. 22), it encounters conditions each winter when the body temperature is so low that normal activities are thought to be suspended. However, field studies of cold-submerged frogs suggest that the animals remain active throughout the winter months (8, 22) and that they rarely, if ever, are to be found hibernating in the severely hypoxic mud at the bottom of lakes and ponds (4, 5). These observations, and others showing that aerobic metabolic rate (O₂) does not decrease greatly during several weeks of cold submersion, have led to the conclusion that frogs do not lower their metabolic rates to any great extent during the overwintering period (25).

Amphibians accumulate substrate reserves during the summer and early autumn to fuel the energetic demands of overwintering and spring breeding (cf. Ref. 13). Aerobic lipid oxidation is considered to be the primary source of fuel for metabolism during hibernation due to the large lipid stores laid down by early autumn and the depletion of these stores during winter and spawning periods (5, 28). Entering a hypometabolic state would confer the advantages of energetic savings and fuel reserve economies during a period of prolonged starvation. Many diving animals are thought to reduce metabolic rate during submergence. The turtle Chrysemys picta exhibits a profound reduction in metabolism in response to several months of submergence at 3°C (17). Indeed, submergence elicits a diving response and causes a decrease in O₂ in both the terrestrial toad Bufo bufo (20) and air-breathing salamander Siren (31).

For the animal to overwinter successfully, it must not only conserve fuel reserves but must also avoid the production of toxic end products of its metabolism. Although submergence provides protection from the terrestrial environment, it presents new acid-base challenges to the animal as it must rely exclusively on cutaneous respiration for all of its gas exchange requirements. Amphibians enter a pronounced metabolic acidosis when they are chronically reliant on anaerobic metabolism to supply their metabolic demands (7). Metabolic rate depression would facilitate the animal remaining aerobic, thereby avoiding the production of the toxic end products of anaerobiosis during long-term normoxic submergence.

It has been proposed that the metabolic rate reduction associated with hibernation in amphibians is entirely due to temperature effects in those species that are not freeze tolerant (25). Clearly, the lowered temperatures associated with overwintering reduce metabolic rate by Q₁₀ effects, but we hypothesize that an additional metabolic reduction not associated with the change of temperature per se would be adaptive. Entering a hypometabolic state would greatly extend an animal's ability to cope with hypothermia and starvation due to lowered rates of fuel and oxygen consumption and thus survive for proportionally longer, using endogenous fuel reserves.

	MATERIALS AND METHODS

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All animals used in these experiments were adult male R. temporaria (25-30 g) collected by commercial suppliers (Blades Biological) from a wild population in Ireland during January 1995. The animals were acclimated in 3°C water over a 4-wk period during which time they had direct access to air. Six animals were then placed in darkened 250-ml water-filled respirometers that were irrigated with normoxic water (mean PO₂ = 155 Torr; 1 mmHg = 0.133 kPa) and maintained in normoxia at 3°C by immersion of both the respirometers and the equilibration towers in a Living Stream (Frigid Units, Cleveland, OH) temperature-controlled recirculated water system.

Water irrigating the frogs was sampled regularly over a time course of 90 days. At each sampling time, the flow through the respirometers was suspended, and the O₂ was determined by measuring the fall in PO₂ (PO₂ × , where PO₂ is the solubility coefficient for oxygen) of the known volume of water over time using Radiometer E5046 oxygen electrodes and meters (Radiometer, Copenhagen, Denmark). The corresponding CO₂ production (CO₂) was determined by measuring the change in total CO₂ concentration using the electrode and cuvette method of Cameron (6). Before every measurement, the CO₂ electrode (Radiometer E5036) was calibrated with gas mixtures of 1 and 6% CO₂, and the system was then calibrated for change in total CO₂ concentration using 1.0 and 3.0 mol/l standards. A 3.30-ml sample of water was withdrawn anaerobically from the respirometers and introduced into the Cameron chamber, which was sealed for 5 min to allow the sample to equilibrate. After equilibration, 50 µl of 0.1 N HCl were added to the Cameron chamber with a Hamilton gas-tight syringe, the chamber was resealed, and another 5 min were allowed for the acidified sample to equilibrate before taking a reading. Subsequent readings taken after 4 and 6 h enabled calculation of the individual O₂ and CO₂. The ratio CO₂/O₂ enabled calculation of the respiratory quotient, "RQ."

In a parallel experiment, four groups of six frogs (n = 24) were acclimated as above and then submerged in a darkened water-filled perspex box maintained at 3°C and supplied with a constant flow of normoxic water at 950 ml/min. At periods of 1 wk, 1 mo, 2 mo, and 4 mo, six animals were individually removed from the chamber and anesthetized in fully aerated 0.18% tricaine methanesulfonate (MS222) solution, buffered to pH 7, and maintained at 3°C. The animals were entirely quiescent throughout the transfer and anesthetic procedure, which lasted ~10 min. A 200-µl blood sample was then withdrawn anaerobically by cardiac puncture. The sample (50 µl) was immediately used for measurement of extracellular pH (pH_e). The remainder of the sample was centrifuged anaerobically (15,800 g), and 50 µl of the true plasma were used for analysis of total CO₂ concentration. The remaining 100 µl were placed in an Eppendorf tube, immediately submerged in liquid nitrogen, and subsequently stored at 80°C before enzymatic analysis of plasma metabolites. Liver and gastrocnemius muscle samples were removed, freeze-clamped between aluminum plates cooled to 196°C, and stored in liquid nitrogen.

pH_e measurements were made on blood plasma using a Radiometer G297/V glass capillary electrode maintained at 3°C and a Radiometer PHM 84 research pH meter. The electrode was calibrated before and after each series of measurements with Radiometer precision buffers S1500 and S1510. Anaerobically centrifuged plasma samples were used to measure true plasma total CO₂ (change in total CO₂ concentration) using a Corning 965 total CO₂ analyzer. Total CO₂ concentrations and pH_e values were used to calculate PCO₂ and concentration ([]) levels in the blood as described previously (3). The solubility of CO₂ and operational pK_i was calculated using the equation of Heisler (16). Intracellular pH (pH_i) was determined using the tissue homogenate method described by Pörtner et al. (26). Briefly, the frozen gastrocnemius muscles were ground into a fine powder under liquid nitrogen, in a mortar immersed in liquid nitrogen using a precooled pestle. Subsamples of gastrocnemius muscle (100-200 mg) were then placed in a preweighed 0.5-ml Eppendorf tube containing 300 µl of chilled metabolic inhibitor solution (6 mmol/l nitrilotriacetic acid; 150 mmol/l potassium fluoride). The tube was immediately reweighed and then filled to the brim with more of the inhibitor solution. The mixture was then rapidly homogenized, capped, and vortexed for a further 10 s. The tube was then centrifuged (15,800 g) for 10 s, and a 200-µl aliquot of the supernatant was used to measure the change in total CO₂ concentration and pH_i as described above. The pH_i, intracellular [] ([]_i), and intracellular CO₂ partial pressures were estimated according to the calculations detailed in Pörtner et al. (26).

The metabolites glucose, glucose 6-phosphate, pyruvate, lactate, ATP, ADP, AMP, phosphocreatine, and creatine were extracted in 7% perchloric acid and neutralized with 2.0 mol/l KOH plus 0.4 mol/l sodium imadizole. Glycogen was extracted according to the procedure described by Bergmeyer (1). Metabolite concentrations were measured using a Hewlett-Packard 8452A ultraviolet spectrophotometer and standard enzymatic techniques (23) using chemicals and enzymes purchased from Sigma Chemicals.

Six control animals were sampled at day 0 in exactly the same manner, with the exception that they always had access to air.

Statistical analysis. Metabolic rate and CO₂ measurements were analyzed using one-way analysis of variance (ANOVA) and Tukey's tests. All other measurements were analyzed using one-way ANOVA and Dunnett's multiple comparison tests. All results were considered statistically significant at P  0.05 and are presented as means ± SE.

	RESULTS

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RQ measurements (Fig. 1A) revealed that submerged frogs initially favored a predominantly lipid or mixed-substrate metabolism for the first 40 days of submergence (RQ  0.82). Over the remaining 50 days of the experiment, RQ values progressively increased to levels indicative of an exclusively carbohydrate-burning metabolism (RQ = 1.01). Figure 1B illustrates the time course of metabolic depression associated with the dormant normoxic state in the frog. It appears that the normoxic animal enters a hypometabolic state at a steady rate for the first 65 days of submergence (Pearson coefficient = 0.88), achieving an O₂ of 38% of control values. However, by 90 days, there is a significant increase in CO₂ (P  0.05) compared with measurements at day 65, with no significant change in O₂. After 3 mo of submergence in fully aerated water, O₂ levels are 61% of those seen before submergence (P  0.05) compared with control values.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Changes in the respiratory quotient [RQ = CO2 production (CO2)/aerobic metabolic rate (O2); A] and reduction in O2 (B) of Rana temporaria over the course of 90 days submergence in normoxic water at 3°C. Values are means ± SE (n = 6 frogs).

After 1 wk of submergence, the frogs demonstrated an extracellular respiratory acidosis (P  0.05), which thereafter subsided as PCO₂ levels were lowered to those seen in the air-breathing animals (Fig. 2). Over the following 15 wk of cold submergence, the extracellular acid-base status was not significantly different from that of the control animals. The initial intracellular response to submergence is a statistically significant respiratory acidosis caused by a significant rise in the intracellular PCO₂. However, unlike extracellular ], the []_i declined over the last 2 mo of the hibernatory period (P  0.05), resetting the acid-base status of the skeletal muscle at a new pH_i-[]_i set point (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   pH- concentration diagram illustrating plasma acid-base status of R. temporaria over the course of 16 wk submergence at 3°C in normoxic water (n = 6). Curved lines represent PCO2 isobars at the prevailing temperature. 1 mmHg = 0.133 kPa. pHe, extracellular pH.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   pH- concentration diagram illustrating intracellular acid-base status of R. temporaria over the course of 16 wk submergence at 3°C in normoxic water (n = 6). Curved lines represent PCO2 isobars at the prevailing temperature. 1 mmHg = 0.133 kPa. pHi, intracellular pH.

Metabolite concentrations for muscle and plasma are tabulated in Table 1. Liver glycogen concentrations are displayed in Fig. 4. Muscle high-energy phosphate homeostasis is maintained over the course of the experiment, with no significant rise or fall in ATP, ADP, AMP, and phosphocreatine. The phosphocreatine-to-creatine ratio remains stable (0.66 ± 0.02) as does adenylate energy charge (0.837 ± 0.008). Lactate-to-pyruvate ratios (3.38 ± 0.20) remain stable, indicating that the cytosolic redox potential is unchanged (n = 30). Glucose 6-phosphate levels are maintained at control levels. Blood glucose levels are significantly depressed after 2 mo of submergence (P  0.01). Glycogen concentration measurements show that liver glycogen is the primary source of carbohydrate during the first 2 mo of submergence. Thereafter, muscle glycogen levels fall significantly (P  0.01), indicating that the frog has begun to utilize muscle glycogen stores to fuel metabolism.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Glycogen concentrations in the liver (A) and gastrocnemius muscle (B) of R. temporaria over the course of 16 wk submergence in normoxic water at 3°C. Values are means ± SE (n = 6).

	DISCUSSION

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Many aquatic ectothermic vertebrates respond to the rigors of the overwintering environment (4, 25) by entering into quiescent states, which are referred to as dormancy, torpor, or hibernation. These states are almost always accompanied by a lowering of standard metabolic rate, which lessens the impact of ATP demand on endogenous energy reserves (2), thereby extending an animals survival time until more ecologically favorable conditions return. This paper shows that the cold-submerged frog adopts an integrative strategy that tailors oxygen demand, fuel consumption, and acid-base status to the stresses imposed upon it while trapped beneath the ice.

Cold-submerged R. temporaria gradually enter a hypometabolic state over the first 9 wk of cold submergence; their O₂ progressively falls to 38% of the normoxic control values after 9 wk (Fig. 1B). The fall in RQ values immediately upon submergence (Fig. 1A) is indicative of an entirely lipid burning and therefore a completely aerobic metabolism. Pasanen and Koskela (22) report that lipase activity is maximally elevated in the liver of frogs caught in the wild at the same time as they enter hibernation, suggesting recruitment of liver triglyceride stores upon submergence. The selective utilization of lipid provides compelling evidence, particularly when combined with the stable muscle and plasma lactate measurements (Table 1), that the animal is not oxygen limited. However, it is unlikely that this equilibrium could have been achieved if the animal had not lowered its O₂ to match oxygen demand with delivery. Entry into a hypometabolic state is clearly a response to submergence rather than being due to temperature effects alone because the animals were acclimated to the experimental temperature for 4 wk before submergence.

The reduction in O₂ allows estimates to be made of how long the frog can extend its survival time during periods of overwintering (Table 2). If the frogs used their endogenous fuel stores at the rates measured immediately before submergence (Table 2), they would have just over 3 mo of fuel reserves before the combined fat body and body glycogen stores were completely exhausted. Our observations that muscle glycogen remains constant over the first half of the 4-mo experiment and that the fall in liver glycogen subsides after 2 mo (Fig. 4) strongly indicate that a reduction in O₂ acts to minimize substrate use. This hypometabolic-induced decrease in respiratory substrate utilization may also have the longer-term consequence of increasing the animal's reproductive fitness. Many northern temperate amphibians engage in costly reproductive behaviors soon after emerging from their overwintering hibernaculum, before having opportunities to feed. Intense levels of muscular activity by males, associated with sexual selection, can raise the resting metabolic rates to levels of sustained metabolic expenditure that are far in excess of any other seasonal activity. As such behaviors occur before the animals have opportunities to feed, they must be fueled by the prehibernatory fuel reserves laid down months in advance. A strategy that slows the rate of substrate depletion (i.e., hypometabolism) is essential not only to allow R. temporaria to survive for up to 8 mo of cold submergence but also to provide adequate energy reserves for the reproductive activities that follow emergence from hibernation. Current optimality hypotheses concerned with the evolution of physiological systems argue that animals are designed such that their structures and functional capacities satisfy, by some margin of safety, maximum physiological requirements or loads (9, 30, 33). Relating this to overwintering frogs, the functional capacity needed to ensure reproductive success is a posthibernatory fuel reserve that can support the physiological load of intense reproductive muscular activities. Maximizing the amounts of fuel stored before hibernation (22, 28) may be one way of ensuring a margin of safety for the adequate provision of energy to reproduction. However, the upper limits on energy reserve capacities for reproductive behaviors in frogs will largely depend on the rates of fuel expenditure during hibernation. In this regard, metabolic suppression is functionally equivalent to a "metabolic savings reserve" and is likely to provide the margins of safety required for the conservation of metabolic fuels.

Our RQ measurements (Fig. 1A) suggest that the frogs burn a mixture of lipid and carbohydrate stores while having access to air, presumably because they have large fat-body reserves of triglyceride (Table 2) and because fat metabolism yields a larger energetic return (i.e., moles ATP) per mole of substrate oxidized than does carbohydrate (12, 22). The reason why the normoxic frog gradually shifts toward an exclusively carbohydrate metabolism from a predominantly lipid metabolism is unclear unless the estimates of fat-body stores (Table 2) do not reflect the amount of free lipid available or unless lipids are being preferentially conserved for posthibernatory activities. Pasanen and Koskela (22) note that fat reserves are preserved in mature frogs but are almost completely exhausted in juvenile frogs over the time course of a winter. They also note that, in adult animals, the fat body lipid reserves are depleted during the spawn and that the fat body lipid esterase Q₁₀ values are at their highest during spawning in sexually mature animals. The progression toward an exclusive respiration of carbohydrate (Fig. 1A) suggests that the mature animal adopts a lipid-sparing strategy in the later stages of hibernation, and the observation that the preservation of lipid stores is peculiar to overwintering adult frogs (22) provides evidence that this could be related to their reproductive activities upon emergence from hibernation.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Plasma pH and PCO2 levels in R. temporaria over the course of 16 wk submergence at 3°C in normoxic water (n = 6). 1 mmHg = 0.133 kPa. Values are means ± SE (n = 6).

The comparatively rapid utilization of endogenous glycogen (Fig. 4A) in the first week of submergence could be explained if the animal was forced to recruit anaerobic sources of energy during this period. However, muscle pyruvate and lactate concentrations (Table 1) indicate an entirely aerobic metabolism throughout the course of the experiment, including the first week of submergence. Moreover, the actual amount of liver glycogen utilized over the course of the first week represents less than one-sixth of the total respiratory substrate required to fuel the hibernating frog's metabolism during this time. The fact that the large reserves of glycogen contained in the total muscle mass of the animal are not recruited until the second month of submergence (Fig. 4B) suggests that anaerobiosis plays little if any role in energy production during normoxic cold submergence. Indeed, phosphocreatine/creatine and pyruvate/lactate ratios as well as adenylate energy charge also remain stable over the entire time course of the experiment (Table 1), which suggests that the muscle remains aerobic throughout hibernation.

The initial extracellular response to overwintering submergence is an apparent respiratory acidosis, i.e., a hypercapnic rather than a hypoxic response, in which PCO₂ increases over the course of the first week, resulting in a corresponding decrease in pH_e (Fig. 5). The developing acidosis indicates that the skin is either unable to compensate for the loss of the lungs in CO₂ exchange or that cutaneous perfusion is actively reduced for other reasons (e.g., to generate a respiratory acidosis or to reduce water uptake across the skin). In any case, blood PCO₂ thereafter decreases and pH_e increases to the levels seen in the air-breathing animal (Fig. 5), presumably due to increased capillary recruitment of the cutaneous vasculature (24). In experiments on Rana catesbeiana, submerged for 8 h at 15°C, Wasser et al. (32) found that pH_e does not play a large role in regulating metabolic depression. Nor did the manipulation of pH_e significantly affect pH_i in the liver, skeletal muscle, or heart of R. catesbeiana. Nevertheless, decreased pH_i has been implicated as a signaling mechanism for metabolic rate reductions in a variety of animals (15, 29). Intracellular acidosis is thought in some cases to be advantageous to hibernating animals by causing a deactivation of intracellular enzymes and thereby directly lowering standard metabolic rate (15). In our experiments, the extracellular acidosis does not appear to alter the acid-base status of the intracellular compartment over the first 2 mo of cold submergence. However, unlike the extracellular compartment, the acid-base balance of skeletal muscle eventually shifts to a lower pH_i- set point (i.e., a relative metabolic acidosis) over the last 8 wk of the experiment. Whether this new acid-base chemistry provides the metabolic context for entry into and/or maintenance of the hypometabolic state has yet to be determined. Intracellular proton buffering capacity is a function of numerous physicochemical parameters. The new acid-base status, in the apparent absence of any lactate production, may be explained if protein buffering decreased over the 4-mo course of the experiment due, for example, to nonreplacement of degraded proteins, as a consequence of decreased protein turnover associated with the hypometabolic state (19). The primary mechanism of pH_i regulation in frog skeletal muscle is thought to be a transmembrane Na⁺/H⁺ exchanger (27) that extrudes H⁺ above a set proton concentration. It is well known that both Na⁺ channel leak and Na⁺-K⁺-ATPase activity are inhibited by decreases in pH_i within the physiological range (10, 21). If ion channel suppression (or arrest; see Ref. 18) is the mechanism by which metabolic demands of the muscle become reduced during hypoperfusion, it may be that Na⁺/H⁺ exchange is inhibited (i.e., due to a reduced sodium leak), leading to an sustained decrease in the pH_i-[]_i set point.

In conclusion, the experiments detailed in this paper were performed to investigate the acid-base status, metabolite concentrations, and the respiratory physiology of submerged hibernating frogs. The results indicate the metabolic conditions required for the submerged animal to enter a hypometabolic state and show that the frog adopts an integrative physiological response whereby the intracellular milieu is preserved at the same time as metabolic rates are being significantly depressed. The avoidance of anaerobic respiration to fuel metabolic demands allows the frog to selectively utilize both lipid and carbohydrate efficiently and therefore conserve substrate reserves, which is of both ecological and reproductive benefit to the animal. The use of the hypoxia-tolerant system of the frog, rather than anoxia-tolerant turtle, to investigate hypometabolism may provide additional clues as to whether metabolic defense mechanisms against oxygen limitation can be transferred to hypoxia-sensitive systems. It seems clear that further identification of the energy-sparing mechanisms evoked in hypoxia-tolerant animals could assist in identifying novel therapeutic targets for the protection of hypoxia-sensitive cells from the catastrophic effects of anoxia and ischemia.