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Am J Physiol Endocrinol Metab 276: E896-E906, 1999;
0193-1849/99 $5.00
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Vol. 276, Issue 5, E896-E906, May 1999

Metabolic adjustments during daily torpor in the Djungarian hamster

Gerhard Heldmaier1, Martin Klingenspor1, Martin Werneyer1, Brian J. Lampi2, Stephen P. J. Brooks2, and Kenneth B. Storey3

1 Department of Biology, Philipps-University, D-35032 Marburg, Germany; 2 Nutrition Research Division, Health Canada, Banting Research Centre, Ottawa K1A 0L2; and 3 Department of Biology, Carleton University, Ottawa, ON, Canada K1S 5B6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Djungarian hamsters (Phodopus sungorus) acclimated to a short photoperiod (8:16-h light-dark cycle) display spontaneous daily torpor with ad libitum food availability. The time course of body temperature (Tb), metabolic rate, respiratory quotient (RQ), and substrate and enzyme changes was measured during entrance into torpor and in deep torpor. RQ, blood glucose, and serum lipids are high during the first hours of torpor but then gradually decline, suggesting that glucose is the primary fuel during the first hours of torpor, with a gradual change to lipid utilization. No major changes in enzyme activities were observed during torpor except for inactivation of the pyruvate dehydrogenase (PDH) complex in liver, brown adipose tissue, and heart muscle. PDH inactivation closely correlates with the reduction of total metabolic rate, whereas in brain, kidney, diaphragm, and skeletal muscle, PDH activity was maintained at the initial level. These findings suggest inhibition of carbohydrate oxidation in heart, brown adipose tissue, and liver during entrance into daily torpor.

Phodopus sungorus; body temperature; metabolic rate; metabolic inhibition; enzymes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SMALL MAMMALS may use daily torpor to reduce their daily energy expenditure. In some species, e.g., mice like Peromyscus leucopus, Peromyscus maniculatus, or Mus musculus, torpor can be facultatively induced by food restriction and moderate cold exposure (25, 49). In strictly photoperiodic species like the Djungarian hamster, Phodopus sungorus, torpor occurs only after several weeks of exposure to a short photoperiod and cannot be provoked by cold exposure and/or moderate food restriction. When maintained in short photoperiods, Djungarian hamsters may enter torpor regularly during their circadian resting period, even when kept at thermoneutrality and fed ad libitum (21, 23, 27). Thus they display torpor spontaneously without any acute shortage of energy supplies and spontaneously reduce their daily energy expenses up to 70% (41, 42). In a long photoperiod they do not display torpor, except after severe food restriction that reduces body mass by >25% (40). This starvation-induced torpor was not considered in the present study.

Daily torpor of small mammals is characterized by a rapid decline of metabolic rate and body temperature (Tb) during the diurnal resting phase. Metabolic rate is lowered to ~30% of the basal metabolic rate (16, 17, 22, 43). To drive this dramatic decrease in metabolic activity, major changes to specific pathways are required (48, 51). At present, three mechanistic processes have been identified that can participate in reducing metabolic pathway flux: covalent or conformational modification of enzymes to lower activity (3, 7, 46, 47), changes in the concentration of enzyme allosteric activators such as fructose 2,6-biphosphate (3, 7, 46), and changes in the degree of enzyme association [particularly phosphofructokinase (PFK)] with subcellular fractions (15, 44).

Although specific flux-reducing mechanisms have been well characterized, their participation in the metabolic processes involved in actively depressing metabolism has not been well investigated. To better characterize the relationship between active metabolic depression and enzyme covalent modification during hibernation, as well as define the processes involved in active metabolic depression, we analyzed the time course of energy-delivering substrates (serum glucose and lipids) during torpor in parallel with metabolic rate and Tb. In addition, the key enzyme activities of the major metabolic pathways of brain, liver, heart, kidney, skeletal muscle, jejunum, and white adipose tissue were measured.

We used glycogen phosphorylase (GP), glycogen synthase (GS), glucokinase (GK), PFK, pyruvate kinase (PK), lactate dehydrogenase (LDH), and pyruvate dehydrogenase (PDH) as indicators for glycogen and carbohydrate metabolism. The enzymes phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphate dehydrogenase (G-6-PDH), malic enzyme (ME), ATP-citrate lyase (ATP-CL), and fatty acid synthetases (FAS) were assayed as indicators for gluconeogenesis, fat metabolism, and NADPH production. These enzymes are known to change with adaptation in other systems. We measured aspartate aminotransferase (Asp-AT), alanine aminotransferase (Ala-AT), glutamate dehydrogenase (GDH), serine dehydratase (SDH), and branched-chain amino acid dehydrogenase (BCAADH) as indicators for amino acid metabolism. For enzymes that are known to be affected by phosphorylation (PFK, PK, PDH, GS), we further analyzed enzyme kinetics (affinity for substrates, effect of inhibitors) and/or the percentage of enzyme present in the activated state. Taken together, these enzymes represent the key enzymes for all the pathways of each of the three fuels, carbohydrates, fatty acids, and amino acids, and should illustrate the enzymic control of metabolic pathways during daily torpor.

Entrance into torpor can only occur when thermoregulatory heat production, like nonshivering thermogenesis (NST), is turned off. Brown adipose tissue (BAT) is a major site for NST, and the rate of thermogenesis as well as its functional state is controlled by sympathetic activity (24, 28, 29, 33). We therefore determined the content of norepinephrine in BAT as well as its activity of lipoprotein lipase (LPL), an enzyme-limiting fatty acid supply of brown fat cells (9, 30). These measures could show whether the fuel supply and the sympathetic activity for NST were depressed during torpor.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Djungarian hamsters were bred and raised in a long photoperiod (16:8-h light-dark cycle) at 23°C ambient temperature (Ta). They were fed ad libitum during the entire study and had free access to water or apple slices. The pelleted food was a special breeding diet (Altromin 7014) containing 23% protein, 5% lipids, 4.2% fiber, 47.8% carbohydrates, 6.5% ash, and 13.5% water. At the age of 3 mo, 60 hamsters were transferred to a short photoperiod (8:16-h light-dark cycle) at 15°C Ta. After 6 wk of exposure to a short photoperiod, the hamsters started to enter torpor. Daily inspection of body surface temperature with an infrared (IR) telethermometer (Heimann, Wiesbaden, Germany) revealed hamsters with the most frequent events of torpor. These were selected and implanted with temperature transmitters (Mini-Mitter Model X, Sunriver, OR). Transmitter calibration, implantation, and measurement of Tb were performed as described in Ref. 22. Tb of hamsters was continuously recorded for 2 wk. Individual hamsters displayed a rather steady pattern of daily torpor, i.e., an individual timing of entry into torpor and duration of torpor episodes. After assessment of this timing, individual hamsters were selected for additional records of metabolic rate.

For measurement of metabolic rate, hamsters were placed in metabolic chambers (volume 1.8 liters, pelleted food and water available ad libitum). O2 consumption (Ametek S3A, Pittsburgh, PA) and CO2 production (Maihak Unor, Hamburg, Germany) were measured by an open flow system as described previously (22). Simultaneous records of Tb and metabolic rate were obtained for 1 wk. To take tissue and blood samples during the course of torpor, we differentiated four different phases: normothermia before onset of torpor (A), early entrance into torpor (B), entrance into torpor with minimum metabolic rate (C), and steady-state level of low metabolic rate in torpor (D). At these different phases hamsters were removed from the metabolic cuvette and killed by cervical dislocation. Blood samples were collected, cooled, and centrifuged. Tissue samples were rapidly dissected and frozen in liquid nitrogen. All samples were stored at -80°C until analysis.

Serum was analyzed for glucose lipid and cholesterol content by use of commercially available kits (Boehringer Mannheim). LPL was reconstituted from acetone-ether dry powder of tissue, and its activity was measured by the release of [14C]oleate from glyceryl-tri-[1-14C]oleate (30). The BAT norepinephrine content was measured by HPLC after extraction of catecholamines with Al2O3, as described previously (33).

GP and GS were measured in crude homogenates after being ground 1:10 in (in mM) 50 imidazole, 0.5 EDTA, 0.5 EGTA, and 50 NaF (pH 7.4) according to Vardanis (50). Activities were measured using either radioactive uridine 5'-diphosphate-[U-14C]glucose (GS) or [U-14C]glucose 1-phosphate (GP) obtained from Amersham (Mississauga, ON, Canada). GS activities were measured in the presence and absence of 2 mM glucose 6-phosphate, and GP activities were measured in the presence and absence of 2 mM AMP and 0.5 M sodium sulfate (45). PDH was measured as described previously (8).

All other enzyme activities were determined in crude homogenates by grinding frozen tissue 1:4 in (in mM) 50 imidazole, 5 EDTA, 5 EGTA, 100 NaF, and 30 beta -mercaptoethanol (pH 7.0). This buffer prevents changes in enzyme phosphorylation by inhibiting phosphatase action (NaF) and by chelating free magnesium and calcium ions. Homogenates were centrifuged for 15 min in an Eppendorf centrifuge (12,000 g), and the supernatant was removed and stored on ice until assay. Enzyme activities were measured at 25°C with a filter microplate reader (Dynatech, Chantilly, VA) to record NAD(P)H production or consumption. All assays included blank reactions (no substrate) to measure background rates. Absorbency values were collected with the software provided by the company (Biolynx version 2.01) and analyzed using a computer program especially designed for this purpose (4). Background rates were subtracted point by point using the analysis software (4). Enzyme kinetic parameters were obtained using a kinetic software package (4). Assay specifics are as follows. Asp-AT, Ala-AT, GDH, SDH, and BCAADH were assayed according to Brooks and Lampi (6). PFK was assayed in 50 mM imidazole-HCl (pH 7.0), 5 mM MgCl2, 0.15 mM NADH, 0.5 mM ATP, 1 U/ml of aldolase, 1 U/ml of triosephosphate isomerase, 2 U/ml of glycerol 3-phosphate dehydrogenase, and varying fructose 6-phosphatase concentrations. Half-maximal inhibitory values for ATP were measured at 3 mM fructose 6-phosphate (F-6-P; liver), 60 mM F-6-P (kidney), 10 mM F-6-P (heart, skeletal muscle), 30 mM F-6-P (brain), 20 mM F-6-P (jejunum, lung), or 50 mM F-6-P (white adipose tissue, brown adipose tissue). PK activity was measured in 50 mM imidazole (pH 7.0), 5 mM MgCl2, 0.15 mM NADH, 2 mM ADP, 1 U/ml lactate dehydrogenase, and varying concentrations of phosphoenolpyruvate concentrations: 1.2 mM (liver), 25 mM (kidney), 0.24 mM (heart), 0.1 mM (brain), 0.34 mM (skeletal muscle), 2.4 mM (jejunum, lung), and 4 mM (white and brown adipose tissues).

Regressions were calculated as least square regressions, and comparisons between groups were performed by ANOVA by use of Sigma-Stat software (Jandel, Düsseldorf, Germany). Significance was assumed for P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

At 15°C Ta, hamsters showed an episode of spontaneous daily torpor about every 2nd day. On normothermic days, their Tb remained at ~36.4°C during nocturnal activity and decreased to 35.5°C during the diurnal resting period. This small circadian amplitude was superimposed by ultradian variations in Tb of 2°C amplitude (Fig. 1). Metabolic rate also showed an ultradian variation, with peaks almost doubling the level of resting metabolic rate (2.64 ± 0.25 ml O2 · g-1 · h-1 at 15°C Ta during the diurnal resting period). On days with torpor episodes, a similar ultradian pattern of Tb and metabolic rate was observed during nocturnal activity. The beginning of daily torpor was characterized by a metabolic peak above 5 ml O2 · g-1 · h-1 that subsequently dropped below the level of resting metabolic rate as well as below basal metabolic rate (2.1 ml O2 · g-1 · h-1 at 23°C Ta). The peak metabolic rate was chosen as starting time for torpor in the present study. All hamsters showed a similar time course of entry into torpor and arousal from torpor, which allowed the calculation of mean values when individual records were matched for the metabolic peak before entry and during arousal from daily torpor (Fig. 2). Within 4.2 h metabolic rate decreased to a minimum of 0.51 ± 0.04 ml O2 · g-1 · h-1 and then slightly increased to 0.7 ml O2 · g-1 · h-1 during prolonged torpor. Tb decreased at a slower rate than metabolic rate and reached its minimum of 20.8 ± 0.3°C ~5.56 h after onset of torpor (Fig. 2). The low metabolic rate and decreased Tb were maintained for several hours until arousal spontaneously occurred. Arousal was characterized by a rapid increase in metabolic rate. After a brief lag phase, Tb rose to the normothermic level of 36°C because of endogenous heat production. Tb as well as metabolic rate increased at a faster rate than they decreased during entrance into torpor. Normothermic Tb was usually reached within 40 min.


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  		Fig. 1.   Body temperature (Tb, bold line), metabolic rate (narrow line), and respiratory quotient (RQ, ) of a Djungarian hamster on 2 consecutive days. On day 1 (top), the hamster remained normothermic. On day 2 (bottom), the same hamster spontaneously entered torpor.



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  		Fig. 2.   Mean values of Tb (open circle ), metabolic rate (triangle ), and RQ () of hamsters during torpor (means ± SE, n = 8). Duration and circadian timing of torpor differed slightly among individual animals. For calculation of mean values, individual curves were time matched for metabolic peaks at beginning and end of torpor. Torpor episodes began, on average, at 0816 and ended at 1725. To match individual curves, torpor episodes lasting longer than 9.15 h were cut 2 h before arousal, and torpor episodes <9.15 h were cut and interpolated 2 h before arousal. Minimum metabolic rate of 0.514 ± 0.046 ml O2 · g-1 · h-1 was reached at 1254, and minimum Tb of 20.85 ± 0.32°C was reached at 1450.

The respiratory quotient (RQ) showed no 24-h variations in normothermic hamsters (Fig. 1). During torpor, the RQ was elevated for the first 2 h and then steadily decreased from 0.95 to 0.79 (Fig. 2). The decrease in RQ was not related to the circadian resting phase, because it was observed only during torpor. This is demonstrated by comparison of two 24-h records of RQ from an individual on two consecutive days with and without torpor (Fig. 1). After arousal, the RQ returned to the pretorpor level. However, this return did not temporally coincide with the process of arousal itself but occurred after a 2-h delay (Figs. 1 and 2). During the arousal period, RQ levels were at their lowest (0.75), indicating that lipids were the major substrate utilized for rewarming. The return of the RQ value to high levels coincided with the second peak metabolic rate after arousal. This is obvious from the original record (Figs. 1 and 3) as well as from mean values (Fig. 2).


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  		Fig. 3.   Blood glucose, triglycerides, and brown adipose tissue (BAT) norepinephrine content and lipoprotein lipase (LPL) activity in different phases of torpor (see METHODS for description of torpor phases) in Djungarian hamsters. * Significant difference from phase A (P < 0.01).

Blood and tissue samples were collected during different phases of a torpor bout (Fig. 3). Serum glucose decreased from 108 ± 4 to 78 ± 6 mg/dl (P < 0.05) during torpor, and a significant decrease was also observed in serum lipid content (175 ± 15 vs. 75 ± 7 mg/dl, P < 0.05). Cholesterol units remained unchanged (phase A 100 ± 6, B 100 ± 5, C 91 ± 4, and D 103 ± 6 mg/dl, not significant). These findings indicate that the major substrates for energy metabolism were gradually removed from circulation during a torpor bout. In addition to changes in RQ, these results suggest that glucose is the primary source of energy during the beginning of a torpor bout but is gradually replaced by an increasing reliance on lipid metabolism.

BAT cells contain lipids in multilocular vacuoles. For thermogenesis, they require a high rate of fatty acid import from circulating lipoproteins. Fatty acids are imported via LPL action that hydrolyzes triglycerides in circulating lipoprotein complexes. Both LPL and the norepinephrine content of BAT (i.e., its sympathetic innervation) remain at a high functional level throughout the course of torpor (Fig. 3).

The activity of enzymes varies considerably among tissues. Skeletal muscle, heart muscle, and liver show the highest glycolytic enzyme activities, e.g., GP activity is ~10 times greater in liver and skeletal muscle than in kidney or white adipose tissue on a gram wet weight basis (Table 1). PFK activity is highest in heart, skeletal muscle, and brain. PK was exceptionally high in the intestine, whereas moderate values were found in liver and skeletal muscle. PDH activity was highest in BAT, brain, heart, and skeletal muscle. LDH activity was highest in skeletal muscle. All these tissue-specific enzyme activities were similar to those observed in other small mammals, with the exception of liver ME, which was ~10 times higher than expected (Table 2).

                              
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  		Table 1.   Activity of glycolytic and gluconeogenic enzymes in Djungarian hamster tissues during course of daily torpor


                              
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  		Table 2.   Enzyme activities of lipid and amino acid metabolism in tissues of the Djungarian hamster during course of torpor

Tables 1 and 2 compare the activities and the kinetic parameters of key regulatory enzymes in euthermic hamsters (phase A), in hamsters just entering torpor (phase B), and in hamsters at the termination of the entrance into torpor and in deep torpor (mixed phases C and D). These measurements showed specific, tissue-associated changes in enzyme activities. Total SDH activity was significantly higher in BAT from torpid animals: it rose to 3.6 times the euthermic level during phase B and was 2.4 times the euthermic level during the mixed phases C and D (Table 2). PDH activity was ~0.6 times that of euthermic animals in heart tissue during all phases of torpor (Fig. 4). GS activity was significantly lower in animals entering torpor in kidney (phase B, 0.5 times euthermic), and white adipose tissue (phase B and mixed phases C and D) was 0.4 times that found in euthermic animals (Table 2). The activities of the remaining metabolic enzymes were unchanged during the experimental time course (Tables 1 and 2).


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  		Fig. 4.   Correlation between the percentage of pyruvate dehydrogenase (PDH) in active form and the total metabolic rate of hamsters in heart muscle, BAT, and liver. Other tissues showed no correlation of PDH activation with metabolic rate. PDH in brain, kidney, and diaphragm maintained almost 100% activity, whereas skeletal muscle PDH remained at ~50% activity through the entire range of metabolic rates.

Metabolic rate and Tb were measured continuously in each hamster up to the moment of tissue sampling. This allowed a direct comparison of enzyme properties with metabolic rate. A comparison between metabolic rate and the percentage of activated PDH in individual hamsters showed a close correlation when data from heart muscle, BAT, and liver were examined (Fig. 4). There was an almost proportional reduction of metabolic rate with the inactivation of PDH. In all other tissues this relationship was missing. In kidney and brain tissue, the PDH remained at a high activation level (close to 100%) during the torpor cycle. In skeletal muscle, activated PDH did not change with torpor but remained at ~50%. The tissue PDH activity levels were slightly different between heart muscle, BAT, and liver (2.79, 3.10, and 1.91 U/g, respectively). Despite this difference in the absolute level of enzyme activity, the inactivation of PDH occurred to a similar extent in all three tissues.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fuel supply and RQ during torpor. The RQ of hamsters at thermoneutrality and at moderately cold temperatures was ~0.92-0.96, as shown here as well as in previous studies (20). At the beginning of torpor, the RQ was slightly elevated and then decreased toward the end of a torpor bout to 0.79. This indicates that, at the start of a metabolic depression episode, the hamsters largely utilized carbohydrate, increasing fat utilization as torpor progressed. During arousal, the RQ decreased further to 0.76, indicating that heat production for rewarming was almost exclusively based on lipid oxidation. The latter is in accordance with arousal from hibernation in golden hamsters, which also relied on lipid utilization during rewarming (34).

This time course of RQ differs from previous data obtained with P. maniculatus (35). In these animals, the RQ value decreased before the onset of torpor to ~0.75. At the onset of torpor, a further transitory decrease of 9- to 12-min duration was observed, with RQ values dropping below 0.7. A similar transitory change was observed during arousal, but during arousal RQ values rose to 1.0. These transitory increases and decreases were interpreted as periods of CO2 retention in body fluids and CO2 release associated with respiratory acidosis during torpor and its removal during arousal (2, 31, 35, 36, 52). The relative metabolic acidosis has even been proposed as an internal signal for metabolic depression (19, 31, 32). The reason for the discrepancies between torpor in P. sungorus and P. maniculatus is not clear. In all previous experiments, torpor was initiated by withdrawal or reduction of food. In the present study, the hamsters were fed ad libitum, which may explain the consistently high levels of RQ before and during the early part of torpor. These high RQ values indicate that glycogen and glucose were available and that glycolytic pathway activities were retained during entrance into torpor. Only during the last hours of torpor were glycolytic pathways gradually replaced by lipid oxidation. Transitory peaks of RQ were never observed in P. sungorus, which may indicate either that species differences in the control of acidosis exist or that metabolic control of spontaneous daily torpor in ad libitum-fed animals is based on different pathways than those in starvation-induced animals. The present findings show that, unlike previous results, spontaneous daily torpor in hamsters can be initiated without major anticipatory changes in CO2 balance and without a shortage of their endogenous energy stores.

The decrease in serum glucose levels during entrance into torpor suggests that glucose and glycogen stores are utilized at this time. During prolonged torpor, an additional small reduction of serum lipids was observed, suggesting an increasing utilization of lipids. These changes in metabolite levels support the decrease of the RQ during prolonged hours of torpor that have been discussed. In a similar study design, Nestler (35, 36) measured metabolic substrate changes during torpor in P. maniculatus. Blood glucose levels had decreased before the onset of torpor, and no further significant changes occurred during torpor. Plasma ketone bodies followed this same trend, suggesting that mice were metabolically starved before the beginning of torpor. This may have been a consequence of food reductions before torpor in this species (37, 38). Our present findings indicate that spontaneous daily torpor in P. sungorus may occur without any anticipatory development of starvation symptoms. Note, however, that these symptoms may develop during prolonged torpor despite reductions in metabolic rate. This suggests that daily torpor cannot simply be regarded as an acute response to shortage in food supply but is the result of deliberate control of metabolic reduction and hypothermia.

The existence of deliberate physiological control during torpor is supported by the fact that all torpid animals aroused before their circadian activity period and raised their Tb to normothermic values by endogenous heat production, without previous food intake. This conclusion is emphasized by the peculiar RQ pattern observed in P. sungorus at the end of torpor. Despite the fact that Tb increased from 20 to 35°C within 40 min, we observed a further slight reduction of RQ associated with endogenous heat production. When hamsters had aroused, the RQ value remained low (lipid oxidation) and only returned to pretorpor levels after ~2 h in the aroused state. This shows that RQ recovery to pretorpor levels is not associated with changes in Tb or with the level of metabolic rate. Djungarian hamsters do not become active immediately after arousal but remain in a resting state for ~2 h (42). The duration of this "after torpor rest period," measured by IR movement detectors, correlated with the duration of the previous torpor bout. We have not directly measured feeding activity after arousal, but the lack of locomotor activity and the high incidence of nonrapid eye movement sleep (from electroencephalographic records) suggest that the hamsters do not feed immediately after arousal (11). The recovery of the RQ value 2 h after arousal correlates with the onset of locomotor activity and is probably associated with food intake. It is obvious that changes in RQ observed during torpor in the Djungarian hamster merely reflect a change in energy metabolism from glucose to lipids and back to glucose and do not indicate a physiological role of substrate availability in initiating or maintaining torpor.

Time course of Tb and metabolic rate. Daily torpor is initiated by a rapid decrease in metabolic rate and Tb. Both physiological functions are linked to each other, i.e., any change in metabolic rate can cause changes in Tb and vice versa. This link provoked a discussion of whether downregulation of metabolic rate or downregulation of Tb is the primary cause of entrance into daily torpor (16, 18, 22, 43). Previous measurements of heat production and heat loss in the Djungarian hamster suggest that downregulation of metabolic rate is of primary significance for the initiation of daily torpor and that hamsters become hypothermic as a consequence of lacking heat production (22). The present findings support this view. Metabolic rate decreases before the development of hypothermia: the minimum metabolic rate is attained before the minimum in Tb. To compare the time course of metabolic rate and Tb during entrance into hibernation, we performed a correlation analysis (r = 0.866 ± 0.017) by shifting both parameters in 4-min steps against each other. The best correlation was obtained when Tb was shifted by 40 min toward the decrease in metabolic rate (r = 0.960 ± 0.016). This indicates that, on average, the decrease in metabolic rate occurred 40 min before the decrease in Tb.

Enzyme activities. A comparison of liver enzyme activities in euthermic hamsters and rats showed similar values (0.5- to 4-fold difference) for many enzymes of glycolytic, fatty acid synthesis, and amino acid-utilizing pathways (Fig. 5). The 10-fold higher ME activity in hamster liver was particularly interesting in light of the theoretical calculations of Flatt (13), who estimated the energy yield for fat synthesis in adipose tissue. Flatt compared ATP yields when a large proportion of reducing equivalents was produced through increased carbon flow through malate dehydrogenase and ME (the malate cycle) or through the pentose phosphate pathway. His calculations revealed that fatty acid synthesis is an energy-yielding step for the cell itself (although not energy yielding overall); the more inefficient cycle is likely to be less regulated than the more efficient cycle (13). The 10-fold higher ME activity in hamster liver may play a significant role in promoting fat accumulation from carbohydrates, because hamsters feed mainly on grass seeds, a diet rich in carbohydrates and only a small amount of lipids (14).


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  		Fig. 5.   Ratio of enzyme activities measured in liver from euthermic Djungarian hamsters (present study) and adult rats maintained on a 10% fat diet (Ref. 5). GP, glycogen phosphorylase; GK, glucokinase; PFK, phosphofructokinase; PK, pyruvate kinase; PDH, pyruvate dehydrogenase; LDH, lactate dehydrogenase; GS, glycogen synthase; PEPCK, phosphoenolpyruvate carboxykinase; G-6-PDH, glucose-6-phosphate dehydrogenase; ME, malic enzyme; ATP-CL, ATP citrate lyase; FAS, fatty acid synthetases; Ala-AT, alanine aminotransferase; Asp-AT, aspartate aminotransferase; GDH, glutamate dehydrogenase; SDH, serine dehydratase; BCAADH, branched-chain amino acid dehydrogenase.

Regulation of metabolic pathways often involves modification of key pathway enzymes to (at least partly) control flux. For example, glycolytic regulation can occur at the start of the pathway via regulation of GP, GK, and PFK (46, 47) or at the end of the pathway by changes in PK or PDH activity. Amino acid utilization may be controlled by changes in GDH, SDH, or BCAADH activity; each enzyme regulates different catabolic pathways. An increase in SDH activity during torpor in BAT may indicate an increase in the capacity for gluconeogenesis. It is more difficult to identify flux-regulating steps for fatty acid synthesis, because many different reactions are required to make fat de novo: reactions that generate carbon units and reactions that generate reducing equivalents are all involved in fatty acid synthesis. However, a few enzymes are common to all reaction pathways: PK and PDH. These enzymes are responsible for entry of carbon units into the tricarboxylic acid cycle and so regulate glycolytic activity and the production of acetyl-CoA (required for fatty acid synthesis). In addition, a large percentage of amino acid oxidation passes through pyruvate to PDH. Both enzymes represent irreversible steps, and both are regulated by reversible phosphorylation in the liver (12, 39). An examination of the potential regulatory enzymes of hamsters during torpor demonstrated the importance of liver, heart, and BAT tissue PDH in regulating metabolic activity (Fig. 4). This was demonstrated by the positive correlation between metabolic rate and PDH activity from liver, heart, and BAT: the metabolic rate of hamsters paralleled PDH activity in these tissues. These findings strengthen the conclusion that Djungarian hamsters actively suppress their metabolic rate, because PDH activity is regulated by reversible phosphorylation and not by temperature. In addition, the close correlation between the metabolic state of the animal and the percentage of active PDH stresses the fact that regulation of PDH precedes a decrease in Tb because the metabolic rate depression precedes the decrease in Tb. The potential role of PDH as a metabolic switch for hypometabolism was recently supported by the finding that PDH kinase mRNA was found elevated in the heart of hibernating ground squirrels (1).

Differential regulation of enzyme activity (see Tables 1 and 2 and Fig. 4) suggests different roles for different organs during torpor. As we have discussed, PDH activity in heart, liver, and BAT correlates with whole body metabolic rate measurements, suggesting an important role of these tissues in regulating metabolic depression during torpor. This conclusion is not novel. BAT is known to be important in NST and regulation of body fat stores in small rodents (see Ref. 24 for review); liver is important in maintaining blood glucose homeostasis, is an important site of lipogenesis, and accounts for a significant fraction of total amino acid metabolism (26); and heart activity is highly regulated during periods of depressed metabolism (see Ref. 51 for review). Downregulation of these tissues may force downregulation of metabolic rate in other tissues. For example, bradycardia during torpor will reduce the supply of substrate to muscular tissues (gut, skeletal tissue), and this may help to reduce their metabolic activity. A differential role of tissues during torpor is also supported by examination of tissue glycogen concentrations in P. leucopus or P. maniculatus: glycogen content decreased in skeletal muscle and increased in heart during torpor (38).

The findings suggest an involvement of glycolytic inhibition in metabolic reduction in torpor, or, more precisely, an inhibition of glycolytic input into the tricarboxylic acid cycle. This is in agreement with other observations in hibernating and torpid animals (for review see Ref. 7), but the enzyme changes differed from species to species. In hibernating Citellus beecheyi, Hand and Somero (19) observed a reduction in PFK activity based on a reversible conversion of the active tetrameric form of PFK to its inactive dimeric state. This was possibly caused by the relative acidosis during hibernation (31, 32). In hibernating ground squirrels, Spermophilus lateralis, Brooks and Storey (7) observed a glycolytic inhibition, e.g., reductions of GP activity and an inactivation of PDH similar to the present study. PFK and PK showed no major changes. These responses differed between tissues such that GP was reduced in liver and kidney and PDH was inhibited in heart and kidney. Another pattern of glycolytic inhibition was observed in hibernating Zapus hudsonicus (46, 47). Here GP, PFK, and PK activities were reduced in the liver. It appears that hibernation-associated low metabolic rates are not brought about through regulation at a single locus but result from a pattern of enzyme conformational changes and activation/inactivation by phosphorylation. These responses differ between tissues, but they all point to a central role for an inhibition of carbohydrate utilization in depressed metabolism. The present results showing PDH inactivation in heart, liver, and BAT are in accordance with this pattern, suggesting a common basis for this inhibition in hibernation as well as in torpor. The possible role of inhibited carbohydrate utilization during torpor is also supported by the observation that injections of deoxyglucose into P. sungorus, which inhibited glycolysis, facilitated the occurrence of daily torpor, whereas blockade of lipid metabolism after mercaptoacetate injection had no apparent effect (10).

All previous studies of hibernators were performed on animals in deep hibernation. These animals had no food for days or weeks and were in steady-state hibernation, with Tb values below 10°C for days. In the present study, hamsters were fed ad libitum, and an inactivation of PDH was observed over the course of several hours during the initial transition from normometabolism to hypometabolism. A similar study of this transition period is lacking in hibernators. Therefore, it is unclear whether the inactivation of PDH in heart, BAT, and liver (with no change in other enzyme activities) characterizes only daily torpor, or other forms of hypometabolism as well. The results further demonstrate that hypometabolism is not due to exogenous or endogenous substrate limitations but may occur in response to downregulation of ATP-generating or ATP-consuming processes in the body.


    ACKNOWLEDGEMENTS

The technical assistance of Sigrid Stöhr and Ralf Liese is gratefully acknowledged.


    FOOTNOTES

This work was supported by grants from the Deutsche Forschungsgemeinschaft (to G. Heldmaier) and the National Sciences and Engineering Research Council of Canada (to K. B. Storey).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: G. Heldmaier, Department of Biology, Philipps-University, D-35032 Marburg, Germany (E-mail: heldmaie{at}mailer.uni-marburg.de).

Received 28 August 1998; accepted in final form 14 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 276(5):E896-E906
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