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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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
-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.
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RESULTS |
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 ( ), metabolic
rate ( ), 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.
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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).
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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
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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.
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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.
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DISCUSSION |
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.
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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.
 |
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