Am J Physiol Regulatory Integrative Comp Physiol
279: R1205–R1214, 2000.
Metabolic depression and enhanced O2 affinity
of mitochondria in hypoxic hypometabolism
JULIE ST-PIERRE, GLENN J. TATTERSALL, AND ROBERT G. BOUTILIER
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom
Received 12 January 2000; accepted in final form 8 May 2000
St-Pierre, Julie, Glenn J. Tattersall, and Robert G.
Boutilier. Metabolic depression and enhanced O2 affinity
of mitochondria in hypoxic hypometabolism. Am J Physiol
Regulatory Integrative Comp Physiol 279: R1205–R1214,
2000.—This study examined whether the steady-state hypometabolism seen in overwintering frogs (Rana temporaria) is
reflected at the mitochondrial level either by a reduction in
their resting (state 4) and active (state 3) respiration rates
and/or by increases in O2 affinity. We isolated mitochondria
from the skeletal muscle of cold-submerged frogs at different
stages during their hibernation in normoxic and hypoxic
water. A modest metabolic depression at the whole animal
level (normoxic submergence) was not associated with a
reduction in mitochondrial state 4 and state 3 respiration
rates. However, mitochondria isolated from frogs that were
submerged for 1 mo manifested an increase in their O2
affinity compared with controls and with animals submerged
for 4 mo. Hypometabolism was more pronounced at the whole
animal level during hypoxic submergence and was accompanied by 1) a reduction in mitochondrial state 4 and state 3
rates and 2) an increase in the O2 affinity of mitochondria.
These findings demonstrate that metabolic depression can be
reflected at all levels of biological organization in hypoxiatolerant animals.
frog; hypoxia; state 3; state 4; P50
cope with harsh overwintering
conditions each year. The common frog, Rana temporaria, hibernates under water, often in ice-covered
ponds (40). Although the selection of an aquatic environment protects against the stresses of freezing and
desiccation, overwintering submergence can last for
several months and is often associated with severe
hypoxia as well as inhibition of normal feeding behavior (2, 5). When R. temporaria are submerged without
air access for 3 to 4 mo at 3°C, so as to mimic the
overwintering conditions under ice cover, they enter
gradually into a state of metabolic depression. The
extent of metabolic depression is greater when frogs
are exposed to hypoxic environments (75% depression
at PO2 ⫽ 60 mmHg; Ref. 14) than in normoxia (39%
depression at PO2 ⫽ 155 mmHg; Ref. 15). The key to
their survival during long periods of cold submergence
in hypoxic environments is their ability to enter slowly
into a hypometabolic state so that their energetic needs
MANY SPECIES OF FROGS
Address for reprint requests and other correspondence: J. StPierre, Dept. of Zoology, Univ. of Cambridge, Downing St., Cambridge CB2 3EJ, United Kingdom (E-mail: [email protected]).
http://www.ajpregu.org
can be met aerobically. Indeed, R. temporaria deplete
their substrate reserves and perish when they are
presented with rapid or prolonged exposure to severe
hypoxia (PO2 ⬍ 40 mmHg) because they lack the capacity to acutely suppress their metabolic rate to
match such drastic reductions in ambient oxygen supply (13).
Metabolic depression is the strategy of choice for
many overwintering ectotherms facing energy limitations because it spares substrate reserves and avoids
the accumulation of toxic end-products from anaerobic
metabolism (31). At the cellular level, metabolic depression may be brought about by decreasing ATP
consuming processes and/or by increasing the efficiency of ATP producing pathways (reviewed in Refs.
24, 26, 28, 30, 44). Among the ATP consuming processes, the rates of protein synthesis and of Na⫹-K⫹ATPase are often reduced in metabolically depressed
ectotherms (24, 26, 28, 30). For example, when R.
temporaria were submerged in normoxic or hypoxic
water, the activity of their skeletal muscle Na⫹-K⫹ATPase was reduced by as much as 50% (16). In addition to a reduced ATP demand for protein synthesis
and for Na⫹-K⫹-ATPase activity, the rates of protein
breakdown, ureagenesis, and gluconeogenesis were
found to be reduced when normoxic turtle hepatocytes
were exposed to anoxia (reviewed in Ref. 30). Most
studies concerning the increased efficiency of energy
production during metabolic depression have focused
on anaerobic metabolism. For example, some invertebrate species display energetically improved fermentative pathways during anoxia (31). In addition, enzyme
phosphorylation events can play important roles in
regulating glycolytic ATP production (reviewed in Refs.
6, 43, 44). However, despite the fact that many organisms entering into metabolic depression maintain an
overall aerobic metabolism, little information exists
concerning the intrinsic properties of mitochondria in
such hypometabolic states.
During prolonged submergence, frogs hyperperfuse
their cutaneous vasculature to facilitate transcutaneous O2 uptake (4). As a consequence of preferentially
redistributing the O2-enriched blood at the skin to the
hypoxia-sensitive central organs, the largely inactive
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PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
and hypoxia-tolerant skeletal muscle mass becomes
hypoperfused (4, 40). It appears, therefore, that the
skeletal muscle of overwintering frogs may become
severely hypoxic for prolonged periods during hibernation. Given that experiments on isolated frog muscle
show that step decreases in perfusate oxygen lead to
step decreases in muscle metabolic rate (3, 49) and
given that 35–40% of the frog’s body mass is skeletal
muscle, we can conclude that blood flow and therefore
O2 flow limitations to the oxyconforming muscle mass
probably serve to bring about a major proportion of the
overall reduction in whole animal metabolic rate (14).
The aim of this study was to test whether the metabolic depression observed in overwintering frogs is
reflected at the mitochondrial level either through
changes in respiration and/or mitochondrial O2 affinity. To do so, we examined the intrinsic properties of
respiration in mitochondria isolated from the skeletal
muscle of frogs at various stages of their hibernation.
MATERIALS AND METHODS
Animals. All animals used in these experiments were
adult male R. temporaria (⬃25–30 g) collected by a local
supplier (Blades Biological) during the winters of 1998 and
1999. At the start of each winter, frogs were acclimated to
3°C water for 4 wk, during which time they had direct access
to air. After this acclimation period, 15 frogs were then taken
for experiments (control groups) while another 30 were submerged in either normoxic water (PO2 ⫽ 155 mmHg; winter
1998) or hypoxic water (PO2 ⫽ 60 mmHg; winter 1999) in a
temperature-controlled recirculated water system (Living
Stream, Frigid Units, Cleveland, OH) maintained at 3°C, as
described previously (14). The normoxic and hypoxic submerged frogs were sampled after 1 and 4 mo.
Isolation of mitochondria. Frogs were killed by concussion
of the brain followed by its immersion in liquid nitrogen
according to Schedule 1 Home Office protocols (UK). Mitochondria were isolated according to a modification of the
methods from Hillman et al. (29). The thigh muscles of three
frogs were pooled for each mitochondrial preparation. Each
thigh muscle was excised rapidly and finely minced with
razor blades. The tissue was then placed in a beaker containing the isolation medium and further homogenized with
microscissors. The isolation medium contained (in mM) 170
mannitol, 55 sucrose, 5 EGTA, 20 HEPES, and 50 units/ml
heparin and 0.5% BSA adjusted to pH 7.3 at room temperature. The homogenate was transferred to a Potter-type glass
homogenizer and ground (200 rpm) consecutively with three
pestles of increasing size (2 strokes with each pestle). The
homogenate was then centrifuged at 755 g for 5 min, and the
supernatant was subsequently filtered through medical
gauze and centrifuged at 9,800 g for 11 min. The resulting
pellet was then washed with isolation medium lacking heparin, resuspended, and centrifuged at 9,800 g for 8 min. The
final pellet was resuspended in isolation medium lacking
heparin at a concentration of 25–30 mg of mitochondrial
protein/ml. Protein concentrations were determined by the
Biuret method (23), and sodium deoxycholate was added to
disrupt the membranes.
High-resolution respirometry. To measure oxygen consumption rates and the O2 affinity of isolated mitochondria,
we used a respirometer (Oroboros Oxygraph, Paar, Graz,
Austria) that enables sensitive measurements of oxygen kinetics at low oxygen partial pressure (22, 27). The air-equil-
ibrated assay medium contained (in mM) 55 mannitol, 24
sucrose, 10 HEPES, 10 K2HPO4, 90 KCl, and 0.5% BSA
adjusted to pH 7.3 at room temperature. The experiments
were carried out at 3 and 20°C. The main purpose of the
experiments carried out at 20°C was to facilitate comparisons
with previous data. The temperature of the Oxygraph was
regulated to ⫾0.05°C by a Peltier heat pump. The oxygen
solubility of the assay medium was considered to be 18.194
and 12.135 ␮M/kPa at 3 and 20°C respectively. Calibration of
the system, including signal correction for electrode response
time, blank controls, internal zero calibration as well as data
acquisition and analyses, was carried out as described elsewhere (22, 27, 37). The signals from the oxygen electrode
were recorded at 1-s intervals on a computer-driven data
acquisition system (DatLab software; Oroboros, Innsbruck,
Austria).
The oxygen concentration in the Oxygraph chamber was
reduced to ⬃20% of air saturation at each experimental
temperature by blowing nitrogen on the surface of the assay
medium before the addition of mitochondria at a final concentration of 1 mg mitochondrial protein/ml. First, malate
(1.2 mM) was added to spark the Krebs cycle followed by the
addition of pyruvate (5 mM). The respiration rate of mitochondria under these conditions is called state 2. The state 3
respiration rate was then obtained by the addition of 0.428
mM ADP. The mitochondria subsequently consumed all of
the oxygen and entered anoxia while still in state 3. After 10
min of anoxia, the oxygen concentration in the chamber was
raised again to roughly 20% of air saturation by lifting the lid
for a few seconds. The state 4 respiration rate was then
reached (state 4 is reached when all the ADP is phosphorylated into ATP) and was recorded until the mitochondria
entered a second anoxic period. The experimental protocol is
represented graphically in Fig. 1. The respiratory control
ratio (RCR) was calculated by dividing the state 3 respiration
rate by the state 4 respiration rate.
Calculations and statistical analyses. All data are presented as means ⫾ SE. Statistical analyses were performed
with SigmaStat (version 2.0). Comparisons of state 3 rates,
state 4 rates, state 3 O2 affinity (1/P50) values, state 4 P50
values, RCR values, and Q10 values between the three groups
of frogs for each year were performed with one-way ANOVA
and the a posteriori test of Tukey. Comparisons of state 3
rates, state 4 rates, state 3 P50 values, state 4 P50 values, and
RCR values between the two groups of control frogs used the
Student’s t-test. Comparisons of RCR values between experimental temperatures and of P50 values between state 4 and
state 3 rates were carried out with Student’s paired t-test.
The level of significance was P ⫽ 0.05.
RESULTS
General characteristics of mitochondria. For the experiments carried out in 1998 (normoxic hibernation),
there were no significant differences in the RCR values
of control, 1-mo-submerged, and 4-mo-submerged
groups of frogs at either of the experimental temperatures (Table 1). However, there was a general tendency
toward higher RCR values at 20°C compared with 3°C,
but this only reached statistical significance for the
1-mo-submerged group of frogs (P ⫽ 0.046; Table 1).
There was no difference in the Q10 values for state 3
and state 4 respiration rates between the three groups
of animals (Table 1).
In the experiments carried out during winter 1999
(hypoxic hibernation), there was no difference in the
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PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
Fig. 1. Graphic illustration of a sample trace of
mitochondrial respiration rates at 3°C. The same
protocol was applied at 20°C. Solid line, oxygen
concentration; dotted line, oxygen consumption
rate. M, mitochondria; m ⫹ p, malate and pyruvate.
RCR values at 3°C between the three groups of animals
(Table 2). However, the mitochondria isolated from
frogs submerged in hypoxic water for both 1 mo and 4
mo displayed lower RCR values at 20°C compared with
those obtained with the mitochondria isolated from the
control group (Table 2). There was no significant difference in the Q10 values for state 3 and state 4 respiration rates between the three groups of frogs (Table 2).
There were some differences between the two groups
of control frogs. The state 4 respiration rates and state
4 P50 values at 20°C were lower in winter 1999 compared with 1998 (Tables 3 and 4). The state 3 P50
values at 3°C were lower in winter 1998 than in 1999,
although it did not reach statistical significance (P ⫽
0.053; Figs. 2 and 4). Moreover, the RCR values at 20°C
were higher in winter 1999 compared with 1998 (Tables 1 and 2). These differences between the two groups
of control animals might be explained by the fact that
physiological parameters can vary considerably between
different groups of frogs brought into the laboratory (14,
15). For that reason, we decided to study a given cohort of
frogs for each year. However, we cannot exclude the
possibility that there were differences in the quality of
the mitochondrial preparations for the two groups of
control frogs that would contribute to, or maybe even
explain, the difference in RCR values obtained at 20°C.
Overall, the general characteristics of the mitochondria were mostly unaltered between the three experimental groups of frogs for both winter 1998 and winter
1999. In addition, the mitochondrial preparations were
of good quality, displaying RCR values above 4 at
almost every sampling period.
Respiration rates and O2 affinity of mitochondria
from normoxic submerged frogs. At 3°C, the active
(state 3) and resting (state 4) oxygen consumption
rates were unchanged between the control, 1-mo-submerged and 4-mo-submerged groups of frogs (Fig. 2).
The P50 values for state 3 and state 4 tended to decrease in the 1-mo-submerged frogs compared with
controls, but the tendency was significant only for the
state 3 P50 values (Fig. 2; P ⫽ 0.077 for P50 values in
state 4). The P50 values for state 3 and state 4 respiration rates from the 4-mo-submerged group were similar to those of the control group (Fig. 2). At 20°C, there
was no difference in the state 3 and state 4 rates or
their corresponding P50 values between the three
groups of animals (Table 3). At both experimental
temperatures and for each group of frogs, the P50
Table 1. RCR and Q10 values of frog skeletal muscle
mitochondria isolated from animals before and at
stages during hibernation in normoxic water at 3°C
Table 2. RCR and Q10 values of frog skeletal muscle
mitochondria isolated from animals before and at
stages during hibernation in hypoxic water at 3°C
RCR
Group
3°C
Control
1 mo
Submerged
4 mo
Submerged
5.0
Q10
20°C
RCR
State 3
State 4
Group
2.5 ⫾ 0.2 (4)
2.3
4.6 ⫾ 0.6 (4) 6.7 ⫾ 0.4* (5)
2.5 ⫾ 0.1 (5)
1.9 ⫾ 0.1 (4)
3.5 ⫾ 0.5 (5) 4.8 ⫾ 0.5 (5)
2.3 ⫾ 0.1 (5)
1.9 ⫾ 0.1 (5)
Control
1 mo
Submerged
4 mo
Submerged
5.6 ⫾ 1.4 (3)
Data are means ⫾ SE. The number of mitochondrial preparations
is indicated in parentheses. * Significant difference from the corresponding 3°C respiratory control ratio (RCR) value: P ⬍ 0.05 (Student’s paired t-test).
3°C
Q10
20°C
6.4 ⫾ 1.2 (4) 10.8 ⫾ 1.3 (5)
State 3
State 4
2.5 ⫾ 0.3 (4) 1.8 ⫾ 0.2 (4)
5.5 ⫾ 0.9 (5)
5.2 ⫾ 0.2* (4) 2.2 ⫾ 0.1 (4) 2.1 ⫾ 0.1 (3)
6.1 ⫾ 1.4 (4)
5.1 ⫾ 0.4* (4) 2.4 ⫾ 0.1 (4) 2.6 ⫾ 0.3 (4)
Data are means ⫾ SE. The number of mitochondrial preparations
is indicated in parentheses. * Significant difference from the corresponding control group: P ⬍ 0.05 (1-way ANOVA and a posteriori
test of Tukey).
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PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
Table 3. Oxygen consumption rate and O2 affinity of skeletal muscle mitochondria from frogs hibernating in
normoxic water under resting (state 4) and active (state 3) conditions at 20°C
Oxygen Consumption Rate,
nmol O 䡠 min⫺1 䡠 mg mitochondrial protein⫺1
P50, kPa
Group
State 3
State 4
State 3
State 4
Control
1 mo Submerged
4 mo Submerged
50.38 ⫾ 10.29 (5)
42.65 ⫾ 11.74 (5)
35.51 ⫾ 7.81 (5)
9.91 ⫾ 1.98 (3)
6.23 ⫾ 1.65 (5)
7.69 ⫾ 1.92 (5)
0.0521 ⫾ 0.0064 (5)
0.0398 ⫾ 0.0064 (5)
0.0509 ⫾ 0.0075 (5)
0.0237 ⫾ 0.0043 (3)
0.0136 ⫾ 0.0010 (5)
0.0151 ⫾ 0.0026 (5)
Data are means ⫾ SE. The number of mitochondrial preparations is indicated in parentheses.
values were lower in the resting state compared with
the active state, but this difference did not reach statistical significance for the group of control frogs at
20°C (Fig. 2 and Table 3).
The kinetics of resting (state 4) and active (state 3)
oxygen consumption rates at 3°C of frog mitochondria
from all groups of frogs in the low oxygen range are
illustrated in Fig. 3. The decrease in state 3 and state
4 P50 values observed in the 1-mo-submerged frogs is
illustrated in Fig. 3 as a shift of the state 3 and state 4
respiration curves to the left compared with the control
and 4-mo-submerged ones (see Fig. 3, insets).
Respiration rates and O2 affinity of mitochondria
from hypoxic submerged frogs. After 1 and 4 mo of
hypoxic submergence at 3°C, the active and resting
oxygen consumption rates were both reduced by ⬃60%
compared with controls (Fig. 4). This decreased active
respiration rate in mitochondria from the hibernating
frogs was paralleled by a ⬃60% diminution of the state
3 P50 value at 1 and 4 mo compared with controls (Fig.
4). On the other hand, there were no significant differences observed for the P50 values at state 4 among the
three different groups of animals (Fig. 4). However, the
state 4 P50 values obtained at 3°C were extremely low
(⬃0.0034 kPa) and probably near the limit of detection
of the apparatus, thus making it difficult to rule out
any possible decrease in mitochondrial P50 during submergence. At 20°C, the state 3 and state 4 respiration
rates were reduced by ⬃70 and ⬃30%, respectively, in
both the 1- and 4-mo-submerged groups compared with
controls (Table 4). Moreover, the state 3 P50 values of
the two submerged groups were ⬃75% lower than the
control values (Table 4). In contrast, the state 4 P50
value after 4 mo of hibernation was higher than those
of both the control and 1-mo-submerged groups of frogs
(Table 4). As was the case for normoxic hibernation,
the P50 values were lower in the resting state compared
with the active state at both experimental temperatures and for each group of frogs, but this difference did
not reach statistical significance for the 1- and 4-mosubmerged groups of frogs at 3°C and for the 4-mosubmerged group of frogs at 20°C (Figs. 4 and Table 4).
The kinetics of mitochondrial state 4 and state 3
respiration rates in the low oxygen range during hypoxic hibernation are represented in Fig. 5. The
matched decrease in state 3 P50 values and state 3
respiration rates for the two groups of hibernating
animals is shown in Fig. 5A, inset, and illustrates that
the shift in P50 values toward higher affinity is much
greater in the animals submerged in hypoxic water
(Fig. 5) than in those submerged in normoxic water
(Fig. 3). Figure 5 also reveals that the mitochondrial
respiration rates of mitochondria from hypoxic submerged frogs are metabolically depressed in both the
resting and active states with respect to control frogs at
any given intracellular PO2.
In conclusion, the mitochondria from frogs submerged in hypoxic water display more profound intrinsic changes in P50 values and respiration rates compared with mitochondria from frogs submerged in
normoxic water.
DISCUSSION
The results presented in this paper show for the first
time that an increase in the in vitro O2 affinity of
mitochondria can occur after chronic in vivo exposure
to cellular hypoxia. Moreover, the results demonstrate
that metabolic depression at the whole animal level
can be reflected at the mitochondrial level. Taken together, these findings illustrate the plasticity of mitochondria under physiological constraints.
The moderate metabolic depression at the whole
animal level in frogs submerged in normoxic water (15)
Table 4. Oxygen consumption rate and O2 affinity of skeletal muscle mitochondria from frogs hibernating in
hypoxic water under resting (state 4) and active (state 3) conditions at 20°C
Oxygen Consumption Rate,
nmol O 䡠 min⫺1 䡠 mg mitochondrial protein⫺1
P50, kPa
Group
State 3
State 4
State 3
State 4
Control
1 mo Submerged
4 mo Submerged
56.40 ⫾ 7.55 (5)
15.66 ⫾ 1.16* (4)
20.60 ⫾ 1.38* (4)
5.20 ⫾ 0.25 (5)
3.04 ⫾ 0.33* (5)
4.12 ⫾ 0.26* (4)
0.1028 ⫾ 0.0266 (5)
0.0303 ⫾ 0.0050† (4)
0.0257 ⫾ 0.0041† (4)
0.0094 ⫾ 0.0012 (5)
0.0120 ⫾ 0.0008 (4)
0.0174 ⫾ 0.0013‡ (4)
Data are means ⫾ SE. The number of mitochondrial preparations is indicted in parentheses. * Significant difference from the respiration
rate of the corresponding control group; P ⬍ 0.05 (1-way ANOVA and a posteriori test of Tukey). † Significant difference from the P50 of the
corresponding control group; P ⬍ 0.05 (1-way ANOVA and a posteriori test of Tukey). ‡ Significant difference from the P50 of the
corresponding 1-mo submerged group; P ⬍ 0.05 (1-way ANOVA and a posteriori test of Tukey).
PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
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Fig. 2. Oxygen consumption rate and P50 of mitochondria under resting (state 4) and active (state 3)
conditions at 3°C for normoxic submerged animals.
Squares, state 4 rates, circles, state 3 rates. The
control, 1-mo-submerged and 4-mo-submerged
groups of frogs are represented by a solid, open, and
shaded filling, respectively. Data are means ⫾ SE.
The number of mitochondrial preparations is indicated in parentheses. * Significant difference from
the P50 of the control group; P ⬍ 0.05 (1-way
ANOVA and a posteriori test of Tukey). ** Significant difference from the P50 of the 1-mo-submerged
group; P ⬍ 0.05 (1-way ANOVA and a posteriori test
of Tukey).
Fig. 3. Kinetics of frog mitochondrial oxygen consumption rates in the low oxygen
range at 3°C for normoxic submerged animals. The kinetics for state 3 (A) and
state 4 (B) are shown. Solid line, control
(C); dashed line, 1-mo-submerged; dotted
line, 4-mo-submerged groups of frogs. The
insets show the P50 for each group of
frogs. These curves were generated using
the equation: V̇O2 (%max)⫽[PO2/(P50 ⫹
PO2)]*100 (21), where V̇O2 represents the
oxygen consumption rate for state 3 or
state 4. The state 3 and state 4 values from
the control group of frogs were assigned to
100%, and the ones obtained from the 1and 4-mo-submerged groups of frogs were
expressed relative to the controls. The
variability in state 3 rates, state 4 rates,
state 3 P50 values and state 4 P50 values is
presented in Fig. 2.
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PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
Fig. 4. Oxygen consumption rate and P50 of mitochondria under resting (state 4) and active (state 3)
conditions at 3°C for hypoxic submerged animals.
Squares, state 4 rates; circles, state 3 rates. The
control, 1-mo-submerged and 4-mo-submerged
groups of frogs are represented by solid, open, and
shaded fillings, respectively. Data are means ⫾ SE.
The number of mitochondrial preparations is indicated in parentheses. * Significant difference from
the P50 of the control group; P ⬍ 0.05 (1-way
ANOVA and a posteriori test of Tukey). † Significant
difference from the respiration rate of the control
group; P ⬍ 0.05 (1-way ANOVA and a posteriori test
of Tukey).
Fig. 5. Kinetics of frog mitochondrial oxygen consumption rates in the low oxygen
range at 3°C for hypoxic submerged animals. The kinetics for state 3 (A) and state
4 (B) are shown. Solid line, control; dashed
line, 1-mo-submerged; dotted line, 4-mosubmerged groups of frogs. The insets
show the P50 for each group of frogs. These
curves were generated using the equation
V̇O2 (%max) ⫽ [PO2/(P50 ⫹ PO2)]*100 (21),
where V̇O2 is the oxygen consumption rate
for state 3 or state 4. The state 3 and state
4 values from the control group of frogs
were assigned to 100%, and the ones obtained from the 1- and 4-mo-submerged
groups of frogs were expressed relative to
the controls. The variability in state 3
rates, state 4 rates, state 3 P50 values, and
state 4 P50 values is presented in Fig. 4.
PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
was not reflected at the mitochondrial level (Fig. 3). In
fact, the active and resting respiration rates of mitochondria stayed more or less constant throughout normoxic submergence. However, the mitochondria from
the 1-mo-submerged group of frogs showed an increase
in their state 3 O2 affinity. This increase in O2 affinity
would act to promote, rather than suppress, aerobic
metabolism in a microoxic intracellular environment
(Fig. 3A, inset). However, when metabolic depression
becomes fully developed after 4 mo of submergence, the
mitochondrial state 3 P50 value reverts to the level
observed in control animals (Fig. 3). The corresponding
state 4 results (respiration rate and P50) reveal similar
trends without reaching statistical significance (Fig.
3B, inset).
The increase in mitochondrial O2 affinity after 1 mo
of submergence in normoxic water might not be physiologically relevant. In fact, we have no indication to
believe that the intracellular PO2 inside frog skeletal
muscle during normoxic submergence are around mitochondrial state 3 or state 4 P50 values. Frogs submerged in normoxic water do not display an increase in
plasma or skeletal muscle lactate levels, thereby indicating that blood supply (i.e., oxygen supply) to the
skeletal muscle, despite being drastically reduced, is
sufficient to maintain aerobic metabolism (15). Moreover, the ATP, ADP, AMP, phosphocreatine, and creatine levels inside the skeletal muscle stay constant
throughout normoxic submergence, indicative of a balance between ATP supply and demand (15).
The O2-dependent properties of mitochondria isolated from hypoxic animals show a more profound
reorganization of function. For example, mitochondria
isolated from frogs submerged in hypoxic water for 1
and 4 mo display reduced resting and active respiration rates and thus metabolic rate at any given intracellular PO2 (Fig. 5). The mechanism responsible for
the decrease in mitochondrial state 4 respiration rates
during submergence in hypoxic water is a reduction in
the activity of the electron transport chain (45). This
reduction in electron transport chain activity is at least
partly, and might be even entirely, responsible for the
decrease in state 3 respiration rates. However, it is also
possible that a reduction in the activity of the phosphorylation system plays a role in the decrease of
mitochondrial state 3 respiration rates. The intracellular PO2 inside frog skeletal muscle during hypoxic
submergence are probably around mitochondrial state
3 and state 4 P50 values and even lower. In fact, frogs
recruit anaerobic metabolism during the first 2 mo of
submergence in hypoxic water as indicated by the
marked increase in plasma lactate concentration (14).
A large proportion of the increase in plasma lactate
concentration is thought to come from the hypoperfused muscle mass (13). The plasma lactate concentration decreases steeply after 1 mo of submergence in
hypoxic water and returns to presubmergence values
after 2 mo when the metabolic rate of the frog is
dramatically reduced compared with controls (14). In
fact, the metabolic depression after 2 mo of submergence is so profound that the energetic needs can now
be met through an entirely aerobic metabolism (14).
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The reduction in the resting and active respiration
rates of frog skeletal muscle mitochondria during hypoxic submergence will lead to a decrease in the rate of
ATP production. Even so, the rate of ATP consuming
processes seems to be reduced accordingly, as indicated
by the maintenance of ATP, phosphocreatine, and
creatine levels similar to controls throughout hypoxic submergence (14). However, there is an increase
in the adenylate energy charge inside the skeletal
muscle during hypoxic submergence owing to significant decreases in ADP and AMP levels (14). This might
indicate lowered ATP demand by the skeletal muscle.
In fact, the activity of the skeletal muscle Na⫹-K⫹ATPase is reduced by 50% during hibernation in hypoxic water (16). The profound metabolic depression
observed in frogs after 2 mo of submergence also has
the advantage of reducing the rate of depletion of
glycogen inside the skeletal muscle (14). Overall, the
steady-state metabolic depression at the whole animal
level during hypoxic submergence (14) is reflected at
the cellular level by a maintenance of energy balance;
a situation similar to that during normoxic hibernation.
Modifications in mitochondrial properties also occur
in mammals during chronic exposure to hypoxia. Hypoxia in mammalian cells is often correlated with a
reduction in cytochrome levels and mitochondrial enzyme activities. In addition, respiration rates decrease
in brain mitochondria of rats and mice exposed to
intermittent hypobaric hypoxia (10, 17, 35, 38). However, the resting and active respiration rates of liver
and heart mitochondria isolated from rats acclimatized
to hypobaric hypoxia did not differ from those of control
rats (11), not even when measured at more physiological low oxygen concentrations (12). Previous studies
concerning the affinity of cells and of mitochondria for
oxygen have produced contrasting results. On the one
hand, studies carried out on liver mitochondria isolated from hypoxic and control rats revealed no change
in P50 values (12, 32). On the other hand, the cellular
P50 values for hepatocytes isolated from hypoxic rats
were lower than those from control rats (32). Such
changes in cellular P50 values have been ascribed to a
redistribution of mitochondria within the cell (11; reviewed in Ref. 32). Similarly, the P50 values of rat
mitochondria isolated from hypoxia-tolerant newborns
are not different than those of their hypoxia-sensitive
adults, whereas hepatocytes isolated from newborn
rats manifest lower P50 values than those of their adult
counterparts (1). Again the differences in cellular P50
values were ascribed to differences in the density and
distribution of mitochondria within the cellular network (32). The differences in mitochondrial and cellular responses to hypoxia in the ectotherm and
mammal may reflect fundamental differences in their
tolerance to hypoxia and/or their relative capacity to
exploit metabolic suppression at the whole animal
level.
Entering into a new viable hypometabolic state implies that energy supply remains balanced with energy
demand. Many studies have looked at cellular adaptations, focusing mainly on the energy demand processes
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PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
that can be turned down during metabolic depression
(reviewed in Refs. 24, 26, 28, 30). However, few studies
have looked at the intrinsic properties of mitochondria
during hypometabolic states. From the information
available, we know that mitochondrial state 3, but not
state 4, respiration rates are reduced in hibernating
mammals (7–9, 19, 34, 36, 39). We also know that
respiration rates of hepatocytes isolated from hibernating ground squirrels are the same as the rates obtained
from hepatocytes isolated from summer “cold-acclimated” animals (41). Indeed, there is no evidence that
state 4 rates become altered during hibernation in
mammals. With regard to ectotherms, citrate synthase
(CS) activities are unchanged during metabolic depression in most tissues of the terrestrial snail, with the
exception of the hepatopancreas (47). Similarly, cytochrome c oxidase activity is considerably reduced in
the hepatopancreas of estivating snails compared
with control animals (46). Also, mitochondrial protein
synthesis is markedly decreased during anoxia-induced quiescence in brine shrimp embryos (33). Other
studies have shown intrinsic reductions in metabolic
rate at the tissue level (18) and at the cellular level
(25). The present study demonstrates metabolic depression at the mitochondrial level (Fig. 5), supporting
the view that hypometabolism can be reflected at all
levels of biological organization in hypoxia-tolerant
animals.
This study reports state 3 and state 4 P50 values
from an ectotherm using high-resolution respirometry.
The average state 3 and state 4 P50 values for the two
control groups of frogs are ⬃0.077 and 0.017 kPa,
respectively, at 20°C. These values are similar to the
state 3 and state 4 P50 values of rat liver mitochondria
(0.057 and 0.020 kPa) and rat heart mitochondria
(0.035 and 0.016 kPa) at 30°C (20). The mitochondrial
P50 values for all groups of frogs increase during transition from a resting state (state 4) to an active one
(state 3). Other studies have shown similar increases
in P50 values in active states in isolated mitochondria
(12, 20, 48). The present study shows that state 3 and
state 4 P50 values can change with metabolic rate. By
varying the mitochondrial concentration in their experimental chamber, Steinlechner-Maran et al. (42)
noted an incidental positive correlation between respiration rate and P50 in endothelial cells. However, the
P50 values of isolated mitochondria were shown to be
constant at various mitochondrial concentrations in
the same experimental chamber as we used (20). This,
in conjunction with the fact that we carried out all our
experiments at the same concentration of mitochondria
(see MATERIALS AND METHODS), supports the conclusion
that the parallel decrease in state 3 respiration rates
and state 3 P50 values during hypoxic submergence
(Fig. 5) was due to different active metabolic states of
the mitochondria.
There is an inverse relationship between the turnover rate of cytochrome c oxidase and the O2 affinity of
mitochondria that can explain the differences in P50
values between state 3 and state 4 respiration rates
and between different types of mitochondria (21). The
turnover rate of cytochrome c oxidase is reduced under
state 4 conditions, which leads to a decrease in P50
value compared with state 3 conditions. Rat heart
mitochondria have a higher excess capacity of cytochrome c oxidase compared with rat liver mitochondria, which leads to a lower turnover rate of cytochrome c oxidase at high flux through the electron
transport chain (state 3) by distributing electron input
flux through a higher number of enzymes. This result
correlates with the higher O2 affinity of heart mitochondria compared with liver mitochondria under
state 3 conditions (21).
Perspectives
During submergence in normoxic water, the state 3
P50 values were lower in 1-mo-submerged frogs compared with control and 4-mo-submerged groups of animals, whereas respiration rates stayed more or less
constant throughout submergence (Fig. 3A). This result suggests an increase in the catalytic efficiency
[maximal velocity (Vmax)/P50] of mitochondria from the
1-mo-submerged group of frogs compared with control
and 4-mo-submerged groups of frogs. On the other
hand, the diminution in the active respiration rates
during submergence in hypoxic water was proportional
to the decrease in state 3 P50 values, indicating that
mitochondria from frogs hibernating in hypoxic water
maintain the same catalytic efficiency as the control
frogs (Fig. 5A). The ratio Vmax to P50, which is based on
the Kcat-to-Km ratio used for isolated enzymes, has
been used in the past as an indicator of the catalytic
efficiency of mitochondria when using a given type of
mitochondria (for a review, see Ref. 21). However,
because we are comparing mitochondria from animals
in different metabolic states, we must interpret this
result cautiously. In fact, the values we measured are
a complex result of different factors (e.g., substrate and
electron transport capacities as well as cytochrome c
oxidase concentration), which can vary between the
three groups of frogs. It will be interesting to see if
these modifications in intrinsic properties of mitochondria during hibernation in frogs are accompanied by
alterations in the number or distribution of mitochondria within the muscle fibers. Mitochondrial and cellular changes could act in concert in the skeletal muscle
of frogs to bring about important functional modifications in face of severe hypoxia. We know very little
about the properties of mitochondria during metabolic
depression. Further studies are required to gain a
better understanding of the role that mitochondria
play in the development of hypometabolic states.
The authors thank Dr. T. G. West and Dr. M. D. Brand for critical
reading of the manuscript.
This work was funded by the Natural Environment Research
Council. J. St-Pierre was recipient of Natural Sciences and Engineering Research Council of Canada (NSERC), Fonds pour la formation
de chercheurs et l’aide à la recherche and Trinity College (UK)
scholarships. G. J. Tattersall was recipient of a 1967 Centennial
NSERC scholarship.
Present address of G. J. Tattersall: Department of Biological
Sciences, Kent State University, Kent, OH 44242
PROPERTIES OF MITOCHONDRIA DURING HYPOXIC HYPOMETABOLISM
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