Adaptive plasticity of skeletal muscle energetics in hibernating frogs: mitochondrial proton leak during metabolic depression
1 Department of Zoology, Downing Street, University of Cambridge, Cambridge
CB2 3EJ, UK
2 MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY,
UK
Present address: Dana-Farber Cancer Institute, One Jimmy Fund Way, SM958,
Boston, MA 02115, USA
* e-mail: rgb11{at}hermes.cam.ac.uk
Accepted 28 May 2002
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Summary |
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Key words: frog, skeletal muscle, mitochondria, proton leak, hibernation, hypoxia, oxyconformation, metabolic depression, Rana temporaria
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Introduction |
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The frog's skeletal muscle is thought to be the most important tissue
contributing to the overall metabolic depression during hibernation because
(i) it makes up the largest proportion of the animal's body mass and (ii) its
metabolic rate conforms to O2 availability such that reduced blood
supply leads to a marked suppression of the rate of oxygen consumption
(Fig. 1). During hibernation,
cold-submerged frogs drastically reduce the blood supply to their skeletal
muscle in order to shunt more blood to the skin for the extraction of oxygen
(Boutilier et al., 1986,
1997
;
Pinder et al., 1992
). Given
that the metabolic rate of isolated frog skeletal muscle decreases with
perfusate oxygen concentration (i.e. the oxyconformation response as shown in
Fig. 1) and that 35-40% of the
frog's body mass is skeletal muscle, the reduction in blood supply (and
therefore O2 supply) to the skeletal muscle mass leads to a
considerable decrease in whole-animal metabolic rate
(Donohoe and Boutilier, 1998
).
The advantage of a metabolic depression during hibernation is that it slows
the rate of utilisation of `on-board' fuels until the environmental conditions
are more favourable and activity can be resumed. Since the mitochondrion is
the major contributor to total energy production during aerobic metabolism and
frog survival over winter depends critically on entry into a hypometabolic
state, the question we asked is whether overwintering frogs will produce
changes in the properties of their mitochondria to preserve or increase their
efficiency of energy production. As very few studies have looked at the
intrinsic properties of mitochondria during metabolic depression, our main
objective was to examine the role that mitochondria play in the development of
hypometabolic states.
|
Recent estimates indicate that 20% of the standard metabolic rate (SMR) in
rats can be accounted for by an intracellular futile cycle of proton pump and
leak across the mitochondrial inner membrane (Brand et al.,
1994,
2000
;
Rolfe and Brand, 1996
;
Rolfe et al., 1999
). This
`proton cycling' or `proton leak' partially uncouples oxygen consumption from
ATP synthesis, thereby leading to less effective energy conservation. When
isolated mitochondria are operating at maximal rates of oxygen consumption in
the presence of saturating amounts of substrate and ADP (i.e. so-called `state
3' respiration), the proportion of respiration used to drive proton cycling is
of the order of 10% (Brand et al.,
2000
). However, in the absence of any ATP production (i.e. when
all the ADP has been used or when the F1Fo-ATPase has
been blocked with the highly specific inhibitor oligomycin), all of the low
residual respiration (state 4) drives the proton leak. Intact cells and
tissues are almost certainly operating closer to `state 4' conditions at SMR,
and this is presumably also the case at minimal metabolic rates (MMRs).
Mitochondrial proton cycling makes up approximately 20% of the cellular
respiration rates of the hepatocytes of the frog, the bearded dragon (reptile)
and the rat and of the hepatopancreas cells of the snail (Brand et al.,
1991
,
2000
;
Bishop and Brand, 2000
;
Bishop et al., 2002
). Thus, the
amount of energy dissipated by proton cycling appears to be very similar
across phylogenetic lines. If proton cycling were to make up 20% of the
standard metabolic rate of an animal such as the frog, which can depress its
metabolic rate by 75%, one could argue that proton cycling would have to be
decreased during metabolic depression. Otherwise, proton cycling would
dominate the residual oxygen consumption, and the efficiency of mitochondrial
energy conservation would fall towards zero. This would effectively negate
most, if not all, of the energy-sparing advantages associated with entry into
a hypometabolic state.
Adenosine triphosphate (ATP) is often referred to as the energy currency of the cell because it is the price to pay to carry out the vast majority of cellular transactions. Most organisms rely on mitochondrial metabolism (aerobic metabolism) to produce energy in the form of ATP. The capacity for producing ATP aerobically can vary throughout an animal's life history according to changes in body mass and/or age as well as in response to environmental conditions such as temperature, oxygen levels and food intake. Modifications in the aerobic capacity of an organism can occur at different levels of the O2 cascade, representing different levels of biological organisation. In the face of acute environmental stress, first lines of physiological defence take place within seconds in order to provide immediate compensation for any adverse effects on cellular functions. If the environmental insult persists, however, more profound changes at the molecular level may be needed to provide for a reorganisation of cellular metabolism. Prolonged physiological stresses, such as those seen during overwintering, can lead to marked changes in the aerobic capacity at the cellular level either (i) by altering the number of mitochondria or (ii) by changing the intrinsic properties of the mitochondria.
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The contribution of mitochondrial proton cycling to standard metabolic rate |
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The extent to which proton leak uncouples cellular ATP synthesis determines
quantitatively the number of moles of ATP produced per mole of oxygen consumed
(i.e. the so-called P/O ratio). To obtain a realistic P/O value, it is
necessary to compute an `effective' P/O ratio by multiplying the `mechanistic'
P/O ratio of the mitochondrial ATP-synthesizing reactions (estimated to be 2.5
for NADH substrate; Rolfe and Brown,
1997) by the fraction of the total tissue O2
consumption rate that is used to drive mitochondrial ATP synthesis. Given that
non-mitochondrial respiration accounts for 10% of mammalian SMR and proton
leak for 20% of SMR, Rolfe and Brown
(1997
) arrive at an effective
whole-body P/O value of 1.8 (i.e. 0.7x2.5). Even lower values of
effective P/O are found in individual tissues. For example, recent estimates
of the contribution of oxidative phosphorylation to resting oxygen consumption
in isolated rat hepatocytes (69%) and perfused hindlimb (57%), predict
effective P/O ratios of 1.7 (i.e. 0.69x2.5) and 1.4 (0.57x2.5) for
liver and skeletal muscle, respectively
(Rolfe et al., 1999
).
On the face of it, proton leak would therefore seem to make cellular
metabolism at SMR rather inefficient, and intense efforts are under way to
discover the functional significance of this so-called `futile cycle'. A
number of physiological functions for proton leak have been proposed, namely
(i) heat generation, (ii) increasing the sensitivity of the oxidative pathway
to effectors, (iii) reducing the rate of production of reactive oxygen species
and (iv) regulation of carbon flow (Rolfe
and Brand, 1997). It is unlikely that the main role of proton leak
is heat generation since ectotherms ranging from snails to lizards seem to
devote the same proportion of their cellular respiration to drive proton leak
as do mammals (Brand et al.,
1991
,
2000
;
Bishop and Brand, 2000
).
It is well known that mitochondrial proton leak in brown adipose tissue is
catalysed by uncoupling protein 1 (UCP1), which diverts energy from ATP
synthesis to heat generation (see Brand et
al., 1999). The more recent discovery of homologues of UCP1,
namely UCP2 and UCP3, has engendered debate as to their probable role in the
regulation of energy balance (Ricquier and
Bouillaud, 2000
). UCP2 is widely expressed in a number of
mammalian organs, whereas UCP3 is mainly expressed in skeletal muscle. Despite
their widespread expression in mammalian tissues, there is little evidence
that their function is tied primarily to thermogenesis. Indeed, the occurrence
of UCP2 and UCP3 in ectotherms (Stuart et
al., 2001
) suggests that functions other than heat generation may
have provided the initial selection advantages for the early evolution of this
class of proteins (Hochachka and Somero,
2002
). Recent evidence indicates that UCPs may play an important
role in decreasing the mitochondrial production of reactive oxygen species
(ROS) such as superoxide (Ricquier and
Bouillaud, 2000
). Greater uncoupling would facilitate increased
rates of electron transfer to molecular oxygen, thereby preventing any back-up
of electrons in the electron-transport chain
(Hochachka and Somero, 2002
).
This would effectively reduce the lifetimes of ROS-generating centres in the
electron-transport chain and ameliorate oxidative damage. Indeed, Echtay et
al. (2002
) have recently shown
that superoxide-induced increases in mitochondrial proton conductance are
effected through UCPs. Speculation continues to be rife as regards the
evolution and adaptive significance of the mitochondrial UCPs.
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Mitochondrial proton leak in metabolic depression |
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If the proton leak rates we have measured in frog skeletal muscle
mitochondria (Fig. 2) were to
produce in intact muscle as large an uncoupling reaction as in mammals
(Rolfe and Brand, 1996;
Rolfe et al., 1999
), the sheer
size of the skeletal muscle mass would make a reduction in proton leak during
deep hypoxic hibernation almost essential. The reason being that, with a 75 %
reduction in whole-animal oxygen consumption during deep hibernation
(Fig. 3A), almost all the
remaining energy budget would have to serve the proton leak pathway at a time
when one might predict that metabolic efficiency needed to be maintained or
even enhanced. For example, whereas the skeletal muscle mass of rat (42 % of
total body mass) makes up 30-40 % of its SMR
(Rolfe and Brand, 1996
), the
35 % fractional muscle mass of the aestivating frog Neobatrachus
kunapalari accounts for 50-65 % of its SMR and up to 85 % of its MMR
during deep metabolic depression (Flanigan
and Guppy, 1997
). With the liver making up an additional 5 % of
the SMR in this frog (Flanigan et al.,
1991
), fully 70 % of the SMR could be accounted for by these two
tissue types and perhaps up to 90 % of the MMR in hypometabolic states. Any
reduction in mitochondrial proton cycling during hibernation would have the
effect of conserving substrate and extending survival time. Thus, to preserve
the efficiency of their aerobic energy production during metabolic depression,
overwintering frogs would have to decrease their proton cycling by the same
proportion as cellular respiration. In the absence of a downregulation of
proton leak rate, hibernating frogs at MMR would be in the undesirable
position of burning more substrates than control animals to produce equivalent
amounts of ATP.
|
|
To test whether proton cycling might be downregulated as a part of a
coordinated response to energy conservation during metabolic depression, we
measured the proton leak rate of mitochondria isolated from the thigh
musculature of frogs at different stages of hibernation
(St-Pierre et al., 2000a),
when whole-animal MMRs are 50-75% lower than SMR
(Fig. 3A;
Donohoe and Boutilier, 1998
).
Although the proton leak rates of isolated skeletal muscle mitochondria were
unaltered in fully aerobic frogs (when MMR is 50% of SMR), those isolated from
animals in chronic hypoxia (when MMR is only 25% of SMR) exhibited a 50%
reduction in their state 4 respiration rate
(Fig. 2).
If the state 4 respiration rates of isolated mitochondria are similar to the respiration rates of mitochondria in intact resting muscle, these results suggest that the proton leak rates of mitochondria in vivo may be reduced during hypoxic submergence. The reduction in proton leak rate during hibernation (Fig. 2) was caused by a decrease in the activity of the electron-transport chain and not by a reduction in the proton conductance of the mitochondrial inner membrane. Indeed, the proton conductance of frog mitochondria was unaltered throughout normoxic and hypoxic submergence. This is illustrated by the fact that there were no significant differences between the respiration rates at any given membrane potential (Fig. 2). However, the state 4 respiration rate achieved by mitochondria from metabolically depressed animals was only 50% of control, and the state 4 membrane potential was also lower, showing that the rates of substrate oxidation were significantly downregulated. Overall, these results suggest that overwintering frogs effectively preserve their metabolic efficiency by reducing mitochondrial proton cycling in parallel with metabolic rate (Fig. 3) and that they do so by decreasing the rate of substrate oxidation and the size of the proton-motive force rather than by decreasing the proton conductance of the mitochondrial membrane.
One might ask how realistic it is for us to expect that mitochondria
isolated in vitro should function the same way as in vivo?
This same question was posed by Bishop and Brand
(2000), who developed an
isolated hepatopancreas cell preparation from the snail Helix aspersa
after earlier studies revealed chronically lower respiration rates of
mitochondria isolated from the kidney of aestivating animals
(Brand et al., 2000
). As was
the case for hibernating frogs, the lower rates of oxygen consumption in
mitochondria isolated from aestivating compared with awake animals were caused
by a decrease in substrate oxidation, not by a decrease in proton conductance
(Brand et al., 2000
;
St-Pierre et al., 2000a
).
Bishop and Brand (2000
) found
that the respiration rate of hepatopancreas cells isolated from aestivating
snails was approximately 30% of that of awake snails. This cellular metabolic
depression was brought about by proportional decreases in mitochondrial and
non-mitochondiral respiration rates. Further investigation of the primary and
secondary causes of the decreased mitochondrial respiration led to studies in
which cellular respiration could be experimentally divided between the
producers of the proton-motive force (substrate oxidation) and the processes
that consume the proton-motive force (i.e. proton leak and ATP turnover).
Bishop et al. (2002
) estimate
that 75% of the total response of mitochondrial respiration to aestivation
occurs through primary changes in the kinetics of substrate oxidation, the
remaining 25% or less being attributed to ATP turnover (proton leak remains
constant). These primary changes resulted in a lower mitochondrial membrane
potential, which led to secondary decreases in the cellular respiration rates
driving ATP turnover and proton leak
(Bishop et al., 2002
). Thus,
both in the isolated mitochondria from hibernating frogs
(Fig. 2) and in the
hepatopanceas cells of aestivating snails, decreases in the rate of substrate
oxidation result in a fall in membrane potential
(Fig. 2;
Bishop et al., 2002
), which
decreases proton leak rates during metabolic depression. As the kinetics of
proton leak did not change in either case of metabolic depression, changes in
proton conductance of the inner mitochondrial membrane do not play a primary
role in the response of mitochondrial respiration to metabolic depression.
Similarities in the metabolic responses of isolated cells and isolated
mitochondria give some credence to our earlier suggestion that the results
obtained using mitochondria isolated from the skeletal muscle of hibernating
frogs may reflect the metabolic responses of intact tissue.
Mitochondrial proton leak during anoxia
The reduction in blood flow to the frog skeletal muscle during
overwintering submergence may also lead to transient periods of ischaemia. In
the absence of oxygen, the mitochondrial F1Fo-ATPase
(the ATP synthase) begins to run backwards as it actively pumps protons from
the matrix in an attempt to maintain the mitochondrial membrane potential. In
this state, mitochondria change from being ATP producers to potentially
powerful ATP consumers (Lisa et al.,
1998). Animals that can withstand anoxia for considerable periods
must have very efficient ways to reduce ATP utilisation by this pathway. If
not, most of the ATP generated by glycolysis could be needed to fuel that one
process alone. Thus, when O2 supplies are exhausted, the
proton-motive force is generated by the F1Fo-ATPase
rather than by the electron-transport chain. Our studies on mitochondria
isolated from frog skeletal muscle have shown that the proton leak is reduced
by a slowing-down of the F1Fo-ATPase and not by a
decrease in the proton conductance of the inner membrane
(Fig. 4;
St-Pierre et al., 2000b
;
Boutilier and St-Pierre, 2000
).
This mechanism is analogous to that observed during hibernation in hypoxia
(St-Pierre et al.,
2000a
,c
).
Thus, whether in the presence or absence of oxygen, the proton leak of
mitochondria isolated from the skeletal muscle of frog is decreased by a
reduction in the rate of the appropriate producer of the proton-motive
force.
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Adaptive increases in the O2-affinity of cells and mitochondria during metabolic depression |
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A number of studies on mammalian tissues have questioned whether the
P50 of mitochondria can be reduced as an adaptation to
hypoxia (i.e. effectively expanding the range over which the rate of
mitochondrial respiration would be independent of
PO2). The results of such studies show that,
while mitochondrial O2-affinity is unchanged during hypoxia,
cellular O2-affinity actually increases. The increased
O2-affinity at the cellular level has been ascribed to a
redistribution of mitochondria within the cells (for a review, see
Jones et al., 1991). We were
interested to determine whether the skeletal muscle mitochondria from
metabolically depressed frogs would modulate their O2-affinity at
the low PO2 levels that characterise the
hypoperfused muscle of hibernating frogs. To this end, we measured the active
(state 3) and resting (state 4) respiration rates of mitochondria isolated
from the skeletal muscle of frogs hibernating for up to 4 months.
When frogs were hibernating in normoxic water, no significant differences
occurred in either the resting or active respiration rates of their skeletal
muscle mitochondria. Although we have no direct estimates of skeletal muscle
PO2 in hibernating frogs, there is no
indication that tissue PO2 levels ever reach
mitochondrial state 3 or state 4 P50 values during
normoxic hibernation (St-Pierre et al.,
2000c). Cutaneous gas exchange during normoxia is evidently
sufficient to maintain all of the animals' aerobic metabolic requirements for
periods of up to 4 months; i.e. haemoglobin O2-saturation remains
high, there is no build-up of lactate, high-energy phosphate levels remain
stable and there is only modest use of glycogen reserves in liver and muscle
(Donohoe et al., 1998
;
Donohoe and Boutilier, 1998
).
In contrast, after 1 month and for up to 4 months of hibernation in hypoxic
water, the isolated muscle mitochondria display reduced resting and active
respiration rates at any given intracellular oxygen partial pressure
(Fig. 5). It seems likely that
the intracellular PO2 values inside frog
skeletal muscle during hypoxic submergence are near state 3 and state 4
mitochondrial P50 values or even lower. Indeed, frogs
recruit anaerobic metabolism during the first 2 months of submergence in
hypoxic water, as indicated by the marked increase in plasma lactate
concentration over this period (Donohoe
and Boutilier, 1998
). Thus, while the skeletal muscle of
overwintering frogs is largely aerobic, it must rely at times on anaerobic
glycolysis for its energy requirements. In fact, frog skeletal muscle might
become transiently ischaemic at various stages during hibernation, especially
during periods of hypoxic stress or heightened activity.
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The mean state 3 and state 4 P50 values for
mitochondria from non-hibernating control groups of frogs (0.077 and 0.017
kPa, respectively, at 20 °C) are similar to those of rat liver (0.057 kPa
and 0.020 kPa, respectively) and rat heart (0.035 kPa and 0.016 kPa,
respectively) mitochondria at 30 °C
(Gnaiger et al., 1998). The
increased mitochondrial P50 values of frog skeletal
muscle, seen during the transition from a resting state (state 4) to an active
one (state 3), mirror those seen in other studies
(Sugano et al., 1974
;
Costa et al., 1997
;
Gnaiger et al., 1998
).
However, our results on hibernating frogs reveal that state 3 and state 4
P50 values of isolated mitochondria can also change with
metabolic rate (i.e. during the transition to hypometabolic states; Figs
3,
5). Unlike earlier studies on
mammals, our data on frogs show that an increase in the in vitro
O2-affinity of mitochondria can occur following chronic in
vivo exposure to cellular hypoxia.
Few studies have focused on the intrinsic properties of mitochondria during
aerobic metabolic depression or on the possible strategies that may have
evolved to increase their efficiency of energy production. From the
information available, we know that mitochondrial state 3, but not state 4,
respiration rates are reduced in hibernating mammals
(Liu et al., 1969;
Pehowich and Wang, 1984
;
Gehnrich and Aprille, 1988
;
Brustovetsky et al., 1989
,
1990
,
1993
). We also know that the
respiration rates of hepatocytes isolated from hibernating ground squirrels
are the same as those obtained from hepatocytes isolated from summer
`cold-acclimated' animals (Staples and
Hochachka, 1997
). Taken together, these results suggest that
proton leak rate is not reduced in mammals during hibernation. Even so, no
studies have reported detailed proton leak titration curves in hibernating
mammals to rule out this possibility. Other studies on ectotherms have shown
intrinsic reductions in metabolic rate at the tissue level (aestivating frogs;
Flanigan et al., 1991
) and at
the cellular level (hepatopancreas cells of aestivating snails;
Bishop and Brand, 2000
;
Guppy et al., 2000
). Our
demonstration that metabolic depression also occurs at the mitochondrial level
(St-Pierre et al., 2000a
,
b
;
Fig. 5) supports the view that
hypometabolism can be reflected at all levels of biological organisation in
hypoxia-tolerant animals.
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Adaptive changes in cellular and mitochondrial enzymes during metabolic depression |
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For example, levels of citrate synthase (CS) and cytochrome c
oxidase (CCO) decrease in the hepatopancreas of aestivating snails compared
with awake controls (Stuart et al.,
1998a,
b
). Even so, there is no
reduction in CS activity in either the heart or kidney of aestivating snails
(Stuart et al., 1998b
). This
suggests that metabolic depression at the whole-animal level might not be
reflected in all tissues by modifications in the intrinsic properties of their
cell metabolism. Similarly, while the in vitro respiration rate of
intact skeletal muscle from aestivating frogs is reduced compared with
controls, no such decreases occur in the intestine, liver, skin or fat
(Flanigan et al., 1991
). The
activity of pyruvate dehydrogenase (PDH), which regulates the entry of acetyl
CoA units in the tricarboxylic acid (TCA) cycle, is also reduced during
aestivation in snails by phosphorylation of the enzyme
(Brooks and Storey, 1992a
).
Phosphorylation of PDH to produce a less active form of the enzyme also occurs
during hibernation (Brooks and Storey,
1992b
) and daily torpor
(Heldmaier et al., 1999
) in
mammals.
Most studies of adaptive plasticity of glycolytic enzymes have focused on
post-translational modifications and the levels of their substrates and
modulators during short-term, anoxiainduced hypometabolic states
(Brooks and Storey, 1997).
Reductions in glycolytic enzyme levels have also been observed during
long-term aestivation in the terrestrial snail and the frog Neobatrachus
pelobatoides. The levels of hexokinase, phosphofructokinase,
glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and lactate
dehydrogenase (LDH) were reduced in the foot muscle of aestivating snails
compared with controls (Brooks and Storey,
1990
). However, aestivating snails did not show a reduction in the
activity of LDH in the kidney, heart and hepatopancreas
(Stuart et al., 1998b
). The
liver of aestivating frogs had lower activities of aldolase and
glyceraldehyde-3-phosphate dehydrogenase compared with controls, but the
activities of both these enzymes remained unchanged during aestivation in the
ventricle, gastrocnemius and brain
(Flanigan et al., 1990
).
Again, these studies emphasise that metabolic depression can have quite
separate and distinctly different effects on different tissues. Taken
together, the results of numerous studies support the idea that changes in
enzyme levels are important during long-term metabolic depression, whereas
alterations in the kinetic properties of enzymes and post-translational
modifications are more consequential during medium- and short-term metabolic
depression (Brooks and Storey,
1990
; Greenway and Storey,
1999
).
We have recently shown that the profound metabolic depression of frogs
hibernating in hypoxia (Donohoe and
Boutilier, 1998) is accompanied by a significant decrease in the
aerobic capacity of their skeletal muscle
(St-Pierre and Boutilier,
2001
), as indicated by the reduction in the activity of key
enzymes of the TCA cycle and of the electron-transport chain
(Fig. 6). Because the decreases
in CS activity at the tissue level were smaller than those observed in
isolated mitochondria, at least some of the decrease in aerobic capacity
during hibernation can be explained by intrinsic changes in aerobic capacity
at the level of the mitochondrion
(St-Pierre and Boutilier,
2001
). The LDH activity of the skeletal muscle of overwintering
frogs was also much lower than in pre-hibernation controls, supporting the
idea of a decreased flux through the glycolytic pathway during hypoxic
hibernation (i.e. the so-called `reversed' Pasteur effect;
Hochachka and Somero, 2002
;
Donohoe and Boutilier, 1998
).
Frogs rely increasingly on a carbohydrate-based metabolism during hypoxic
hibernation (Donohoe and Boutilier,
1998
) and metabolic depression is associated with homeostatic
concentrations of ATP, phosphocreatine and creatine inside the skeletal
muscle, indicating that the decreased ATP demand is matched by a reduced ATP
supply from the glycolytic pathway (glycolysis and mitochondrial
pathways).
|
Overall, these results indicate that an important reorganisation of
ATP-producing pathways occurs during long-term metabolic depression to match
the lowered ATP demand. However, recent studies on the aestivation-induced
responses of mitochondrial respiration in isolated hepatopancreas cells of
snail suggest that the initiation of metabolic depression is through ATP
production pathways and not through the pathways of ATP utilisation
(Bishop et al., 2002). These
authors estimate that changes in the kinetics of substrate oxidation are three
times more important than changes in the kinetics of ATP turnover for the
response of mitochondrial respiration to aestivation. The implication here is
that the processes we normally consider to dominate ATP demand (e.g.
ion-motive ATPases and protein synthesis) may in fact not be very important in
causing metabolic depression.
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Concluding remarks |
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Acknowledgments |
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References |
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