Metabolic enzyme activities across an altitudinal gradient: an examination of pikas (genus Ochotona)
Department of Biology, Mount Union College, Alliance, OH 44601, USA
e-mail: sheafobr{at}muc.edu
Accepted 13 January 2003
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Summary |
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Key words: hypobaric hypoxia, citrate synthase, ß-hydroxyacyl CoA dehydrogenase, lactate dehydrogenase isozymes, lactate removal, Ochotona, pika
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Introduction |
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In general, one of two approaches has been taken in the examination of
enzyme modification in response to low oxygen levels. The first approach has
been to examine the effects of acute hypoxia on organisms that ordinarily
encounter normoxic environments. Laboratory rats and humans are most commonly
used in this type of experiment. Short-term exposure to low oxygen levels
through either exercise (in rats: Constable
et al., 1987; in humans:
Holloszy, 1967
;
Baldwin et al., 1973
;
Terrados et al., 1990
),
normobaric hypoxia (in rats: Daneshrad et
al., 2000
; in humans:
Desplanches et al., 1993
) or
hypobaric hypoxia (in humans: MacDougall
et al., 1991
; Hoppeler and
Desplanches, 1992
; Howald et
al., 1990
) has produced mixed results. Previous studies have
demonstrated that activities of oxidative marker enzymes can be elevated (in
humans: Holloszy, 1967
;
Desplanches et al., 1993
;
Terrados et al., 1990
),
decreased (in humans: Hoppeler and
Desplanches, 1992
; Howald et
al., 1990
) or show no change (in rats:
Daneshrad et al., 2000
; in
humans: Holloszy, 1975
) during
the process of hypoxic acclimation. Activities of enzymes that catalyze
glycolytic and anaerobic reactions have been shown to either decrease (in
rats: Constable et al., 1987
;
in humans: Baldwin et al.,
1973
; Holloszy,
1975
; Terrados et al.,
1990
) or remain constant (in humans:
Holloszy and Oscai, 1969
;
Howald et al., 1990
) when
animals are acclimated to hypoxia. Despite the inconclusive results of
previous experiments, researchers have inferred that a modification of enzyme
activity is inherently adaptive and, therefore, advantageous to organisms
subjected to chronic hypoxia, such as animals living at high altitude
(Holloszy, 1975
;
Pette and Dölken, 1975
;
MacDougall et al., 1991
).
However, it has not been demonstrated which, if any, of these alterations
impart functional, long-term benefits to animals that are continually exposed
to low oxygen levels. Furthermore, since the experimental subjects examined
(e.g. laboratory rats or humans) are not known to have been exposed to chronic
hypoxia at any time in their genetic history, there is no reason to expect
that they have evolved the appropriate responses needed to cope with this
phenomenon.
The second method for determining the effects of hypoxia on metabolic
enzyme activity has been to investigate the biochemical makeup of animals that
inhabit hypoxic environments, such as guinea pigs
(Harris et al., 1970;
Mensen de Silva and Cazorla,
1973
; Barrie et al.,
1975
) or high altitude ungulates
(Hochachka et al., 1982
). It
is assumed that animals exposed to a hypoxic environment over an evolutionary
time frame have, through natural selection, developed and maintained
characters that are beneficial to maintaining ATP synthesis despite
continually limited oxygen availability. These studies have reported an
elevated level of oxidative enzyme activity in all cases and a depressed
(Mensen de Silva and Cazorla,
1973
; Hochachka et al.,
1982
) or unchanged
(Reynafarje, 1962
) anaerobic
enzyme activity level when compared with animals living at low altitude.
However, the low altitude species used for comparison in these studies (e.g.
laboratory rats or rabbits) do not share a close phylogenetic relationship
with the high altitude animals to which they are compared.
The shortcomings associated with comparing two distantly related species
and drawing adaptive conclusions have been well described by Garland and
Adolph (1994). In short, the
conclusions made in these studies assume that, were it not for the effects of
the environmental parameter being analyzed, the two species would be
physiologically identical. However, the genetic differentiation that occurs
during speciation can affect many physiological traits. Which changes are due
to a particular environmental factor and which are due to the countless other
selective pressures that an organism encounters are unclear. Therefore, to
demonstrate adaptation to hypoxia convincingly, a comparison among closely
related species that have similar selective pressures but differ in oxygen
availability is needed.
Pikas, small lagomorphs of the Family Ochotonidae, are ideal animals for
the study of physiological adaptation to high altitude. In North America,
these mammals are restricted to the rocky, talus fields of tundra/alpine
ecosystems found above treeline. In the lower latitudes of their distribution,
treeline occurs only at high altitude. However, as one moves towards the
poles, this ecosystem exists at elevations as low as sea level. As pikas can
be found throughout this latitudinal gradient, it is possible to examine their
physiology over a range of altitudes. Environmental temperatures over this
range are similar, which eliminates the confounding effects of differing
ambient temperatures on metabolism
(Greenland, 1989;
Barry et al., 1981
). As a
result, it is possible to compare closely related species in environments that
differ markedly in the amount of oxygen available but are comparable in other
environmental parameters (Table
1).
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Pikas are extremely active foragers year-round and have high metabolic
rates (MacArthur and Wang,
1973) with no form of metabolic reduction such as daily torpor,
aestivation or hibernation (Krear,
1965
; MacArthur and Wang,
1973
). Thus, their demand for a high rate of ATP turnover is not
decreased by metabolic reduction strategies. While most mammals at high
altitude have adequate oxygen for sustaining resting metabolism, oxygen
quantities are frequently not sufficient to support activity and exercise at a
level comparable with that achieved at low altitude
(West, 1982
). High altitude
pikas have a scope of activity comparable with their low altitude counterparts
and other low altitude mammals (Table
1) and therefore must have some way to compensate for their
hypoxic surroundings. Alterations in the activities of metabolic enzymes or in
the proportions of metabolic isozymes are likely sites for this compensation
and are explored in this paper. It is hoped that this information will present
an insight into the crucial physiological modifications that allow an organism
to maintain ATP synthesis in a continuously hypoxic environment.
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Materials and methods |
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Ochotona princeps is the high altitude species investigated in this study. Eight adult O. princeps (mean body mass, 148.41 g; range, 145.9170.7 g) were live-trapped using Tomahawk live traps baited with apples. All animals were trapped at the University of Colorado Mountain Research Station on Niwot Ridge, Colorado (40°02' N, 105°35' W; 3350 m). Nine adult O. collaris (mean body mass, 125.37 g; range, 113.6130.8 g) were similarly live-trapped from Eagle Summit, Alaska (65°29' N, 145°25' W; 1070 m) and are considered to be low altitude animals in this study. Heart tissue from four adult O. hyperborea was obtained from the frozen tissue collection at the University of Alaska at Fairbanks. O. hyperborea were snap-trapped 18 km north of Magadan, Russia (59°45' N, 150°53' E; 0 m). Tissues were immediately removed from animals upon trapping and frozen in liquid nitrogen. In this study, O. hyperborea is designated a sea level species. All pikas were trapped between the months of June and August.
Tissue extraction
Five muscle tissues were examined in this study. Three skeletal muscles
were chosen (vastus lateralis, gastrocnemius and soleus) in order to compare
muscles with varying compositions of oxidative and glycolytic muscle fibers.
Diaphragm and ventricular cardiac muscles were also investigated because they
are intimately involved with oxygen delivery and are excellent indicators of
an animal's overall metabolic status.
After capture, O. princeps and O. collaris were anesthetized on site with methoxyflurane and carried to a four-wheel drive vehicle outfitted to operate as a mobile laboratory. Pikas were then injected with pentobarbital intraperitoneally (50 mg kg1). As soon as the animal was fully under anesthesia, a 50200 mg sample was taken of each tissue and frozen on dry ice. Identical procedures were performed in the laboratory on eight adult laboratory rabbits (Oryctolagus cuniculus). Rabbit data are used as an intrafamiliar outgroup control.
O. hyperborea cardiac muscle was removed upon trapping, placed in 2 ml cryovials and frozen in liquid nitrogen until it could be stored in a 70°C freezer at the University of Alaska at Fairbanks.
All tissues remained frozen until they were homogenised in 19 volumes (w/v) of homogenisation buffer (175 mmol l1 KCl, 2 mmol l1 EDTA, 10 mmol l1 Tris, pH 7.0) using refrigerated ground glass tissue homogenisers. Between three and six separate aliquots of the 1/20 homogenate were placed into 0.7 ml Eppendorf tubes for each tissue. Homogenates were stored at 70°C until immediately before analysis of enzyme activities, when they were thawed and diluted with homogenisation buffer to the desired concentrations. Assays performed on fresh homogenates showed no difference in enzyme activity when compared with assays using frozen homogenates.
Protein concentrations were determined spectrophotometrically using a
modification of the Lowry protein assay
(Peterson, 1977). Bovine serum
albumin was used as a standard and the 1/20 homogenate was used as the tissue
source. Protein assays showed no significant difference in protein content
between homologous tissues.
Enzyme activity
Enzyme activities were measured on a Gilford 260 spectrophotometer
(Oberlin, OH, USA) that was kept at a constant temperature by a circulating
water bath (Isotemp Model 900; Fisher Scientific, Pittsburgh, PA, USA). All
reactions took place in pre-warmed, quartz cuvettes and had a final volume of
1 ml. Enzyme activities were measured at both 38°C (rabbit operating body
temperature) and at 40°C (pika operating body temperature) for all
species. Analysis of enzyme Q10 showed no differences among
species. Therefore, comparisons were made at 40°C.
Citrate synthase (CS; EC 4.1.3.7) and ß-hydroxyacyl CoA dehydrogenase
(HOAD; EC 1.1.1.35) activities were measured using a final dilution of 1/1000
for heart and diaphragm tissues and 1/200 for skeletal muscles. For CS, the 1
ml reaction volume contained 0.3 mmol l1 acetyl CoA, 0.1
mmol l1 DTNB 5,5'-dithio-bis(2-nitrobenzoic acid), 100
mmol l1 Tris-HCl buffer at pH 8.0 and 100 µl diluted
homogenate. The reaction was initiated by adding 100 µl of 0.5 mmol
l1 oxaloacetate. Absorbance was measured at 412 nm
(Srere, 1969). For HOAD, the
final 1 ml reaction volume contained 0.5 mmol l1 EDTA, 0.23
mmol l1 NADH, 100 mmol l1
triethanolamine-HCl buffer at pH 7.0 and 100 µl diluted homogenate. The
reaction was started by the addition of 100 µl of 0.1 mmol
l1 acetoacyl-CoA. Absorbance was measured at 340 nm
(Bass et al., 1969
). Total
lactate dehydrogenase activity (LDH; EC 1.1.1.27) was measured using a final
dilution of 1/1000 for all tissues. The 1 ml reaction volume consisted of 0.25
mmol l1 NADH, 100 mmol l1 potassium
phosphate buffer at pH 7.0 and 50 µl diluted homogenate. The addition of
100 µl of 10 mmol l1 sodium pyruvate initiated the
reaction. The disappearance of NADH was measured at 340 nm
(Gleeson and Harrison,
1986
).
This study assumes that, for homologous tissues, CS activity g1 fresh tissue produces a measure of relative overall oxidative capacity, HOAD activity g1 fresh tissue yields an estimation of relative fatty acid oxidative capacity, and LDH activity g1 fresh tissue provides a measure of relative anaerobic metabolic capacity.
Isozyme analysis
LDH isozymes were separated by native polyacrylamide gel electrophoresis on
a vertical gel electrophoresis system (Model V16, Bethesda Research
Laboratories, Gaithersburg, MA, USA). Gels consisted of a separating gel of
7.5% acrylamide in 1.5 mol l1 Tris-HCl (pH 8.5) and a
stacking gel of 5% acrylamide in 0.5 mol l1 Tris-HCl (pH
6.8). Homogenates were diluted with a sample buffer of 62.5 mmol
l1 Tris-HCl, 10% glycerol and 0.002% bromophenol blue. A
running buffer of 250 mmol l1 Tris-HCl and 190 mmol
l1 glycine was used in all trials. Electrophoresis was
carried out for 12 h at 4°C using a constant current power supply (Model
3-1500; Haake-Buchler, Saddleback, New Jersey) set at 15 mA. Gels were stained
with a solution containing 0.33 mmol l1 NAD+,
0.10 mmol l1 phenazine methosulfate, 0.27 mmol
l1 nitro blue tetrazolium, 25.0 mmol l1
lactic acid and 20 mmol l1 Tris-HCl (pH 8.2).
Quantification of relative isozyme activity followed the protocol of Klebe
(1975). For each of the five
muscle tissues examined, samples from three animals of each species were
tested. Serial twofold dilutions of samples were electrophoretically separated
in consecutive lanes and then stained until a visual endpoint was reached for
each tetramer. The relative percentage of activity produced by a given
tetramer was calculated by dividing the dilution factor of the last visible
band by the sum of the dilution factors for all five tetramers. The
contribution of each isozyme to the relative percentage of activity produced
by a tetramer was calculated by multiplying the relative percentage by the
proportion of each isozyme within the tetramer (e.g. M1:H3=0.25 M-type and
0.75 H-type). Values for isozyme activity were calculated by multiplying the
total isozyme contribution by the total LDH activity.
Measurements of enzyme and isozyme activities were compared among species by simple one-way analysis of variance (ANOVA) and a post-hoc Scheffe's multiple range test. Statistical analyses were computed using SPSS statistical programs (SPSS Inc., Chicago, IL, USA).
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Results |
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ß-hydroxyacyl CoA dehydrogenase activity also showed a positive relationship with altitude in cardiac tissue for the three species of pikas (Table 2; Fig. 1). HOAD activity in the cardiac and diaphragm tissue of O. princeps was significantly greater than that of O. collaris. As with citrate synthase, HOAD activity in skeletal muscle showed little correlation with altitude.
Within pika species, total lactate dehydrogenase activity showed a positive
correlation to altitude in all tissues examined
(Table 2; Fig. 1). All tissues showed a
significant difference between high and low altitude pikas, with the high
altitude animals having higher LDH activity. Within each pika species, LDH
activity levels were similar across all skeletal muscles. Rabbits followed the
expected mammalian pattern of high LDH activity in vastus lateralis, moderate
activity in gastrocnemius and low activity in soleus muscle
(Pagliassotti and Donovan,
1990).
Isozyme analysis
Lactate dehydrogenase isozyme activities for O. princeps, O. collaris,
O. hyperborea and O. cuniculus are reported in
Table 3. In all tissues, H-LDH
activity was significantly greater in O. princeps when compared with
the other species (Table 3;
Fig. 2).
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In heart muscle, O. princeps had H-LDH activity that was nearly 2.5 times greater than cardiac tissue values in any other species examined. Cardiac M-LDH activity for O. princeps was significantly lower than in other pika species. High-altitude-adapted pikas had diaphragm tissue values that were greater in both H-LDH and M-LDH activity when compared with both pikas and rabbits adapted to low altitude (Table 3; Fig. 2).
H-LDH activity in skeletal muscle was up to 3.8 times greater in O. princeps than in O. collaris and was significantly greater in all skeletal muscles examined. O. princeps had significantly higher H-LDH activity in all skeletal muscles when compared with rabbits. M-LDH activity in skeletal muscle was greater in O. princeps when compared with O. collaris in vastus lateralis and gastrocnemius, but there was no activity difference between pika species in soleus muscle (Table 3). In all skeletal muscles examined, O. princeps had greater H-LDH activity than M-LDH activity within a muscle, while O. collaris had more M-LDH than H-LDH activity (Table 3; Fig. 2).
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Discussion |
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Efficient synthesis of ATP is critical in heart and diaphragm muscles that are continuously active. Therefore, oxidative rather than glycolytic pathways are favored, creating a high oxidative demand in these tissues. In order for an animal at high altitude to maintain adequate oxidative metabolism in these tissues, particularly during exercise, modifications in the ability of the tissues to utilize available oxygen are necessary to sustain function at a level comparable with that achieved under normoxic conditions. Skeletal muscle, on the other hand, can be rested and may rely to a greater extent on the less economical glycolytic pathway for a portion of its energy production. Due to the lower oxidative demands on skeletal muscle tissues, modification of oxygen utilization may not be imperative to maintain a reasonable scope of activity.
Citrate synthase activities provide a measure of relative overall oxidative
capacity. The site of oxidative metabolism is within the mitochondria and,
therefore, an increase in CS activity is considered to be an indication of an
increase in mitochondrial density and/or mitochondrial size. A positive
relationship between an increase in CS activity and mitochondrial density has
been demonstrated during acclimation to hypoxia via exercise
(Holloszy, 1967;
Gollnick and King, 1969
),
normobaric hypoxia (Desplanches et al.,
1993
) and hypobaric hypoxia
(Ou and Tenney, 1970
). Under
hypoxic conditions, a greater mitochondrial density is thought to be
advantageous due to the increased probability that an oxygen molecule will
come in contact with an oxidative enzyme site (i.e. cytochrome c
oxidase) in a short time interval and, subsequently, increase the ability of
the cell to utilize available oxygen (Ou
and Tenney, 1970
). Therefore, it is not surprising to find that
tissues with a high oxidative demand, such as heart and diaphragm, show a
correlation between altitudinal hypoxia and CS activity
(Table 2; Figs
1,
2). By increasing CS activity
and/or mitochondrial density in tissues with large oxidative demands, high
altitude pikas allow the tissues to maintain aerobic metabolism at a
sufficient level without a reliance on glycolytic means of ATP synthesis in
tissues critical for oxygen transport. Skeletal muscle in pikas is not used
for sustained activities but is used in short bursts during foraging forays
and predator avoidance. These tissues have a low oxidative demand and can rely
on glycolytic energy production in the face of low oxygen availability.
Therefore, changes in skeletal muscle CS activity and mitochondrial density
may not be obligatory in pikas found at altitude
(Table 2;
Fig. 2). An alternative
explanation would be that high-altitude-adapted pikas increase ventilation and
heart rate in order to maintain oxygen transport despite reduced oxygen
availability. The increase of ventilation and cardiac output might lead to a
training effect in heart and diaphragm, which would increase oxidative enzyme
activity in these tissues.
HOAD activity indicates relative fatty acid oxidation capacity. While this
is a gauge of oxidative metabolism, it does not give an indication of total
oxidative capacity as does CS but measures only the portion derived from fat
oxidation. Pikas feed on leaves and flowers from alpine/tundra plants and have
low levels of fats in their natural diet
(Dearing, 1997). Subsequently,
they do not rely heavily on lipid metabolism, which is reflected in their
relatively low HOAD activities when compared with laboratory animals
(Table 2). However, among pika
species, HOAD activity corresponds to altitude in the highly oxidative heart
and diaphragm tissues but has no relationship to altitude in skeletal muscles
(Table 2). This pattern is
similar to that found in CS activity and further emphasizes an adaptive upward
scaling of oxidative enzyme activity in animals at altitude, particularly in
tissues that rely heavily on oxidative pathways.
Anaerobic function
One possible response that an animal exposed to hypoxia could develop in
order to maintain cellular function is reliance upon anaerobic means of ATP
synthesis. This strategy is employed by some bacteria
(Gottschalk, 1979), yeast
(Hochachka and Somero, 1984
)
and invertebrates (De Zwaan and Wijsman,
1976
; Collicutt and Hochachka,
1977
; Saz, 1981
)
that encounter hypoxic environments. However, anaerobic pathways are not
extensively used in terrestrial vertebrates because fermentation by-products
such as lactate can induce a decrease in cell function through pH imbalance
and inhibition of glycolysis (Hochachka
and Mommsen, 1983
). Therefore, it is not surprising that increases
in anaerobic capacity have not been demonstrated in mammals acclimated to
hypoxic conditions (Constable et al.,
1987
; Howald et al.,
1990
; Terrados et al.,
1990
). It has been proposed that skeletal muscles exposed to
hypoxia are modified to function more like heart tissue, confining ATP
synthesis to oxidative pathways and limiting glycolytic energy production in
order to minimize lactate accumulation
(Baldwin et al., 1973
;
Holloszy, 1975
).
Studies comparing mammals adapted to high altitude with lowland species
have reported similar reductions in anaerobic capacities
(Mensen de Silva and Cazorla,
1973; Hochachka et al.,
1982
,
1991
). A downregulation of
anaerobic metabolic capacity and the ensuing reduction in lactate accumulation
in response to hypoxic conditions, dubbed the lactate paradox
(West, 1986
;
Hochachka, 1988
), have been
observed in all high altitude species examined thus far. The effect is thought
to be caused by a more efficient coupling between ATP demand and ATP supply,
allowing for a more effective integration between glycolysis and oxidative
metabolism (Hochachka, 1988
,
1994
). However, pikas do not
follow the lactate paradox pattern. Lactate dehydrogenase activity, an
indicator of relative anaerobic metabolic capacity, shows a positive
correlation with altitude in pikas rather than the negative relationship
associated with the lactate paradox (Table
2; Fig. 2). This
indicates that the lactate paradox is not necessarily a required modification
for success at altitude.
The maintenance of a high anaerobic potential in pika tissues may be
dictated by their ecology. Pikas are heavily preyed upon by a variety of
animals and avoid predation by sprinting from foraging grounds to the safety
of nearby rock piles (Broadbooks,
1965; Krear, 1965
;
Ivins and Smith, 1983
). This
makes survival dependent upon the ability to generate burst activity for short
periods of time. The rapid ATP synthesis derived through glycolytic rather
than oxidative means largely powers burst activity. Therefore, pikas may not
be able to reduce their anaerobic capacity and still maintain the sprinting
ability necessary for survival in their specific environment. In other words,
it may be beneficial for a pika to contend with lactate build-up in order to
avoid becoming a meal.
LDH isozyme activity
While predatory avoidance may explain why it is beneficial for all pikas to
maintain a high anaerobic capacity, it does not explain why high altitude
animals have a higher total LDH activity than do low altitude animals. Since
burst activity is equally necessary at any altitude and is not dependent on
oxygen availability, anaerobic capacities should be comparable among pika
species. Yet, in all tissues examined, O. princeps have LDH
activities that are over twofold higher than those of O. collaris
(Table 2). The phenomenon
appears to be explained by differences in LDH isozyme ratios between the two
species.
H-LDH is the isozyme found predominantly in mammalian heart and liver
tissue. It preferentially converts lactate to pyruvate, and high levels of
H-LDH in cardiac tissue would allow lactate to be used as a metabolic
substrate, which removes the possibility of cardiac muscle fatigue due to pH
imbalances (Hochachka and Somero,
1984). M-LDH is found predominantly in mammalian skeletal muscle
and preferentially converts pyruvate to lactate. High levels of M-LDH are
necessary for the maintenance of redox balance within tissues anaerobically
synthesizing ATP.
LDH isozyme patterns in O. princeps suggest that muscle tissues in high altitude pikas are behaving in a fashion similar to cardiac muscle. In all tissues examined, O. princeps had higher H-LDH activity than M-LDH activity. In O. collaris, H-LDH activity was only greater than M-LDH in highly oxidative heart and diaphragm. All skeletal muscles in O. collaris had higher M-LDH than H-LDH activity (Table 3).
Rabbit tissues follow more characteristically mammalian LDH isozyme
patterns when compared with pikas
(Pagliassotti and Donovan,
1990; Tables 2,
3). Highly oxidative tissue
such as heart, diaphragm, and soleus have a high H-LDH to M-LDH ratio, while
glycolytic tissue such as vastus has a great deal more M-LDH than H-LDH
activity. The gastrocnemius muscle of the rabbit is composed of both oxidative
and glycolytic fibers and, consequently, has similar H-LDH and M-LDH
activities.
It has been proposed that the high H-LDH isozyme activities found in the
skeletal muscles of animals such as hummingbirds function to reduce lactate
accumulation during prolonged exercise
(Hochachka et al., 1992).
Because pika muscle is rarely involved in sustained activity, high levels of
H-LDH are more likely to be a mechanism for accelerating the reconversion of
acquired lactate after anaerobic bouts of activity rather than as a means of
reducing accumulation of lactate in tissues. Hypoxia impedes the rate at which
lactate can be oxidized and increases the time it takes animals at altitude to
clear lactate from tissues where it has been produced
(Hochachka and Mommsen,
1983
).
An increase in skeletal muscle H-LDH subunits would facilitate lactate
reconversion and, consequently, reduce the time that an animal would be
required to remain inactive while lactate is eliminated. In normoxic mammals,
the majority of accumulated lactate is thought to be removed through the
lactate shuttle (Brooks, 1986).
In this process, lactate is transported via the circulatory system
from its production site to tissues with high respiratory rates where it is
utilized as a metabolic substrate. This removal strategy may not be sufficient
for pikas at altitude because, even at rest, tissues such as heart that
typically utilize lactate as a substrate are hypoxically restricted and cannot
contribute substantially to lactate removal. Skeletal muscle does not
typically play a major role in lactate removal in mammals. For example, <7%
of lactate in rat skeletal muscle has been shown to be oxidized in
situ (McLane and Holloszy,
1979
). Therefore, the high levels of H-LDH in pika skeletal muscle
may facilitate lactate reconversion to pyruvate that can then be oxidized at
the site of production. Excess pyruvate could also be converted to glucose
through glyconeogenic pathways in situ
(Guppy et al., 1979
;
McLane and Holloszy, 1979
) and
glyconeogenic capabilities should be further analyzed. Hepatic and renal
conversion of lactate to glucose through the Cori cycle is a prevalent site of
lactate removal in mammals and should be investigated in future
experiments.
The findings presented in this paper allow for both general and specific conclusions to be drawn about the modification of metabolic enzyme activity in response to hypoxia. The upward scaling of oxidative metabolic enzyme activity appears to be a physiological response general to mammals in low oxygen environments. This shift is prominent in tissues that are highly aerobic and requires constant, steady ATP synthesis but may not be evident in all tissues. Increased oxidative capacities in tissues with high metabolic demands serve to facilitate oxygen utilization when oxygen availability is low. While a downregulation of anaerobic enzyme activity has been viewed as a common physiological response of hypoxic mammals, this study demonstrates that, at least for pikas, the opposite is true. Pikas show a distinct positive correlation between total LDH activity and hypoxia. However, the majority of the increase in total activity is due to an increase in H-LDH isozyme, which may function to expedite the removal of lactate in muscle tissue and allow pikas to return to resting levels within a short time interval. Although this strategy for maintaining energy production in a hypoxic environment may be specific to pikas, it demonstrates that there are many ways in which metabolic processes can be modified in response to environmental stress.
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Acknowledgments |
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References |
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Baldwin, K. M., Winder, W. W., Terjung, R. L. and Holloszy, J.
O. (1973). Glycolytic enzymes in different types of skeletal
muscle: adaptation to exercise. Am. J. Physiol.
225,962
-966.
Barnard, J. R. and Peter, J. B. (1971). Effects
of exercise on skeletal muscle III. Cytochrome changes. J. Appl.
Physiol. 31,904
-908.
Barrie, E., Heath, D., Arias Stella, J. and Harris, P. (1975). Enzyme activities in red and white muscles of guinea-pigs and rabbits indigenous to high altitudes. Environ. Physiol. Biochem. 5,18 -26.[Medline]
Barry, R. G., Courtin, G. M. and Labine, C. (1981). Tundra climates. In Tundra Ecosystems: A Comparative Analysis (ed. L. C. Bliss, J. B. Cragg, D. W. Heal and J. J. Moore), pp. 81-114. Cambridge: Cambridge University Press.
Bass, A., Brdiczka, D., Eyer, P., Hofer, S. and Pette, D. (1969). Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 10,198 -206.[Medline]
Broadbooks, H. E. (1965). Ecology and distribution of the pikas of Washington and Alaska. Am. Mid. Nat. 73,299 -335.
Brooks, G. A. (1986). Lactate production under fully aerobic conditions: the lactate shuttle during rest and exercise. Fed. Proc. 45,2924 -2929.[Medline]
Collicutt, J. M. and Hochachka, P. W. (1977). The anaerobic oyster heart: Coupling of glucose and aspartate fermentation. J. Comp. Physiol. 115,147 -157.
Constable, S. H., Favier, R. J., McLane, J. A., Fell, R. D.,
Chen, M. and Holloszy, J. O. (1987). Energy metabolism
in contracting rat skeletal muscle: adaptation to exercise training.
Am. J. Physiol. 253,C316
-C322.
Corbet, G. B. (1978). The Mammals of the Palaearctic Region: a Taxonomic Review. London, Ithaca: British Museum (Natural History) and Cornell University Press.
Daneshrad, Z., Garcia-Riera, M. P., Verdys, M. and Rossi, A. (2000). Differential responses to chronic hypoxia and dietary restriction of aerobic capacity and enzyme levels in the rat myocardium. Mol. Cell. Biochem. 210,159 -166.[CrossRef][Medline]
Dearing, M. D. (1997). Effects of Acomastylis rossii tannins on a mammalian herbivore, the North American pika, Ochotona princeps. Oecologia 109,122 -131.[CrossRef]
Desplanches, D., Hoppeler, H., Linossier, M. T., Denis, C., Claassen, H., Dormois, D., Lacour, J. R. and Geyssant, A. (1993). Effects of training in normoxia and normobaric hypoxia on human muscle ultrastructure. Pflügers Arch. 425,263 -267.[Medline]
De Zwaan, A. and Wijsman, T. C. M. (1976). Anaerobic metabolism in Bivalvia (Mollusca). Comp. Biochem. Physiol. B 54,313 -324.[CrossRef][Medline]
Garland, T., Jr and Adolph, S. C. (1994). Why not to do two-species comparative studies: limitations on inferring adaptation. Physiol. Zool. 67,797 -828.
Gleeson, T. T. and Harrison, J. M. (1986). Reptilian skeletal muscle: fiber-type composition and enzymatic profile in the lizard, Iguana iguana. Copeia 1986,324 -332.
Gollnick, P. D. and King, D. W. (1969). Effect
of exercise and training on mitochondria of rat skeletal muscle.
Am. J. Physiol. 216,1502
-1509.
Gottschalk, G. (1979). Bacterial Metabolism. New York: Springer-Verlag.
Greenland, D. (1989). The climate of Niwot Ridge, Front Range, Colorado, USA. Arctic Alpine Res. 21,380 -391.
Guppy, M., Hulbert, W. C. and Hochachka, P. W. (1979). Metabolic sources of heat and power in tuna muscles. II. Enzyme and metabolite profiles. J. Exp. Biol. 82,303 -320.[Medline]
Gureev, A. A. (1964). Lagomorpha. Fauna USSR, Mammals. Moscow: Science Publishing House.
Hall, E. R. (1981). The Mammals of North America. Second edition. New York: John Wiley & Sons.
Harris, P., Castillo, Y., Gibson, K., Heath, D. and Arias-Stella, J. (1970). Succinic and lactic dehydrogenase activity in myocardial homogenates from animals at high and low altitude. J. Mol. Cell. Cardiol. 1, 189-193.
Hochachka, P. W. (1988). The lactate paradox: analysis of underlying mechanisms. Ann. Sports Med. 4, 184-189.
Hochachka, P. W. (1994). Muscles as Molecular and Metabolic Machines. Boca Raton: CRC Press.
Hochachka, P. W., Stanley, C., Merkt, J. and Sumar-Kalinowski, J. (1982). Metabolic meaning of elevated levels of oxidative enzymes in high altitude adapted animals: an interpretive hypothesis. Respir. Physiol. 52,303 -313.[CrossRef]
Hochachka, P. W. and Mommsen, T. P. (1983). Protons and anaerobiosis. Science. 219,1391 -1397.[Medline]
Hochachka, P. W. and Somero, G. N. (1984). Biochemical Adaptation. Princeton: Princeton University Press.
Hochachka, P. W., Stanley, C., Matheson, G. O., McKenzie, D. C.,
Allen, P. S. and Parkhouse, W. S. (1991). Metabolic
and work efficiencies during exercise in Andean natives. J. Appl.
Physiol. 70,1720
-1730.
Hochachka, P. W., Stanley, C., McKenzie, D. C., Villena, A. and Monge, C. (1992). Enzyme mechanisms for pyruvate-to-lactate flux attenuation: study of Sherpas, Quechuas, and hummingbirds. Int. J. Sports Med. 13,S119 -S123.[Medline]
Holloszy, J. O. (1967). Biochemical adaptations
in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory
enzyme activity in skeletal muscle. J. Biol. Chem.
242,2278
-2282.
Holloszy, J. O. (1975). Adaptation of skeletal muscle to endurance exercise. Med. Sci. Sports 7, 155-164.[Medline]
Holloszy, J. O. and Oscai, L. B. (1969). Effect
of exercise on -glycerophosphate dehydrogenase activity in skeletal
muscle. Arch. Biochem. Biophys.
130,653
-656.[Medline]
Hoppeler, H. and Desplanches, D. (1992). Muscle structural modifications in hypoxia. Int J. Sports Med. 13,S166 -S168.[Medline]
Howald, H., Pette, D., Simoneau, J. A., Hoppeler, H. and Cerretelli, P. (1990). Effects of chronic hypoxia on muscle enzyme activities. Int. J. Sports Med. 11,S10 -S14.[Medline]
Ivins, B. L. and Smith, A. T. (1983). Responses of pika (Ochotona princeps: Lagomorpha) to naturally occurring terrestrial predators. Behav. Ecol. Sociobiol. 13,277 -287.
Klebe, R. J. (1975). A simple method for the quantification of isozyme patterns. Biochem. Gen. 13,805 -812.[Medline]
Krear, H. R. (1965).An ecological and ethological study of the pika (Ochotona princeps saxatilis Bangs) in the Front Range of Colorado. Ph.D. Thesis. Boulder: University of Colorado, Boulder.
MacArthur, R. A. and Wang, L. C. H. (1973). Physiology of thermoregulation in the pika, Ochotona princeps. Can. J. Zool. 51,11 -16.[Medline]
MacDougall, J. D., Green, H. J., Sutton, J. R., Coates, G., Cymerman, A., Young, P. and Houston, C. S. (1991). Operation Everest II: structural adaptations in skeletal muscle in response to extreme simulated altitude. Acta Physiol. Scand. 142,421 -427.[Medline]
McLane, J. A. and Holloszy, J. O. (1979). Glycogen synthesis from lactate in the three types of skeletal muscle. J. Biol. Chem. 254,6548 -6553.[Medline]
Mensen de Silva, E. and Cazorla, A. (1973).
Lactate, -GP, and Krebs cycle in sea-level and high-altitude native
guinea pigs. Am. J. Physiol.
224,669
-672.
Ou, L. C. and Tenney, S. M. (1970). Properties of mitochondria from hearts of cattle acclimatized to high altitude. Respir. Physiol. 8,151 -159.[CrossRef][Medline]
Pagliassotti, M. J. and Donovan, C. M. (1990).
Role of cell type in net lactate removal by skeletal muscle. Am. J.
Physiol. 258,E635
-E642.
Peterson, G. L. (1977). Amplification of the protein assay method of Lowry et al., which is more generally applicable. Anal. Biochem. 83,346 -356.[Medline]
Pette, D. and Dölken, G. (1975). Some aspects of regulation of enzyme levels in muscle energy-supplying metabolism. Adv. Enz. Reg. 13,355 -375.[CrossRef][Medline]
Reynafarje, B. (1962). Myoglobin content and enzymatic activity of muscle and altitude adaptation. J. Appl. Physiol. 17,301 -305.
Saz, H. J. (1981). Energy metabolism of parasitic helminths. Ann. Rev. Physiol. 43,323 -341.[CrossRef][Medline]
Smith, A. T. and Ivins, B. L. (1986). Territorial intrusions by pikas (Ochotona princeps) as a function of occupant activity. Anim. Behav. 34,392 -397.
Srere, P. A. (1969). Citrate synthase. Methods Enzymol. 13,3 -5.
Terrados, N., Jansson, E., Sylvén, C. and Kaijser, L.
(1990). Is hypoxia a stimulus for synthesis of oxidative enzymes
and myoglobin? J. Appl. Physiol.
68,2369
-2372.
West, J. B. (1982). Diffusion at high altitude. Fed. Proc. 41,2128 -2130.[Medline]
West, J. B. (1986). Lactate during exercise at extreme altitude. Fed. Proc. 45,2953 -2957.[Medline]
Weston, M. L. (1981). The Ochotona alpina complex: a statistical re-evaluation. In Proceedings of the World Lagomorph Conference (ed. K. Myers and C. D. MacInnes), pp. 73-89. Guelph, Ontario: Guelph University Press.
Youngman, P. M. (1975). Mammals of the Yukon Territory. Ottawa: National Museum of Natural Sciences, National Museums of Canada, Publications in Zoology.