Cost of transport is increased after cold exposure in Monodelphis domestica: training for inefficiency
1 Department of Biological Sciences, Physiology and Functional Morphology
Group, Northern Arizona University, Flagstaff, AZ 86011, USA
2 Center for Cardiovascular Research and Department of Medicine, Washington
University School of Medicine, St Louis, MO 63110, USA
3 Department of Molecular Biology and Pharmacology, and Department of
Pediatrics, Washington University School of Medicine, St Louis, MO 63110,
USA
* Author for correspondence (e-mail: pschaeff{at}im.wustl.edu)
Accepted 17 May 2005
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Summary |
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Key words: oxygen consumption, locomotion, thermogenesis, uncoupling protein 3, muscle mechanics, marsupial, Monodelphis domestica
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Introduction |
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It has been generally accepted that Ct is constant
within an individual (Holloszy and Coyle,
1984); however, there is a lack of consensus on this issue. There
have been numerous studies exploring the effect of exercise training on
Ct, testing the hypothesis that training would result in a
reduced Ct. The results have been somewhat equivocal, with
some evidence supporting the traditional view that Ct is
not responsive to training (e.g. Patch and
Brooks, 1980
; Bailey and Messier, 1991), while others have found a
significant, albeit small, decrease in the aerobic demand of running in elite
humans (Morgan et al.,
1995
).
Although studies of the malleability of cost of transport are equivocal,
numerous studies have demonstrated that the muscle metabolic system that
supplies energy substrates to power locomotion is highly plastic. Alterations
in activity result in profound adaptive changes in the oxidative machinery
within the muscle fibers (Hoppeler,
1986; Rome and Lindstedt,
1997
). Similarly, expression of contractile proteins is highly
responsive to changes in the mechanical activity of the muscle
(Schiaffino and Reggiani,
1996
; Goldspink,
1999
). The cost of transport is relatively invariant in the face
of these adaptive responses, which suggests that although the cellular
components of energy supply and utilization are very responsive to changing
activity patterns, both appear to be maintained in close functional coupling.
The basic pattern is set by body size constraints, as indicated by the similar
body size scaling of the time-dependent properties of these systems
(Lindstedt and Thomas,
1994
).
In addition to locomotion, skeletal muscle is an important source of
metabolic heat during cold stress. There has been little attention given to
the effects of muscle thermogenesis upon the energetics of locomotion. Like
exercise, chronic cold exposure is a cause of increased metabolic demand, to
support thermogenesis and maintain temperature homeostasis. In small placental
mammals, brown adipose tissue (BAT) is responsible for the majority of the
increased thermogenic capacity (Foster and
Frydman, 1976). However, only placental mammals weighing less than
10 kg possess this tissue (Heldmaier,
1971
), thus many animals, including adult humans, must rely upon
other tissues for thermogenesis. At approximately 40% of body mass
(Lindstedt and Thomas, 1994
),
skeletal muscle is pre-adapted to supply considerable metabolic heat. In many
species lacking BAT, cold acclimation has led to structural alterations in
muscle resembling those resulting from exercise. Specifically, cold
acclimation in ducks led to increased capillary density and a shift to
slow-oxidative muscle fiber types (Duchamp
et al., 1992
). Increased cytochrome oxidase activity was observed
in ducks (Barre et al., 1987
),
king penguins (Duchamp et al.,
1991
) and pigs (Berthon et al.,
1996
). We previously reported an increase in muscle mitochondrial
volume in short-tailed opossums, Monodelphis domestica
(Schaeffer et al., 2003
). In
contrast, in rodents possessing BAT, cold acclimation does not lead to
alterations in muscle structure (Barre et
al., 1987
, Hoppeler et al.,
1995
).
Functionally, whole animal running at maximum rate of oxygen consumption
O2max is greater
in both cold-exposed goats (Schaeffer et
al., 2001
) and Monodelphis domestica
(Schaeffer et al., 2003
)
compared to animals maintained in thermoneutral conditions. Winter-acclimated
deer mice (which possess BAT) showed an increase in thermogenic capacity, but
only a slight increase in running
O2max
(Hayes and Chappell, 1986
).
Thus while alterations of the metabolic system occur in skeletal muscle in
response to cold exposure, little is known about the response of the
contractile system. We recently reported that cold-acclimated goats had
apparently higher Ct (measured at
O2max;
Schaeffer et al., 2001
),
suggesting that increased capacity for muscle thermogenesis may be acting as
`training for inefficiency'. This observation could be explained in part if
mitochondrial uncoupling processes play a role in muscle thermogenesis.
Uncoupling protein 3 (UCP3) is highly expressed in skeletal muscle and,
although the physiological function of UCP3 is debated (e.g.
Porter, 2001
;
Nedergaard and Cannon, 2003
),
in species lacking BAT, a role for UCP3 as a metabolic uncoupler in skeletal
muscle may be unveiled.
To investigate the comparative roles of cold acclimation and endurance
exercise training on whole animal running efficiency, we utilized the
short-tailed opossum Monodelphis domestica, a small (100 g)
marsupial lacking BAT. We recently demonstrated that these animals are reliant
upon skeletal muscle thermogenesis following cold exposure, increasing both
whole animal
O2max and
O2summit
(maximum
O2
during cold exposure) as well as muscle mitochondrial volume density
(Schaeffer et al., 2003
). We
thus utilized these animals to ask the following questions. (i) How do muscle
responses to exercise training and cold exposure impact cost of transport?
(ii) To what extent do alterations of muscle contractile properties in
response to cold exposure resemble or contrast those associated with exercise?
(iii) Is there any activation of gene expression in response to exercise
training or cold exposure of an uncoupling protein 3 homologue in skeletal
muscle of Monodelphis domestica?
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Materials and methods |
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Experimental design
For the experimental protocol, animals were divided into four groups, which
were treated as follows. Two groups were maintained at thermoneutral ambient
temperature (28°C; Dawson and Olson,
1988), one of which was exercise-trained (see below) on a
motorized treadmill. These groups are designated `Thermoneutral, sedentary'
(TnS) and `Thermoneutral, trained' (TnT). The second two groups were
cold-exposed (see below) and again, one of these was exercise-trained on a
motorized treadmill. These groups are designated `Cold, sedentary' (CS) and
`Cold, trained' (CT).
Exercise training was performed at constant speed while running up a 10% incline. During the first week, every animal (including sedentary groups) ran on the treadmill at 10 m min1 for 5 min each day for 3 days to gain familiarity with the apparatus, necessary for subsequent testing. Thereafter, sedentary animals ran once (again at 10 m min1 for 5 min) during the eighth week of the experimental period. The animals that underwent endurance exercise training ran for 30 min at 10 m min1 for 5 days during the second week. This was increased to 45 min at 15 m min1 for 5 days during the third week and then to 45 min at 20 m min1, 5 days/week for the remaining 6 weeks duration of the experiment.
Cold exposure entailed being housed in a cold room at 19°C for 1 week. The temperature was then decreased to 16°C at the beginning of the second week, to 12.5°C for the third week, to 9°C for the fourth through sixth weeks and finally to 12°C for the final 3 weeks.
Measurement of oxygen consumption
Upon completion of the experimental procedures, we measured rates of oxygen
consumption O2
while running on a level, motorized treadmill at 10, 15, 20 and 25 m
min1 at ambient temperature of 28°C using an open flow
system, as described previously (Schaeffer
et al., 2003
). Animals performed these tests beginning the day
following completion and within 5 days of the end of the experimental
protocol, running at one speed per day. During this period, animals were
maintained at their acclimation temperature and also performed the
O2max and
O2summit tests
described in Schaeffer et al.
(2003
). Each exercise test
consisted of
20 min running at a constant speed and energetic cost of
transport (Ct) was determined using the average value from
the final 5 min of steady state recording. Ct was
calculated as the ratio of rate of oxygen consumption (in ml O2
kg1min1) over treadmill speed (m
min1), yielding the oxygen consumed per unit of mass per
distance traveled (ml O2 kg1
m1). Treadmill speed was calibrated before every run and the
O2 system was calibrated after every second or third run using the
N2 dilution method of Fedak et al.
(1981
). All measurements of
oxygen consumption took place at an elevation of 2100 m (ambient
PBAR=600 torr; 1 torr=133.3 Pa) and data are reported
after correction to STPD values.
Isolated muscle preparation
Within 24 days after completion of the Ct
measurements (during which animals were maintained at acclimation
temperatures), animals were anesthetized with isoflourane and the
semitendinosus muscle was removed from one leg. Prior to muscle removal, silk
suture was tightly tied about the two ends of the muscle. Upon removal, the
muscle was suspended in a Krebs solution (118 mmol l1 NaCl,
4.7 mmol KCl, 2.5 mmol CaCl2, 1.18 mmol MgSO4 and 1.18
mmol KH2PO4, with 2.1 g l1
NaHCO3 and 2.0 g l1 dextrose added before use),
which was aerated with 95% O2 and 5% CO2. The muscle and
solution were held within a double-walled testing chamber maintained at
32°C (normal body temperature for this species) with a refrigerated
constant temperature circulating water bath (Model 1165, VWR, West Chester,
PA, USA). The muscle was anchored to a bar in the lower part of the testing
chamber and to a transducer system above, containing both a force and
displacement gauge (Cambridge Technology Inc., Cambridge, MA, USA; Model
6650). Muscles were activated using a field stimulator (Grass Telefactor, West
Warwick, RI, USA; Model S48) with the signal passing through a stimulus
isolation unit (Grass Telefactor; Model S1U5) to platinum electrodes (1
cmx2 cm) placed across the muscle. After the semitendinosus had been
removed, the animals were killed by exsanguination and skeletal muscle samples
removed and frozen in liquid nitrogen as previously described
(Schaeffer et al., 2003).
Measurement of mechanical properties
After the muscle was suspended within the apparatus, we first found the
minimum voltage that elicited peak force generation (maximal recruitment) of a
single twitch, then set the remainder of the stimulations to this voltage plus
20 V (typically about 100 V). We then determined the optimal length and
frequency of stimulation for the muscle, both measured when a single twitch
generated maximal force. These data were monitored using a digital
oscilloscope (Tektronix Inc., Beaverton, OR, USA; TDS 340A) and the muscle was
allowed to recover for at least 15 s between each stimulation. For both twitch
and tetanus data, the transducer output was processed through an Aurora
Scientific Muscle Lever System (Aurora Scientific Inc., Aurora, Ontario,
Canada; Model 305B) and recorded digitally using SuperScope2TM, ver. 2.17
sampling at 2000 Hz.
The muscle was then subjected to 46 twitches, with the lever set to
maximum resistance to motion (isometric contractions), each a 2 ms single
square wave pulse, for determination of time to peak tension (TPT), and
half-relaxation time (RT) of a single twitch. To determine the time
required to generate each unit of force, as well as the time required for each
unit of force decay, we divided TPT and
RT by the peak force (in g)
for each twitch. These data are reported in ms g1.
Next, the stimulator delivered a single 200 ms train of 2 ms pulses at 200
Hz to elicit a tetanus. Two to three tetani were imposed to determine peak
tetanic tension (P0). At this point, optimal length was
checked and adjusted if necessary. We then collected a series of isotonic
tetani with decreasing load to determine maximal contraction velocity;
typically this generated 1015 points. The muscle was allowed to recover
for at least 2 min between each tetanus. The recordings of tetanic
contractions from P0 to minimal loading were used to
calculate vmax using the Hill equation described in Roy et
al. (1984). Force and speed
(dL/dt) were determined for each point, then plotted as
y=(P0P)/v vs x=P,
where P is the measured force and v is the calculated speed. A
regression line was fit to the data with less than 35% P0.
vmax was calculated as P0 divided by the
y-intercept extrapolated from the regression analysis.
Finally, the muscle was subjected to a fatigue test under isometric
conditions consisting of repeating trains of stimuli at 1 s1
at 40 Hz for 2 min, with forces being recorded in real time from the
oscilloscope. Each train lasted 330 ms, thus the muscle was activated for
s and allowed to recover for
s. The highest force generated
(usually the 4th6th tetanus) was recorded, as was
the force generated by the last tetanus. The muscle fatigue index (a measure
of muscle endurance) was calculated as the ratio of final force generation
divided by peak force generation during the fatigue test. This value was
multiplied by 100 to give the percent of force generation that remained after
the stimulation protocol.
Amplification and sequencing of the Md-UCP3 cDNA
Total RNA was isolated from Monodelphis domestica hindlimb muscle
using the RNAzol method (Tel-Test, Inc., Friendswood, TX, USA). Following
reverse transcription, PCR was performed using primers designed based on
sequence homology with the UCP3 gene of cow, pig, dog and rat as follows:
sense, 5'-GGCCCCCGCAGCCCCTACAACGG-3'; antisense,
5'-CTGGACTTTCATCAAGGCCCGTTTCA-3'. PCR cycles were as follows:
95°C for 5 min; 95°C for 1 min, 52°C for 2 min, 72°C for 3
min, 30 cycles. The product was electrophoresed, gel isolated and cloned into
the TOPO-TA plasmid (Invitrogen, Carlsbad, CA, USA). The plasmid was
transformed into E. coli cells, amplified and sequenced by the
Protein and Nucleic Acid Chemistry laboratory at Washington University School
of Medicine using the Big Dye cycle sequencing kit (Applied Biosystems, Foster
City, CA, USA). The resultant cDNA fragment is designated Md-UCP3.
RNA blot analyses
15 µg total RNA was electrophoresed in a denaturing agarose gel,
transferred to a GeneScreen membrane (PerkinElmer Life Sciences, Boston, MA,
USA) and fixed by UV radiation. Northern blot analysis was performed with
QuickHyb (Stratagene, La Jolla, CA, USA) using random primed
32P-labeled cDNA probes, derived from the PCR fragment of Md-UCP3.
Bands were detected by phosphor-imaging using a GS 525 Molecular Imager System
(Bio-Rad Laboratories, Hercules, CA, USA).
Statistics
Cost of transport was analyzed using analysis of covariance (ANCOVA), with
running speed as the covariate. Muscle contractile parameters were tested for
statistical significance using two-way analysis of variance (ANOVA), with
temperature and training status as the dependent variables, using Sigma-Stat
(version 2.03, SPSS Inc., Chicago, IL, USA). For those parameters that showed
significant differences, pairwise comparisons were run using the
StudentNeumanKeuls method. The level of significance was set at
P<0.05 in all cases. Data are reported as means ± standard
error of the mean (S.E.M.).
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Results |
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Twitch parameters
Force generation of isolated, perfused semitendinosus muscles during a
single twitch (Pt) was significantly lower in the
cold-acclimated animals, and lowest in the CS group (two-way ANOVA,
temperature effect P<0.01, training effect P=0.17;
Table 1). We therefore divided
the time to peak tension (TPT) and the half relaxation time (RT) by
Pt to determine the time of force generation and relaxation
per gram of force developed (TPTg and
RTg,
respectively, in ms g1). The TPTg was
significantly longer in the cold-acclimated but not the exercise-trained
animals (two-way ANOVA, temperature effect P<0.05, training effect
P=0.10). The TnS and TnT groups did not differ. The CS group did not
differ from either warm group or from the CT group
(Fig. 2A).
|
|
Similarly, cold-acclimated animals had significantly prolonged
RTg (two-way ANOVA, temperature effect P<0.01,
training effect P=0.27). The pairwise comparisons showed the same
pattern of differences as with TPTg, with the TnS and TnT groups
nearly identical, while the CS group was intermediate and did not differ from
either warm group or from the CT group
(Fig. 2B).
Maximal contraction velocity
Muscle maximal contraction velocity (vmax) of the
semitendinosus muscle, expressed as muscle lengths per second (ML
s1), was significantly lower with both treatment effects
(two-way ANOVA, temperature effect P<0.01, training effect
P<0.05). There was a trend toward lower vmax
with either exercise or cold acclimation alone, although neither differed
significantly from the TnS animals. Combined cold acclimation with exercise
training resulted in a significantly slower muscle contraction
(Fig. 3).
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Muscle fatigability
The fatigue index was measured as the ratio of the final force generated by
semitendinosus muscles after a series of tetanic contractions over the peak
force measured: this is thus a measure of the endurance, or fatigue
resistance, of the muscle. The fatigue index was significantly greater
following cold acclimation, but not exercise training (two-way ANOVA,
temperature effect P<0.01, training effect P=0.14). The
low fatigue index observed in the semitendinosus muscle of the TnS animals
indicated that only a small fraction of their force-generating capacity was
maintained. Both exercise-trained and cold-acclimated animals showed slightly,
but non-significantly, higher fatigue indices. The fatigue index of the CT
group was highest, thus their semitendinosus muscles possessed significantly
more fatigue resistance than either warm-exposed group
(Fig. 4).
|
|
Northern blot analysis revealed an increase of expression of Md-UCP3 in skeletal muscle of cold-acclimated animals. Md-UCP3 was undetected in thermoneutral-acclimated animals, both sedentary and exercise-trained, but present in the muscle of all cold-acclimated animals, demonstrating a dramatic upregulation of gene expression (Fig. 5).
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Discussion |
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We did not distinguish between shivering and non-shivering thermogenesis in
these experiments and although skeletal muscle structure and function is
clearly altered in cold-acclimated animals, the liver may also contribute to
thermogenesis (Villarin et al.,
2003). The importance of non-shivering thermogenesis in rodents is
demonstrated by mice lacking ucp1 gene expression and thus BAT
function. Upon acclimation to cold, rodents no longer rely upon shivering, but
when ucp1 null animals are kept at 4°C, shivering thermogenesis
continues and is sufficient to maintain body temperature for several weeks,
although the animals fail to survive long term
(Golozoubova et al., 2001
).
Thus during long-term cold exposure, shivering thermogenesis is insufficient
to sustain life. As BAT supplies the majority of thermogenic effort, in deer
mice running
O2max is only
slightly increased by cold exposure while
O2summit is
greatly increased. Surprisingly, Chappell and Hammond
(2004
) recently reported that
cold-acclimated deer mice show very little increase in
O2max when
running at very cold temperatures, arguing for a competition between
locomotion and thermoregulation. This competition may be of importance to
Monodelphis domestica as well, given their reliance upon muscle for
thermogenesis.
The Ct reported here for Monodelphis domestica
acclimated to thermoneutral conditions is similar to that reported for similar
sized kangaroo rats (Taylor et al.,
1970). Further, the calculated minimum cost of transport of
Monodelphis domestica (
1.5 ml O2
kg1 m1), is also very close to that
predicted for a 100 g mammal (Taylor et
al., 1970
). In contrast, Didelphis virginianum, the North
American opossum, has a higher than predicted Ct, due to
adaptations for arboreal locomotion
(Fournier and Weber, 1994
).
However, Monodelphis domestica are primarily terrestrial, thus the
elevated Ct of Didelphis virginianum is likely a
derived characteristic typical of arboreal marsupials while Monodelphis
domestica represents a more basal condition. The elevation in
Ct with cold acclimation was greatest at slower speed and
decreased with increased running speed such that the calculated minimum
Ct (at top speed) was similar to thermoneutral-acclimated
animals. The reduction in difference in Ct with increased
running speed suggests that inefficient oxidative respiration is of greater
magnitude at lower overall respiration rates. This is in agreement with the
observation that the control of respiration in skeletal muscle shifts from
proton leak to greater coupling of ATP synthesis and utilization during
periods of higher demand (Kunz,
2001
). The elevated Ct observed in these
animals after cold acclimation could be due to numerous potential mechanisms,
including increased ATP consumption rates at terminal ATPases without a
corresponding work increase (futile cycles), uncoupling of oxygen consumption
from ATP production in the mitochondria via either increased proton
leak or uncoupling protein action, and cycling of metabolic substrates without
progression to ATP production.
Our measurements of single twitch kinetics and vmax in
isolated semitendinosus demonstrate that cold acclimation served as a strong
stimulus for fast-to-slow transition in mechanical properties. Both
Ca2+ cycling costs (Clausen et
al., 1991) and myosin ATPase activity
(Barany, 1967
;
Rome and Lindstedt, 1997
) are
lower in slow twitch fibers, suggesting that those muscles have acclimated
toward a reduction in peak rates of energy use. Thus the mechanical properties
of the cold-acclimated muscles argue for decreased Ct,
i.e. the opposite of what was observed. However, endurance exercise training
also leads to a fast-to-slow shift in mechanical properties
(Booth and Baldwin, 1997
), and
endurance training has not been associated with alterations in whole organism
locomotor efficiency. That the difference in Ct decreases
with increased running speed further suggests that inefficient energy
transduction at the primary terminal ATPases is not the principal cause of
increased oxygen consumption during locomotion following cold acclimation.
We observed greater fatigue resistance in the isolated semitendinosus
muscles, in accordance with our previous report that cold acclimation led to
increased mitochondrial volume in skeletal muscle of Monodelphis
domestica (Schaeffer et al.,
2003). While mitochondrial biogenesis alone does not lead to
altered Ct, it does provide the structural machinery
essential for thermogenesis. Uncoupling of oxygen consumption from ATP
production in the mitochondria, whether due to uncoupling proteins
(Rolfe and Brown, 1997
) or
increased proton leak (Brand et al.,
1994
), during locomotion should result in an upward shift in
O2 at any given
speed, consistent with the pattern observed in
Fig. 1.
We identified a homologue of UCP3 in Monodelphis domestica
(Md-UCP3) whose expression is upregulated in skeletal muscle in response to
cold acclimation. UCP3 has been identified as the major uncoupling protein
isoform expressed in skeletal muscle (Boss
et al., 1997; Vidal-Puig et
al., 1997
). UCP3 has been implicated in uncoupled metabolism and
may play a role in the increased cost of transport observed in these animals;
however, the capacity of UCP3 for uncoupling metabolic activity is
controversial (Porter, 2001
;
Nedergaard and Cannon, 2003
).
UCP3 is able to facilitate proton translocation in isolated skeletal muscle
mitochondria, albeit at low rates, leading to the suggestion that its primary
role is in protection against reactive oxygen species
(Echtay et al., 2002
). UCP3 is
also induced in muscle in response to fasting
(Millet et al., 1997
;
Gong et al., 1997
;
Boss et al., 1998
), a condition
in which uncoupled respiration would be expected to be diminished. It has been
previously reported that UCP3 is not upregulated in rodent skeletal muscle
following cold acclimation (Boss et al.,
1997
,
1998
), but Toyomizu et al.
(2002
) reported that UCP3
expression is induced in the skeletal muscle of cold-exposed chickens. More
recently, UCP3 was not induced in Antechinus flavipes, a similar
sized Australian marsupial, although the period of cold exposure was short
(Jastroch et al., 2004
). Thus
although equivocal, a role for UCP3 in muscle thermogenesis may be better
elucidated in organisms in which skeletal muscle plays a primary role in
thermogenesis.
We recently reported that the respiratory exchange ratio was decreased in
Monodelphis domestica following cold acclimation
(Schaeffer et al., 2003). Thus
increased UCP3 expression in these animals is associated with both increased
oxygen consumption and lipid oxidation. Further experiments will be required
to assess whether induction of UCP3 expression in cold acclimation functions
to facilitate greater lipid oxidation and protection from reactive oxygen
species or directly leads to metabolic uncoupling during locomotion. Increased
lipid oxidation may also be associated with substrate cycling of
triacylglycerol and fatty acids
(Newsholme, 1978
), leading to
increased thermogenesis. This process can be activated by the hormone leptin,
leading to an increase in metabolic rate comparable to that observed in
cold-acclimated Monodelphis domestica during locomotion
(Reidy and Weber, 2002
).
These data demonstrate that the efficiency with which metabolic and
mechanical power output are coupled is responsive to experimental
manipulations targeting metabolic function. Metabolic power output is altered
by cold acclimation, resulting in increased whole animal aerobic capacity
(Schaeffer et al., 2003), but
decreased efficiency of energy utilization during locomotion. This `training
for thermogenesis' led to an apparent reduction in the coupling of oxygen
consumption and ATP production during locomotion, but not increased ATP
utilization via decreased efficiency. Reduction of efficiency of the
terminal ATPases would not be expected to accompany the robust fast-to-slow
fiber type transition seen in all contractile parameters measured. These
results are in agreement with prevailing evidence, which indicates that the
coupling of most metabolic reactions does not change, with the exception of
the uncoupling of mitochondrial ATP synthesis
(Rolfe and Brown, 1997
). Thus,
as observed in whole animal studies, training for mechanical efficiency
appears to have little potential to influence Ct, while
cold acclimation serves as a potent stimulus of muscle plasticity, altering
muscle function in the absence of BAT thermogenesis. Cold acclimation of
skeletal muscle represents a `training for inefficiency' that must balance the
need to maintain contractile function with the demands of thermogenesis,
apparently driving a profound fast-to-slow fiber type shift and enhancing
energy-wasting metabolic processes. We propose that Monodelphis
domestica, in which marked induction of endogenous skeletal muscle UCP3
occurs with a cold acclimation, is an excellent model for the assessment of
UCP3 function.
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List of abbreviations |
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Acknowledgments |
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Footnotes |
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References |
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