Substrate utilization during graded aerobic exercise in rainbow trout
1 Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S
4K1
2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8S
4K1
* Author for correspondence (e-mail: richarjg{at}mcmail.cis.mcmaster.ca )
Accepted 18 April 2002
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Summary |
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Key words: Swimming, red muscle, white muscle, pyruvate dehydrogenase, lipid, carbohydrate, lactate shuttling, malonyl-CoA, rainbow trout, Oncorhynchus mykiss
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Introduction |
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In addition to the spatial separation and different recruitment patterns of
red and white muscle, the two muscle types have different substrate
preferences. Red muscle contraction relies heavily on ATP generated from
mitochondrial oxidative phosphorylation (maximum ATP production of 29 µmol
ATP g-1 wet tissue min-1;
Moyes and West, 1995).
Mitochondria isolated from red muscle of carp (Cyprinus carpio) have
a very high capacity to oxidize pyruvate, fatty acids and some amino acids
(Moyes et al., 1989
).
Conversely, white muscle mitochondrial ATP production is very low (approx. 3.5
µmol ATP g-1 wet tissue min-1;
Moyes et al., 1992
),
therefore, white muscle relies primarily on creatine phosphate (CrP)
hydrolysis and glycolysis (i.e. substrate level phosphorylation) for ATP
production. However, during recovery from high-intensity exercise, white
muscle mitochondria oxidize lipids to fuel CrP and glycogen synthesis
(Moyes et al., 1992
;
Richards et al., 2002a
). Thus,
it is generally thought that at swimming speeds
70-80%
Ucrit red muscle contraction and swimming will be
supported primarily by oxidative utilization of fuels, and as swimming speed
increases there will be greater reliance on ATP production by substrate level
phosphorylation in white muscle.
Despite the variety of substrates oxidized by red muscle mitochondria,
considerable debate surrounds the pattern of substrate selection in fish
swimming at speeds Ucrit (see
Moyes and West, 1995
).
Classically, protein and lipids were considered to be the major fuels oxidized
during sustained swimming and carbohydrate utilization was considered to be
minimal (Dreidzic and Hochachka, 1978;
Jobling, 1994
). However, Lauff
and Wood (1996
), using
respirometry, demonstrated that (i) juvenile rainbow trout primarily oxidize
lipid during swimming at 55% and 85% Ucrit, (ii)
carbohydrate oxidation is the next most important, and (iii) protein oxidation
is minimal. White muscle glycolytic utilization of carbohydrate contributes to
ATP turnover during the swimming fast-start
(Wokoma and Johnson, 1981
) and
at speeds
70% Ucrit
(Burgetz et al., 1998
). In
addition, whole body oxidative utilization of carbohydrate increases as
swimming speed approaches Ucrit
(Lauff and Wood, 1996
).
The objective of the present study was to determine the biochemical
pathways involved in carbohydrate and lipid utilization by red and white
muscle of rainbow trout while swimming at speeds corresponding to 30, 60 and
90% Ucrit. Furthermore, insights into the regulation of
lipid and carbohydrate oxidation were gained through measurements of pyruvate
dehydrogenase (PDH) and malonyl-CoA. Previously, we demonstrated the integral
role of PDH transformation in regulating carbohydrate and lipid metabolism in
white muscle during a bout of high-intensity exercise
(Richards et al., 2002b) and
during recovery from exhaustive exercise
(Richards et al., 2002a
). In
the present study we measured the activity of PDH in both red and white
muscle, and changes in oxidative metabolites (e.g. acetyl-CoA and carnitine)
and glycolytic intermediates in red muscle during graded swimming, in an
attempt to determine whether lipid or carbohydrate was oxidized during graded
swimming.
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Materials and methods |
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Critical swimming speed (Ucrit)
In order to establish the swimming capacity of the fish under study,
critical swimming speed (Ucrit) was determined on all fish
at least 2 weeks before experimentation, according to the methodology outlined
by Brett (1964). Briefly,
groups of 4-6 fish were introduced into a Beamish-style swim tunnel (volume
1561) and allowed to acclimatize for 1 h at a linear water velocity of 10 cm
s-1 at 15 °C. This water velocity caused the fish to orient
into the current, but all fish maintained station at the bottom of the tunnel
with only periodic tail movements. After the acclimatization period, swimming
speed was increased by 10 cm s-1 every 30 min until each fish
fatigued. Fatigue was established when a trout fell against the back screen of
the swim tunnel three consecutive times after being manually re-introduced
into the current. Ucrit was then calculated by
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Experimental protocol
On the day of an experiment, 2-3 fish were transferred into the swim tunnel
and allowed to acclimatize for 1 h at a swimming speed of 10 cm
s-1. At the end of the acclimatization period, water velocity was
increased over a 5 s period to one of three swimming speeds, corresponding to
30% (20 cm s-1), 60% (41 cm s-1) and 90% (62 cm
s-1) Ucrit. Fish swum at 30% and 60%
Ucrit for 2, 15 or 240 min (4 h), but at 90%
Ucrit for only 2, 15 and 45 min, because at the high speed
they fatigued between 50 and 80 min (N=9). During swimming at 90%
Ucrit, periodic prodding was required to prevent fish from
resting against the rear grid of the swim tunnel. Control fish were swum at 10
cm s-1 for either 2 min or 240 min after the initial
acclimatization period. There were no differences between trout sampled at
either control time, therefore in all figures and tables the controls are
combined into a single point.
To sample fish while swimming, 25 ml clove oil (Sigma-Aldrich;
Anderson et al., 1997) was
introduced into the 1561 swim tunnel, resulting in anaesthesia after 2-4 min,
when the fish fell against the back screen. Clove oil anaesthesia is an
effective fish anaesthetic that has no detrimental effects on
Ucrit in juvenile or adult trout
(Anderson et al., 1997
). Fish
continued to swim during the onset of anaesthesia and only fell against the
back screen within the final 10-25 s. Any fish that struggled were discarded.
At complete anaesthesia, the fish were manually removed from the tunnel and a
section of trunk extending from posterior to the dorsal fin to the anterior
side of the anal fin was excised from each fish and immediately freeze-clamped
between two metal plates pre-cooled in liquid N2. Muscle sampling
took less than 15 s. All samples were stored at -86 °C until analysis.
Analytical techniques
Frozen muscle was broken into small pieces (50-100 mg) under liquid
N2 in an insulated mortar and pestle. Care was taken to obtain only
white muscle from the area dorsal to the lateral line and red muscle along the
lateral line. Several pieces of red and white muscle were stored separately in
liquid N2 for determination of PDH activity by radiometric assay,
as previously described (Richards et al.,
2002a). The remaining broken muscle was lyophilized for 72 h, then
red and white muscle fibers were separated by careful dissection, cleaned of
blood, bone and connective tissue, and each stored dry at -86 °C for
subsequent analysis.
For extraction of metabolites from red and white muscle, samples of
lyophilized muscle (approx. 10 mg for red muscle, 20 mg for white muscle) were
weighed into borosilicated tubes, 1 ml of ice-cold HClO4 (1 mol
l-1) was added, and the suspension was homogenized at the highest
speed of a Virtis hand-held homogenizer for 20 s at 0 °C. Red and white
muscle homogenates were vortexed and 200 µl of homogenate slurry was
removed, placed into a 1.5 ml centrifuge tube and frozen at -86 °C for
determination of glycogen. The remaining homogenate was centrifuged for 5 min,
4,800 g at 4 °C and the supernatant was neutralized with 3
mol l-1 K2CO3. The neutralized extract was
centrifuged for 5 min at 20 000 g at 4 °C and the
supernatant was analyzed immediately for ATP, CrP and lactate. Red muscle
extracts were further assayed fluorometrically for glucose, glucose
6-phosphate (Glu 6-P), fructose 6-phosphate (Fru 6-P), glucose 1-phosphate
(Glu 1-P), glycerol 3-phosphate (Gly 3-P) and glycerol. All these assays
followed methods described (Bergmeyer,
1983) that were modified for fluorometry. Red muscle extracts were
also analyzed for acetyl-CoA, free CoA (CoA-SH), and acetyl-, free-,
short-chain fatty acyl- (SCFA), long-chain fatty acyl- (LCFA) and total
carnitine by radiometric methods previously described
(Richards et al., 2002a
). Red
muscle intramuscular triacylglycerol (IMTG) was determined on lyophilized
tissue as described (Keins and Richter,
1996
). Malonyl-CoA was determined by high-performance liquid
chromatography on a separate extraction as described
(Richards et al., 2002a
).
Respirometry
To determine oxygen consumption
(o2) in control fish and
fish swimming at 30 % and 60 % Ucrit, we used an identical
acclimatization and swimming procedure as outlined above. Oxygen consumption
was not determined on fish swimming at 90 % Ucrit because
our metabolic data indicated that the fish were not in steady state at this
swimming speed. In view of the high volume of the swim tunnel it was necessary
to swim fish in groups to achieve measurable changes in water
Po2. Briefly, four fish were introduced into the swim
tunnel and allowed to acclimatize for 1 h at a flow speed of 10 cm
s-1. At the end of the acclimatization period, water speed was
either increased to 30 % or 60 % Ucrit or left at the
control speed. After 2 h of swimming at the pre-determined speed, the swim
tunnel was sealed from the atmosphere and an initial water sample was taken.
Fish were swum for an additional 1.5-2 h, at which point another water sample
was taken. Water samples (4 ml) were taken into gas-tight syringes and
Po2 was measured using a Radiometer E5046 oxygen electrode
thermonstatted to 15 °C and attached to a Cameron Instruments OM-200
meter. Oxygen consumption, in µmol kg-1 wet mass
min-1, was calculated according to the Fick Principle:
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Data presentation and statistical analysis
All data are presented as means ± S.E.M. (N, number of fish
for all metabolite data or
o2 measurements). All
muscle metabolite concentrations determined on lypohilized tissue were
converted back to wet masses by taking into account a wet:dry ratio of 4:1
(Wang et al., 1994
).
Statistical analysis was a one-way analysis of variance (ANOVA) followed by a
least-significant difference method of pairwise multiple comparisons. Results
were considered significant at P<0.05.
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Results |
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Red muscle
Creatine phosphate and adenylates
Red muscle [CrP] did not change in trout swum at 30 %
Ucrit for up to 240 min
(Fig. 2A). In trout swimming at
60 % Ucrit, there was a 26 % drop in [CrP] at 2 min, but
by 15 min levels had returned to control values and remained unchanged for up
to 240 min. At 90 % Ucrit, there was a 54 % decrease in
[CrP] at 2 min, which remained depressed compared to control values for up to
45 min. Swimming speed or duration had no effect on red muscle [ATP]
(Fig. 2B).
|
Glycogen, glycolytic intermediates and intramuscular triacyl
glycerol
Swimming at 30 % Ucrit for 240 min or at 60 %
Ucrit for up to 15 min had no effect on red muscle
[glycogen] (Fig. 3A). However,
after 240 min of swimming at 60 % Ucrit there was a
significant 34 % decrease in [glycogen] compared to levels in control trout.
At 90 % Ucrit, there was a significant 46 % decrease in
[glycogen] after 15 min of swimming, which remained depressed by 57 % compared
to control trout at 45 min. Red muscle [lactate] did not change in trout swum
at 30 or 60 % Ucrit for up to 240 min
(Fig. 3B). However, after
swimming for 2 min at 90 % Ucrit, red muscle [lactate] had
increased 3.6-fold, and remained elevated and stable compared to levels in
control trout for up to 45 min. The rise in red muscle [lactate] (approx. 1.6
µmol g-1 wet tissue) was slight relative to the fall in
[glycogen] (approx. 9 µmol glucosyl units g-1 wet tissue = 18
µmol lactate units g-1 wet tissue).
|
Red muscle [glucose] remained constant compared to controls in trout swimming at 30 and 60 % Ucrit for up to 240 min (Fig. 4A). At 90 % Ucrit, muscle [glucose] remained at control values for the first 2 min, then increased by 1.9-fold by 15 min and remained elevated for up to 45 min. There was no effect of swimming at 30 % Ucrit on red muscle [Glu 6-P] (Fig. 4B). At 60 % Ucrit, muscle [Glu 6-P] increased 2.2-fold within 2 min, but returned to control values by 15 min and remained low until 240 min. In trout swimming at 90 % Ucrit, [Glu 6-P] increased 6.2-fold and gradually returned to control values by 45 min. Swimming at 30 and 60 % Ucrit for up to 240 min did not affect red muscle [Fru 6-P] except for a small, but significant, decrease after 240 min of swimming at 30 % Ucrit (Table 1). Swimming at 90 % Ucrit caused an initial 2.3-fold increase in [Fru 6-P], which returned to control values by 15 min and remained low at 45 min. Red muscle [Glu 1-P] was below detection in all fish regardless of swimming speed or duration (data not shown). Red muscle [Gly 3-P] did not change in trout swimming at 30 % Ucrit for up to 240 min (Table 1). However, at 60% Ucrit there was a significant 27-fold increase in [Gly 3-P] after 15 min of swimming, which decreased to control values by 240 min. In trout swimming at 90% Ucrit there was a 29-fold increase in [Gly 3-P], which further increased to 55-fold at 45 min. Swimming speed or duration had no effect on red muscle [glycerol] except in fish swimming at 90% Ucrit for 45 min, where [glycerol] was 2.6-fold higher than in control fish. Swimming speed or duration had no effect on red muscle IMTG levels (Table 1).
|
|
Pyruvate dehydrogenase activity
During the first 2 min of swimming, red muscle PDH activity increased by 4,
8 and 12-fold in trout swimming at 30, 60 and 90% Ucrit,
respectively (Fig. 5). In trout
that continued swimming at 30 and 60% Ucrit, PDH activity
returned to values that were not significantly different from those in control
fish, and remained low for the full 240 min of swimming. However, in trout
that continued swimming at 90% Ucrit, PDH activity
remained approximately 10- to 12-fold higher than in control trout throughout
the swimming period.
|
Acetyl group accumulation and carnitine
In general, swimming speed and duration had no effect on red muscle CoA-SH
levels except at 15 min, where trout swimming at 60 and 90%
Ucrit exhibited significantly lower (approx. 30%) CoA-SH
levels compared to control trout and trout swimming at 30%
Ucrit (Table
1). During the first 2 min of swimming, red muscle [acetyl-CoA]
increased by 1.5-, 2.6- and 3.6-fold in trout swimming at 30, 60 and 90%
Ucrit, respectively, and remained at these elevated levels
throughout the swimming period (Fig.
6A). Similarly, during the first 2 min of swimming, there were
2.1-, 6.0- and 7.5-fold increases in [acetyl-carnitine] in trout swimming at
30, 60 and 90% Ucrit, respectively
(Fig. 6B). These elevations in
[acetyl-carnitine] remained constant for the duration of the swimming.
|
Swimming speed and duration had no effect on red muscle total carnitine concentration, except for a minor, but significant elevation after 2 min of swimming at 60% Ucrit (Fig. 7A). Red muscle free-carnitine concentration decreased by 18%, 56% and 59% in trout swimming for 2 min at 30, 60 and 90% Ucrit, respectively, and remained close to these values throughout the swimming period (Fig. 7B). Swimming speed or duration had no effect on red muscle SCFA-carnitine concentration, except that trout swimming at 90% Ucrit for 15 min had significantly lower SCFA-carnitine levels than trout swimming at 30% Ucrit (Table 1). [LCFA-carnitine] remained constant compared to the levels in control trout for the first 15 min of swimming at 30% Ucrit, then increased 1.7-fold by 240 min (Fig. 7C). At 60% Ucrit, there was an initial 1.6-fold increase in [LCFA-carnitine] at 2 min, and a 2.5-fold increase by 240 min. Swimming at 90% Ucrit for 45 min did not affect [LCFA-carnitine].
|
Red muscle [malonyl-CoA] did not change within the first 2 min of swimming at any of the three swimming speeds (Fig. 8). However, by 15 min, there were significant 55% decreases in [malonyl-CoA] in fish swimming at 30 and 60% Ucrit. [Malonyl-CoA] remained at these levels for up to 240 min of swimming at these two speeds. In contrast, in trout swimming at 90% Ucrit, there was a gradual decrease in muscle [malonyl-CoA] to values that were 36% lower than in control fish at 45 min.
|
White muscle
White muscle [CrP] did not change in trout swimming at 30%
Ucrit for up to 240 min or in trout swimming at 60%
Ucrit for up to 15 min
(Fig. 9A). At 240 min of
swimming at 60% Ucrit, there was a 43% decrease in [CrP].
At 90% Ucrit, muscle [CrP] decreased by 33% at 2 min and
67% at 45 min. White muscle [ATP] did not change in trout swimming at 30 or
60% Ucrit for up to 240 min
(Fig. 9B). At 90%
Ucrit, white muscle [ATP] remained at control values for
the first 15 min of swimming, but decreased by 46% at 45 min.
|
White muscle glycogen levels remained relatively stable in trout swum at 30% Ucrit, except for a small, but significant decrease at 15 min, which recovered by 240 min (Fig. 10A). There was an initial 18% decrease in white muscle [glycogen] at 2 min of swimming at 60% Ucrit, which decreased by a further 34% by 240 min. At 90% Ucrit, there was a 33% decrease in [glycogen] at 2 min, which continued to decrease (by 73%) at 45 min. There were no changes in white muscle [lactate] in trout swum at 30 and 60% Ucrit for 240 min (Fig. 10B). Trout swum at 90% Ucrit for 2 min showed a 3.8-fold increase in white muscle [lactate], which further increased at 15 min to 7.3-fold the control values, then remained constant until 45 min. This rise in white muscle [lactate] at 90% Ucrit (approx. 8 µmol g-1 wet tissue) only accounts for approx. 30% of the relative fall in white muscle [glycogen] (approx. 13 µmol glycosyl units g-1 wet tissue =approx. 26 µmol lactate units g-1 wet tissue).
|
Swimming speed or duration had no effect on white muscle PDH activity: mean PDH activity was 39.8±4.4 nmol g-1 wet tissue min-1 (N=35; data not shown).
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Discussion |
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Our data generally agree with previous studies suggesting that mainly red
muscle is used to power swimming at speeds up to 70% or 80%
Ucrit, beyond which both red and white muscle contribute
(Hudson, 1973;
Johnson, 1981
;
Wilson and Egginton, 1994
;
Burgetz et al., 1998
). At all
swimming speeds, there were some changes in red muscle metabolites (e.g. Figs
2A,
6A,B) and PDH activity
(Fig. 5), indicating that red
muscle contributes to power production during swimming. During sustained
swimming, there were moderate decreases in white muscle [CrP] after 240 min at
60% Ucrit (Fig.
9A), and small decreases in white muscle [glycogen] at 2 min and
240 min at 60% Ucrit, and at 15 min at 30%
Ucrit (Fig.
10A). These minor decreases in [CrP] and [glycogen] suggest that
there is an early but minor recruitment of white muscle during sustainable
swimming, possibly to power bursts. The larger depletions of white muscle
[CrP] observed over 45 min at 90% Ucrit
(Fig. 9A), along with decreases
in white muscle [ATP] (Fig. 9B)
and [glycogen] (Fig. 10A),
point to a substantial recruitment of white muscle during non-sustainable
swimming. These results are in general agreement with those of Burgetz et al.
(1998
) who demonstrated a
significant contribution of white muscle `anaerobic' metabolism in trout at
70% Ucrit. The recruitment of white muscle probably
acts to enhance swimming performance by providing greater power output, but
may also play a role in supplying lactate to the red muscle for oxidation,
particularly at high swimming speeds
(Moyes and West, 1995
). Note
that lactate buildup was less than glycogen depletion in white muscle at 60
and 90% Ucrit (Fig.
10A,B), suggesting that lactate was exported for oxidation
elsewhere.
Sustainable swimming
During sustainable swimming at 30% and 60% Ucrit there
were increases in
O2 and thus the
oxidative generation of ATP (Fig.
1). Our
O2 data agree
well with those previously reported for trout at rest (90 µmol
kg-1 body mass min-1) and during swimming at 45% and 75%
Ucrit (107 and 158 µmol kg-1 body mass
min-1, respectively; Kieffer et
al., 1998
). Similarly, Lauff and Wood
(1996
) found resting rainbow
trout had an
O2
of 92 µmol kg-1 body mass min-1, which increased to
150 µmol kg-1 body mass min-1 at 55%
Ucrit. This increase in
O2 with
increasing swimming speeds probably reflects an increase in red muscle ATP
turnover from oxidative phosphorylation, which occurs in the absence of
changes in red muscle [ATP] (Fig.
2B) and with only very minor changes in red muscle [CrP]
(Fig. 1A) and [glycogen]
(Fig. 3A).
Substrate utilization at 30% and 60% Ucrit can be
divided into two phases: an initial acclimatization period that relies on
carbohydrate oxidation, followed by a prolonged period that is characterized
by enhanced lipid oxidation. During the first 2 min at 60%
Ucrit there is an increase in glycolytic flux, indicated
by the accumulation of Glu 6-P (Fig.
4B), and a small, non-significant decrease in [glycogen]
(Fig. 3A). By analogy with the
data of Richards et al.
(2002b), this increase in
glycolysis was probably mediated through the transformation of the
rate-limiting enzyme glycogen phosphorylase into its active form. Enhanced
glycolysis, without an increase in lactate production
(Fig. 2B), yields sufficient
substrate for the speed-dependent increase in PDH activity
(Fig. 5). This increase in PDH
activity observed at 30 and 60% Ucrit indicates an
increase in the catalytic rate of PDH and tricarboxylic acid (TCA) cycle flux
and oxidative phosphorylation.
Pyruvate dehydrogenase is the rate-limiting enzyme for entry of
glycolytically derived pyruvate into the TCA cycle for ATP production
via oxidative phosphorylation. In mammals, PDH activity is regulated
by reversible covalent modification and by product inhibition
(Weiland, 1983). At the onset
of exercise, Ca2+ release from the sarcoplasmic reticulum probably
acts as the initial cue to activate red muscle PDH via a stimulation
of PDH phosphatase, which dephosphorylates and activates PDH. The increase in
PDH activity at 2 min of swimming (Fig.
5) resulted in product accumulation as acetyl-CoA
(Fig. 6A). However,
[acetyl-CoA] was kept relatively low within the mitochondria through the
formation of acetyl-carnitine (Fig.
6B), which can be transported from the mitochondrial matrix to the
cytoplasm. Total acetyl group accumulation accounted for only 7 and 19% of the
total PDH activity during the first 2 min at 30 and 60%
Ucrit, respectively, indicating a large increase in
acetyl-CoA oxidation.
During sustained swimming (15-240 min) at 30 and 60%
Ucrit, PDH activity
(Fig. 5) returned to control
values, indicating a relative reduction in carbohydrate oxidation and a shift
toward lipid oxidation. Inactivation of PDH after 15 min was probably
via a mechanism similar to that proposed by the glucosefatty
acid cycle in mammals (van der Vusse and
Reneman, 1996; Randle,
1998
). We have previously demonstrated that the regulation of PDH
transformation in fish white muscle during high-intensity exercise
(Richards et al., 2002b
) and
recovery (Richards et al.,
2002a
) is similar to that described in mammals. During submaximal
exercise, increase in ß-oxidation causes a sustained elevation in
acetyl-CoA/CoA-SH ratio (cf. Fig.
6A, Table 1) and an
increase in [NADH]/[NAD+]. These increases in acetyl-CoA and NADH
levels may override the stimulatory effects of Ca2+ on PDH
phosphatase. They may also reduce the transformation of PDH by stimulating PDH
kinase, resulting in the phosphorylation and deactivation of PDH. Increased
PDH kinase activity has been proposed to explain reduced carbohydrate
oxidation in rat red quadriceps muscle during sustained exercise
(Denyer et al., 1991
).
Sustained elevations of muscle [acetyl-CoA] would also inhibit PDH activity
in vivo through product inhibition.
During sustained exercise at 60% Ucrit (240 min) there
was a small, but significant, decrease in red muscle [glycogen]
(Fig. 3A), suggesting that some
carbohydrate utilization persisted. Based on measured red muscle PDH activity
observed during sustained swimming (15-240 min), and taking into account the
fact that red muscle constitutes 7% of the body mass
(Moyes and West, 1995),
carbohydrate oxidation at 30 and 60% Ucrit could account
for 47 and 44% of the
O2,
respectively. If we assume in the present study that 20% of the
O2 is due to
protein oxidation (from Lauff and Wood,
1996
), then the remaining increase in
O2 observed at
30 and 60% Ucrit (33 and 36% of
O2,
respectively) must be due to increased lipid oxidation. Thus, sustained
swimming is supported by the oxidation of approx. 45% carbohydrate, 35% lipid
and 20% protein. These relative increases in
O2 due to lipid
oxidation at 30 and 60% Ucrit can be supported by the
oxidation of only 4.4 and 7.3 µmol g-1 wet mass palmitate over
the swimming period (15-240 min). Oxidation of these low concentrations of
palmitate would yield changes in red muscle IMTG of only 1.5-2.4 µmol
glycerol units g-1 red muscle from 15 to 240 min, concentrations
that are well within the error of IMTG measurement
(Table 1). Therefore, in the
present study, no changes in IMTG were expected or observed.
During sustained swimming, however, there were small, but significant
increases in LCFA-carnitine levels at 240 min of swimming at 30 and 60%
Ucrit (Fig.
7C). In muscle, carnitine plays two major roles
(Brass, 2000). First, carnitine
acts to transport long-chain fatty acids into the mitochondria for
ß-oxidation. Second, excess acetyl groups from acetyl-CoA are bound to
carnitine, forming acetyl-carnitine, which keeps [acetyl-CoA] relatively low
within the mitochondria in order to sustain flux through ß-oxidation. In
trout red muscle, there is a clear reliance on carnitine to bind acetyl groups
from acetyl-CoA production (Figs
6,
7). However, the significant
increase in [LCFA-carnitine] observed during sustained swimming
(Fig. 7C) implicates long-chain
fatty acid oxidation as the source of acetyl groups. Short-chain fatty
acyl-carnitines are also oxidized at high rates in mitochondria isolated from
carp red muscle (Moyes et al.,
1989
), but we observed no increase in SCFA-carnitine levels during
exercise (Table 1). This does
not preclude the oxidation of short-chain fatty acids, however, because their
movement across the inner mitochondrial membrane is not necessarily dependent
on carnitine (van der Vusse and Reneman,
1996
).
The binding of long-chain fatty acids to carnitine is catalyzed by
carnitine palmitoyltransferase-1 (CPT-1), which is thought to be the
rate-limiting step in fatty acid oxidation
(van der Vusse and Reneman,
1996). Based on mammalian research, CPT-1 is thought to be
regulated in vivo by malonyl-CoA production
(Ruderman et al., 1999
).
Malonyl-CoA is the first committed step in fatty acid synthesis and is formed
by the acetyl-CoA carboxylase carboxylation of acetyl-CoA. During sustained
exercise at 30 and 60% Ucrit (
15 min), malonyl-CoA
concentrations decreased in trout red muscle
(Fig. 8) despite a sustained
elevation in acetyl-CoA (Fig.
6A). These decreases in [malonyl-CoA] would relieve the resting
inhibition of CPT-1 during sustained exercise and enhance fatty acid oxidation
through an increase in carnitine-dependent transport of fatty acids into the
mitochondria. In rat muscle, submaximal exercise reduces the concentration of
malonyl-CoA, as seen in trout red muscle
(Fig. 8), and relieves
inhibition of CPT-1. However, the response of CPT-1 to malonyl-CoA is not
consistent between species. In the human, submaximal exercise at 60%
VO2max does not cause a decrease in muscle
[malonyl-CoA], despite the fact that this exercise intensity is characterized
by enhanced lipid oxidation (Romijn et
al., 1993
). In trout, malonyl-CoA may play different roles in red
versus white muscle. We have previously demonstrated that during
recovery from high-intensity exercise, a period characterized by enhanced
white muscle lipid oxidation (low PDH activity, elevated acetyl-CoA, decreased
IMTG and increased LCFA-carnitine levels), white muscle [malonyl-CoA]
increases rather than decreases (Richards
et al., 2002a
). This increase in [malonyl-CoA] observed in white
muscle during recovery from high-intensity exercise may act to elongate fatty
acids for oxidation, as proposed by Richards et al.
(2002a
). These apparent
differences in the regulation of CPT-1 by malonyl-CoA in red and white muscle
of trout merit further research.
In the present study, we provide indirect biochemical evidence for lipid
oxidation during sustained exercise. Our results agree well with those of
Lauff and Wood (1996), who
assessed substrate use in juvenile trout by respirometry. However, direct
evidence for enhanced lipid oxidation during sustained exercise remains to be
demonstrated. Bernard et al.
(1999
) demonstrated that
triacylglycerol:fatty acid cycling is not enhanced during endurance exercise
in trout. However, basal rates of triacyglycerol:fatty acid cycle are high in
trout so could not limit fatty acid utilization during exercise.
Quantification of the absolute rates and relative contributions of endogenous
and exogenous lipids in fuelling sustained swimming needs to be addressed in
isolated muscles using pulse-chase techniques
(Dyke et al., 1997
).
Non-sustainable swimming
During swimming at 90% Ucrit, there was a sustained
elevation of red muscle PDH activity (Fig.
5) and a large depletion of red muscle glycogen and accumulation
of lactate (Fig. 3A,B),
indicating that trout rely heavily on carbohydrate as the substrate for
high-speed swimming. In particular, the high PDH activity indicates that
oxidative phosphorylation of carbohydrate is a major source of ATP at 90%
Ucrit. In addition, there was a significant contribution
of white muscle CrP (Fig. 9A)
hydrolysis and glycolysis (Fig.
10A,B) to total power output and a potential role of the white
muscle supplying substrate, as lactate, to the red muscle.
The contributions of lipid oxidation during swimming at 90%
Ucrit are likely to be smaller than at 30 and 60%
Ucrit. In the present study, we did not observe changes in
[LCFA-carnitine] in red muscle of trout swimming at 90%
Ucrit for 45 min and only demonstrated decreased red
muscle malonyl-CoA concentrations at 45 min of swimming. However, given the
large ATP turnover generated by a small amount of lipid oxidation, we cannot
rule out a contribution of lipid oxidation to non-sustainable exercise. These
data agree with those of Lauff and Wood
(1996), indicating a greater
reliance on carbohydrate as the carbon source during high-speed swimming at
80% Ucrit and a lower, but sustained utilization of lipid
fuels.
In trout swimming at 90% Ucrit, approx. 25% of the
substrate required to support the measured red muscle PDH activity could have
been supplied by endogenous glycogen, the remaining substrate being supplied
by hepatic glucose release or by white muscle lactate production
(Moyes and West, 1995).
However, Shanghavi and Weber
(1999
) have recently
demonstrated that hepatic glucose release does not contribute significantly to
red muscle ATP turnover in trout swimming at approx. 70%
Ucrit. Numerous studies have proposed that the large store
of white muscle glycogen could contribute substrate to red muscle during
steady-state exercise (e.g. Wokoma and
Johnson, 1981
). In the present study, the observed decrease in
white muscle glycogen at 60 and 90% Ucrit could not be
accounted for solely by white muscle lactate accumulation. Taking into account
the fact that white muscle PDH activity did not change with swimming speed or
duration (PDH activity was approx. 40 nmol g-1 wet mass
min-1), the difference between glycogen depletion and combined
lactate accumulation and oxidation reveals a discrepancy of 2.0 and 15.6
µmol g-1 white muscle lactate at 60 and 90%
Ucrit, respectively. Based on whole body calculations, if
this lactate were transported from the white muscle to the red muscle for
oxidation, the discrepancy could account for an additional 37% (0.06 µmol
g-1 red muscle min-1) and 211.5% (2.96 µmol
g-1 red muscle min-1) of the total substrate needed for
the measured red muscle PDH activity at 60 and 90% Ucrit,
respectively. However, based on estimates of lactate turnover in trout plasma
during swimming at 85% Ucrit (9.7 µmol kg-1
min-1; Weber,
1991
), the primary circulation has the capacity to shuttle 10% of
the substrate required. Clearly, more work is needed to resolve this apparent
discrepancy.
In conclusion, the present biochemical evidence indicates that sustained swimming in trout (30 and 60% Ucrit) is primarily supported by red muscle contraction where carbohydrate serves as the initial substrate for ATP production. During extended periods of sustained swimming there is a partial shift in substrate utilization away from carbohydrate to an activation of long-chain fatty acid oxidation. Non-sustainable swimming at 90% Ucrit relies extensively on carbohydrate as the substrate for ATP production, through both red muscle oxidative phosphorylation and white muscle glycolysis. The likely contributions of lipid oxidation to ATP production during swimming at 90% Ucrit are lower than at 30 and 60% Ucrit, but may still be important.
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