Download Postexercise recovery of skeletal muscle malonyl-CoA, acetyl

Postexercise recovery of skeletal muscle malonyl-CoA,
acetyl-CoA carboxylase, and AMP-activated protein kinase
B. B. RASMUSSEN, C. R. HANCOCK, AND W. W. WINDER
Department of Zoology, Brigham Young University, Provo, Utah 84602
carnitine palmitoyl transferase-1; postexercise substrate utilization
rate of oxidation of long-chain fatty acids to increase.
Credence to this postulated sequence was recently
gained by using the rat perfused hindlimb preparation
(17). When AMPK is activated by the 58-AMP analog,
5-aminoimidazole-4-carboxamide-1-b-D-ribofuraosyl 58monophosphate (ZMP), the consequent decrease in
ACC activity and malonyl-CoA is accompanied by an
increase in the rate of palmitate oxidation. This is in
agreement with previous studies on liver and heart
that have shown an inverse correlation of malonyl-CoA
content and fatty acid oxidation rate (1, 15, 23).
At submaximal work rates, the absolute rate of fatty
acid oxidation increases during exercise (11, 22, 28). In
human subjects and in rats exercised for prolonged
time periods, fatty acid oxidation remains elevated
above resting levels in the postexercise period (4, 33).
We hypothesized that if muscle malonyl-CoA concentration does not recover rapidly after exercise, CPT-1
would remain active, allowing fatty acids to be utilized
as an energy source during the time when muscle
glycogen is being replenished. The purpose of this study
was to determine the time course of malonyl-CoA, ACC
activity, and AMPK activity in skeletal muscle after
exercise.
MATERIALS AND METHODS
CARNITINE PALMITOYL-TRANSFERASE-1
(CPT-1) is the enzyme required to move long-chain fatty acyl-CoA molecules from the cytoplasmic to the intramitochondrial
compartment where oxidation can occur (15). MalonylCoA, an inhibitor of CPT-1, has been demonstrated to
decrease in muscle in response to exercise (21, 30–32)
and in response to electrical stimulation (14, 20, 29).
The decrease in malonyl-CoA is accompanied by increases in AMP-activated protein kinase (AMPK) activity and by decreases in acetyl-CoA carboxylase (ACC)
activity (14, 29, 32). This mechanism has been postulated to couple muscle contraction with an increase in
the rate of fatty acid oxidation by the muscle during
exercise. The following sequence of events has been
proposed (30, 32). 1) Muscle contraction increases
58-AMP concentration in the muscle. 2) The increase in
58-AMP activates AMPK allosterically and also activates AMPK kinase. 3) The AMPK kinase phosphorylates AMPK, resulting in further activation. 4) AMPK
phosphorylates ACC, resulting in a decrease activity. 5)
The decrease in ACC results in a decrease in malonylCoA, which decreases inhibition of CPT-1, allowing the
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Animal care. Male Sprague-Dawley rats (Sasco, Omaha,
NE) were housed in individual cages at a temperature of
19–21°C in a light-controlled room (12:12-h light-dark cycle)
and were fed a normal diet (Harlan Teklad rodent diet,
Madison, WI) and water ad libitum. Rats were run on a
rodent treadmill (20–40 m/min) up a 5–15% grade (5–10
min/day) for 5–10 days to accustom them to running on the
treadmill and to handling procedures. While rats were anesthetized with ether, jugular catheters were implanted 3–4
days before the final exercise test. The rats weighed 357 6 3 g
when they were killed at the end of the experiment.
Exercise test. Rats were anesthetized by injection of pentobarbital sodium via the catheter at rest or after running for 5
min on the treadmill at 40 m/min up a 5% grade (sprinting
group) or running for 30 min at 21 m/min up a 15% grade
(submaximal group). Prior studies have demonstrated that
malonyl-CoA decreases significantly after sprinting for 5 min
(21) and after submaximal endurance exercise of 30 min (31,
32). At 90–180 s after rats were anesthetized, the red region
of the quadriceps was removed rapidly from both hindlimbs
and was frozen between stainless steel block tongs at liquid
nitrogen temperature. Blood was removed via the abdominal
aorta. An aliquot was heparinized for collection of plasma to
be used for determination of free fatty acids (FFA) (19). A
perchloric acid extract of blood was made for measurement of
glucose (3) and lactate (7).
Muscle assays. Muscle samples were kept in liquid nitrogen until they were analyzed. Red quadriceps muscles were
ground to a powder while they were in liquid nitrogen. For
ACC and AMPK assays, the frozen powder was weighed and
then homogenized in a buffer containing (in mM) 100 manni-
8750-7587/98 $5.00 Copyright r 1998 the American Physiological Society
1629
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Rasmussen, B. B., C. R. Hancock, and W. W. Winder.
Postexercise recovery of skeletal muscle malonyl-CoA, acetylCoA carboxylase, and AMP-activated protein kinase. J. Appl.
Physiol. 85(5): 1629–1634, 1998.—Previous studies have
demonstrated that oxygen consumption and fat oxidation
remain elevated in the postexercise period. The purpose of
this study was to determine whether malonyl-CoA, an inhibitor of fatty acid oxidation, remains depressed in muscle after
exercise. Rats were sprinted for 5 min (40 m/min, 5% grade)
or run for 30 min (21 m/min, 15% grade). Red quadriceps
malonyl-CoA returned to resting values by 90 min postexercise in the sprinting rats and remained significantly lower at
least 90 min postexercise in the 30-min exercise group.
AMP-activated protein kinase activity remained significantly
elevated (P , 0.05) for 10 min after exercise in both groups.
The most rapid rate of glycogen repletion was in the first 30
min postexercise. The respiratory exchange ratio decreased
from a nonexercise value of 0.87 6 0.01 to an average 0.82 6
0.01 during the 90-min period after 30 min of exercise. Thus
muscle malonyl-CoA remains depressed and fat oxidation is
elevated for relatively prolonged periods after a single bout of
exercise. This may allow fat oxidation to contribute more to
muscle energy requirements, thus leaving more glucose for
replenishment of muscle glycogen.
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POSTEXERCISE RECOVERY OF MALONYL-COA
RESULTS
Figure 1 shows that malonyl-CoA in sprinting and
endurance-run rats decreases significantly with exercise (P , 0.05). Malonyl-CoA remains significantly
decreased for 60 min after exercise in the sprinting
group and for at least 90 min in the 30-min exercise
group.
ACC activity is reported in Fig. 2 and Table 1. ACC
activity at the physiological citrate concentration (0.2
mM) remains significantly lower (P , 0.05) for 30 min
after exercise in the sprinting group and for 90 min
postexercise in the 30-min exercise group. Table 1 gives
a more detailed presentation of the enzyme kinetics of
ACC. Vmax in the sprinting group remains significantly
decreased (P , 0.05), and the Ka for citrate (calculated
from the Hill equation) was significantly elevated for 30
min after the 5-min sprinting exercise. Similar results
were observed for the rats that were run for 30 min.
AMPK activity is significantly increased (P , 0.05)
for 10 min after a bout of sprinting or submaximal
exercise (Fig. 3) and then rapidly returns to values near
resting levels for both groups. This return to resting
Fig. 1. Effect of sprinting for 5 min (40 m/min) or running for 30 min
(21 m/min) on recovery time course of malonyl-CoA in red quadriceps
muscle. Values are means 6 SE; n 5 6–7 rats. Resting values are left
of zero time point of postexercise axis. Zero time point represents
values from rats immediately after exercise. * Significantly different
from resting muscle (P , 0.05).
values after 10 min parallels the changes in Ka for
citrate activation of ACC.
Figure 4 shows the time course of red quadriceps
glycogen content from 0 to 90 min after bouts of
sprinting or submaximal exercise. Glycogen content is
significantly lower (P , 0.05) for 15 min postexercise
after a 5-min bout of sprinting exercise and remains
significantly decreased (P , 0.05) for 90 min after a
30-min bout of submaximal exercise. The glycogen
content of the red quadriceps is higher at the 90-min
postexercise time point vs. the 15-min time point (P ,
0.05).
Plasma FFA concentration did not change with a
5-min bout of sprinting exercise but was significantly
elevated (P , 0.05) for 5 min after the 30-min run
Fig. 2. Effect of sprinting for 5 min (40 m/min) or running for 30 min
(21 m/min) on recovery time course of acetyl-CoA carboxylase (ACC)
activity at 0.2 mM citrate in red quadriceps muscle. Values are
means 6 SE; n 5 6–7 rats. See Fig. 1 legend for additional details.
* Significantly different from resting muscle (P , 0.05).
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tol, 50 NaF, 10 Tris, 1 EDTA, 10 mercaptoethanol, pH 7.5, and
proteolytic enzyme inhibitors (5.0 ml/l aprotinin, 5 mg/l
leupeptin, and 5 mg/l antitrypsin). The homogenate was
immediately centrifuged at 48,000 g for 20 min. The ACC and
AMPK were precipitated from the supernatant by addition of
ammonium sulfate (144 mg/ml) and by stirring for 30 min on
ice. The precipitate was collected by centrifugation at 48,000
g for 20 min. The pellet was dissolved in 10% of the original
volume of the homogenate buffer and was centrifuged again
to remove insoluble protein.
The supernatant was used for determination of ACC and
AMPK activity. ACC activity was determined at citrate
concentrations that varied between 0 and 20 mM by measuring the rate of incorporation of [14C]bicarbonate into malonylCoA, an acid-stable compound, at 37°C for 10 min as previously described (32). The data were fitted to a modified Hill
equation by using the Grafit program (Sigma Chemical),
which allows determination of maximal velocity (Vmax ) as a
function of citrate concentration and determination of the
activation constant for citrate (Ka ). AMPK activity was determined by using the substrate SAMS peptide by the method
described previously (5, 32).
Neutralized perchloric acid extracts of the red quadriceps
muscles were used for the determination of malonyl-CoA (16).
Glycogen content of the red quadriceps muscles was determined by using the anthrone method (9).
The respiratory exchange ratio (RER) was determined for
three consecutive 30-min intervals for rats at rest and for the
same rats (on a different day) after running on the treadmill
for 30 min at 21 m/min up a 15% grade. The latter measurement was started 5 min postexercise. Rats were placed in a
Plexiglas chamber. Air was passed through the chamber and
into an airtight bag. After each 30-min interval, the volume of
air in the bag was determined, along with the carbon dioxide
and oxygen content (mass spectrometer). The RER was then
calculated.
Results are expressed as means 6 SE. Analysis of variance,
followed by Fisher’s least significant differences test, was
used to determine statistical differences (P , 0.05) between
treatment groups.
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POSTEXERCISE RECOVERY OF MALONYL-COA
Table 1. Time course of postexercise recovery of Vmax
and Ka of red quadriceps acetyl-CoA carboxylase
Postexercise,
min
ACC Vmax ,
nmol · g21 · min21
ACC Ka
Rats sprinted for 5 min
Rest
0
5
10
15
30
60
90
35.4 6 1.7
22.4 6 1.0*
21.9 6 1.8*
25.4 6 1.8*
27.0 6 3.1*
30.9 6 1.9
31.4 6 1.1
34.3 6 2.4
4.1 6 0.4
12.2 6 1.5*
9.9 6 0.9*
9.6 6 1.6*
8.9 6 0.9*
7.4 6 1.3*
5.0 6 0.7
4.9 6 0.6
Rats run for 30 min
40.3 6 2.6
21.9 6 1.6*
25.5 6 2.7*
28.4 6 2.4*
28.3 6 1.0*
29.2 6 2.5*
33.3 6 1.5*
33.8 6 2.9*
4.1 6 0.4
9.7 6 0.7*
7.4 6 0.5*
6.5 6 0.4*
5.6 6 0.4*
5.1 6 0.6
4.3 6 0.4
4.1 6 0.4
Values are means 6 SE from 5 to 7 rats. Vmax , maximal velocity;
ACC, acetyl-CoA carboxylase activity; Ka , activation constant. * Significantly different from resting group (P , 0.05).
(Table 2). Blood lactate was much higher (P , 0.05)
after 5 min of sprinting than at rest (Table 2). Lactate
also increased (P , 0.05) in the rats run for 30 min and
remained elevated for 10 min postexercise (P , 0.05).
Table 3 shows that the RER is significantly decreased
in the postexercise period in rats run for 30 min at 21
m/min. From these RER values, it was estimated that,
with no prior exercise, the rats derived an average of
41% of calories from fat oxidation. After 30 min of
treadmill exercise, the same rats derived 63, 60, and
53% of calories from fat oxidation during the periods
5–30, 30–60, and 60–90 min postexercise.
Fig. 4. Effect of sprinting for 5 min (40 m/min) or running for 30 min
(21 m/min) on recovery time course of glycogen content in red
quadriceps muscle. Values are means 6 SE; n 5 6–7 rats. See Fig. 1
legend for additional details. * Significantly different from resting
muscle (P , 0.05).
DISCUSSION
Wolfe et al. (33) have shown that the rate of fatty acid
oxidation remains elevated above resting values after
exercise. Their work in human subjects indicates that
fatty acid oxidation is ,50% higher than at rest for at
least 2 h after 4 h of treadmill exercise at 40% maximal
oxygen consumption. Brooks and Gaesser (4) previously observed a significant decrease in RER in rats
after prolonged exhausting exercise. The decrease in
RER in the postexercise period in the current study is
consistent with results of previous reports and provides
further evidence of increased fat oxidation in the
postexercise period. In addition, Treuth et al. (26) have
Table 2. Time course of postexercise recovery of plasma
FFA, blood glucose, and blood lactate
Postexercise,
min
FFA,
mM
Glucose,
mM
Lactate,
mM
Rats sprinted for 5 min
Rest
0
5
10
15
30
60
90
0.22 6 0.03
0.22 6 0.03
0.21 6 0.01
0.23 6 0.05
0.27 6 0.03
0.23 6 0.03
0.21 6 0.03
0.22 6 0.03
Rest
0
5
10
15
30
60
90
0.26 6 0.03
0.39 6 0.02*
0.35 6 0.04*
0.34 6 0.03
0.29 6 0.03
0.26 6 0.03
0.31 6 0.03
0.32 6 0.03
6.3 6 0.3
8.6 6 0.3*
8.3 6 0.4*
7.8 6 0.3*
7.8 6 0.2*
7.4 6 0.3*
7.4 6 0.4*
7.0 6 0.3
1.5 6 0.2
7.2 6 1.0*
4.4 6 0.8*
3.1 6 0.2
2.3 6 0.3
2.3 6 0.5
2.9 6 0.8
2.5 6 0.7
Rats run for 30 min
Fig. 3. Effect of sprinting for 5 min (40 m/min) or running for 30 min
(21 m/min) on the recovery time course of AMP-activated protein
kinase (AMPK) activity in red quadriceps muscle. Values are means
6 SE; n 5 6–7 rats. See Fig. 1 legend for additional details.
* Significantly different from resting muscle (P , 0.05).
6.9 6 0.2
8.1 6 0.4*
8.6 6 0.2*
8.1 6 0.6*
7.9 6 0.5
8.0 6 0.4
8.1 6 0.5*
6.7 6 0.2
1.8 6 0.2
4.2 6 0.5*
3.5 6 0.5*
3.7 6 0.5*
2.7 6 0.5
2.5 6 0.9
1.7 6 0.3
1.3 6 0.3
Values are means 6 SE from 5 to 7 rats. FFA, free fatty acids.
* Significantly different from resting group (P , 0.05).
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Rest
0
5
10
15
30
60
90
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POSTEXERCISE RECOVERY OF MALONYL-COA
Table 3. Respiratory exchange ratio at rest with no
prior exercise and at rest after 30 min of running
at 21 m/min up a 15% grade
Treatment
5–30 min
30–60 min
60–90 min
No exercise
After running 30 min
0.88 6 0.01
0.81 6 0.02*
0.86 6 0.02
0.82 6 0.03*
0.88 6 0.01
0.84 6 0.02
Values are means 6 SE; n 5 8 rats. * Significantly different from no
exercise trial; P , 0.05 (paired t-test).
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recently demonstrated an increase in energy expenditure over a 24-h period with high-intensity exercise.
The data presented in the present study provide one
possible explanation for a mechanism that allows this
increase in fatty acid oxidation in the postexercise
recovery period. Malonyl-CoA content continued to
remain at a low concentration for 60 min in animals
that sprinted for 5 min and remained depressed for at
least 90 min in animals run at a submaximal rate for 30
min. ACC and AMPK activity had returned to resting
values by 90 min, thus implying that malonyl-CoA
would eventually return to resting values. The decrease in malonyl-CoA would be expected to relieve
inhibition of CPT-1 and to allow fatty acids to be
oxidized in the postexercise period. It should be emphasized that only those muscle fibers involved in the
exercise would exhibit a decrease in malonyl-CoA under these conditions. A previous study (31) indicates
that malonyl-CoA does not decrease in the white region
of the quadriceps muscle of rats until after at least 30
min of exercise and that the decrease is small compared
with changes observed in the red region of the quadriceps. In addition, the change in RER in the red quadriceps is likely to be much larger than the change in whole
body RER, the latter being diluted by absence of change in
nonexercising muscle fibers and other tissues.
Malonyl-CoA inhibition of CPT-1 is probably only one
of several factors regulating the substrate oxidation
mix in muscle. Other factors (11–13, 18, 25, 27, 28) may
include: 1) plasma FFA concentration, 2) regulation of
FFA uptake into the muscle cell via a plasma membrane fatty acid-binding protein, 3) control of fatty
acid-binding proteins and fatty acyl-CoA-binding proteins in the transport and release of lipid to the
mitochondria, 4) glucose availability, 5) carnitine availability, and 6) intramitochondrial acetyl-CoA concentration. The concentration of FFA in plasma was observed
to increase during 30 min of exercise but was not
markedly elevated in the postexercise period. However,
increased rate of turnover of plasma FFA and/or supply
of fatty acids from intramuscular lipolysis could also
contribute to the increased rate of fat oxidation. The
rate of oxidation of fatty acids, even in the muscle fibers
involved in contraction, would be expected to be limited
by the low plasma fatty acid concentration. In more
prolonged bouts of exercise that continue until liver
glycogen is depleted, insulin is depressed, and glucagon
is elevated, plasma fatty acid concentration would
be expected to be elevated for much longer after exercise (2).
Glycogen replenishment appeared to occur quite
rapidly in the red region of the quadriceps muscle
during the postexercise period in this study. Liver
glycogen would not have been expected to be depleted
by these relatively short bouts of exercise. Thus glucose
would be readily available to the muscle fibers requiring replenishment. A recent study from this laboratory
(17) demonstrated that 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR), an analog of adenosine, can be taken up into muscle and phosphorylated
to form ZMP, an analog of 58-AMP. ZMP activates
AMPK similarly to 58-AMP. Perfusion of isolated rat
hindlimb with AICAR thus can mimic the effect of
exercise on AMPK, ACC, and malonyl-CoA. AICAR
causes a decrease in malonyl-CoA, with a resultant
increase in palmitate oxidation with no change in
oxygen consumption, thus indicating a change in the
substrate oxidation mix. That experiment also yielded
the exciting observation of a twofold increase in glucose
uptake in AICAR-infused muscle. This result led to the
hypothesis that activation of AMPK not only increases
availability of intramitochondrial long-chain fatty acids for oxidation but also increases availability of
glucose (possibly by phosphorylation of undefined target proteins involved in translocation of GLUT-4 to the
sarcolemma) to the working muscle. The contractioninduced increase in glucose transport (see Refs. 6, 10,
and 11), which we postulate is due to AMPK activation,
would be important for replenishment of glycogen
stores after exercise. This fits well with the suggestion
that AMPK serves as a fuel gauge of the mammalian
cell (8), signaling the need for increased glucose uptake
and increased fatty acid oxidation. The prolonged depression of malonyl-CoA in the postexercise period
would allow these muscle fibers to rely more on fatty
acid oxidation for energy needs, thus diverting more of
the available glucose into glycogen.
Malonyl-CoA does not always mirror changes in ACC
activity (24) because of allosteric control (inhibition of
ACC by long-chain acyl-CoA and activation by citrate).
Citrate dependence of ACC activity is measured simply
to detect changes due to covalent modification (phosphorylation). The allosteric regulators are not retained
in the final enzyme preparation. Saha et al. (24)
reported malonyl-CoA to be elevated in isolated skeletal muscle in response to incubation with glucose,
insulin, and acetoacetate, with no concurrent change in
ACC activity. When ATP citrate lyase was inhibited
with hydroxycitrate, the increase in malonyl-CoA was
attenuated, implying that the increase in malonyl-CoA
under these conditions was due to increases in cytosolic
citrate.
The applicability of the results of the current study to
human muscle is unclear at this time. Odland et al. (20)
previously reported very low levels of malonyl-CoA in
resting human muscle (compared with rat muscle) and
no significant change during exercise. One of the challenges in studying biopsies from human muscle is the
difficulty in obtaining a relatively homogenous sample
of type IIa fibers. We have previously noted that
POSTEXERCISE RECOVERY OF MALONYL-COA
The authors express appreciation to Emily Kurth for technical
assistance and to J. Bly and Drs. G. Fisher and P. Kelly for assistance
with the RER measurements.
This research was supported by the National Institute of Musculoskeletal and Skin Diseases Grant AR-41438.
Address for reprint requests: W. W. Winder, 545 WIDB, Dept. of
Zoology, Brigham Young Univ., Provo, Utah 84602 (E-mail:
[email protected]).
Received 6 April 1998; accepted in final form 22 June 1998.
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malonyl-CoA content is much less in IIb fibers than in
IIa fibers and that a decrease in IIb fibers is observed
only after 30 min of exercise. Even at relatively high
work rates, no change was seen in white quadriceps
(composed of type IIb fibers) malonyl-CoA after a 5-min
bout of exercise. Thus dilution of type IIa with IIb fibers
in a biopsy would diminish the resting malonyl-CoA
concentration measurement and would also reduce the
magnitude of the decrease in malonyl-CoA. The data
from the current study also suggest that care must be
taken to avoid muscle contraction for an extended
period before taking the biopsy of resting muscle. At the
present time, however, it is not possible to draw any
certain conclusion regarding the role of this control
system in human muscle.
Vavvas et al. (29) electrically stimulated the gastrocnemius muscle group in rats and found that 60–90 min
were required for ACC activity to return to resting
values. The present study demonstrates that malonylCoA and ACC activity remain depressed in muscle for
relatively prolonged periods after short bouts of highintensity exercise and longer bouts of submaximal
exercise in the rat. These results are consistent with
the hypothesis that a reduction in muscle malonyl-CoA
in the postexercise period allows increased fatty acid
oxidation, thus diverting a greater proportion of the
glucose taken up into muscle into the glycogen synthesis pathway.
1633
1634
POSTEXERCISE RECOVERY OF MALONYL-COA
29. Vavvas, D., A. Apazidis, A. K. Saha, J. Gamble, A. Patel,
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