Download Activation by Exercise of Human Skeletal Muscle Pyruvate

Clinical Science (1982) 63,81-92
87
Activation by exercise of human skeletal muscle pyruvate
dehydrogenase in vivo
G. R . W A R D * , J . R . S U T T O N , N . L. J O N E S A N D C . J . T O E W S
Department of Medicine, McMaster University Health Science Centre, Hamilton, Ontario, Canada
(Received 9 November 1981; accepted 12 February 1982)
Summary
1. The activity of pyruvate dehydrogenase in
its active and inactive forms was measured in
biopsy samples obtained from the vastus lateralis
muscle of healthy subjects before and after
exercise.
2. At rest, 40 ? 4% (mean & SEM) of the
enzyme was in the active form.
3. After progressive aerobic exercise to exhaustion (n = 3,88 ? 2.3% was in the active
form.
4. After intermittent supramaximal short-term
exercise (1 min exercise, 3 min rest) to exhaustion
(n = 6), 60 k 2.2% was in the active form.
5 . After isometric maximal exercise of 65 f
3.6 s duration (n = 3), only 39
1% of the
enzyme was in the active form.
6. Muscle glycogen depletion was greatest
with intermittent exercise and least with isometric maximal exercise; in contrast, the increase
in muscle lactate was least with progressive
exercise (1.3 to 9.4 pmollg), intermediate in
intermittent maximal exercise (1 2 to 13 1
pmol/g) and greatest after isometric exercise (1.8
to 17.6 pmol/g). There were no significant
differences between the three studies in the
changes in lactate/pyruvate ratios.
7. In three subjects who exercised with one
leg, activation of the enzyme was twice as great in
the exercised as in the inactive leg.
-
* Present address: Variety Village Sport Training
and Fitness Centre, 3701 Danforth Avenue, Scarborough, Ontario, Canada.
Correspondence: Professor C. J. Toews, Department of Medicine, McMaster University Health
Sciences Centre, 1200 Main Street West, Hamilton,
Ontario L8S 459, Canada.
8. The ratio of active to total enzyme in
biopsies of resting muscle was greater in four
well-trained athletes than in four untrained
control subjects (70% compared with 41%
respectively).
9. The activation of pyruvate dehydrogenase
appears to play an important part in regulating
the use of glycogen and glucose during exercise in
man.
Key words: adenosine diphosphate, adenosine
triphosphate, aerobic exercise, anaerobic exercise, creatine, glycogen, lactate, phosphocreatine, pyruvate.
Introduction
The use of glycogen and glucose for fuels in
muscular exercise is regulated by a number of
nonequilibrium enzymes. The pyruvate produced
by glycolysis undergoes oxidation in the mitochondria to acetyl-CoA, the reaction being
catalysed by the pyruvate dehydrogenase
complex:
Pyruvate
+ CoA + NAD+
acetyl-CoA + NADH + H+ + CO,
+
The enzyme complex is subject to two types of
regulation: product inhibition (acetyl-CoA and
reduced nicotinamide-adenine
dinucleotide,
NADH) 111 and by the interconversion of an
inactive phosphorylated form and an active
non-phosphorylated form [21. Linn and colleagues [31 showed that the interconversion
between the two forms is regulated by an
Mg-ATP-linked
kinase and a specific
phosphatase. The activation of the inactive form
of the enzyme is probably brought about by a
0143-5221/82/070087-06$01.50 @ 1982 The Biochemical Society and the Medical Research Society
88
G . R . Ward et al.
combination of stimulation of the phosphatase
and inhibition of the kinase. In mitochondria1
preparations of heart and skeletal muscle, both
Mgz+ and CaZ+are important activators of the
enzyme [41, among other metabolites [21.
The energy required for the regeneration of
ATP during heavy exercise is largely derived
from muscle glycogen, with only a minor proportion coming from fats and amino acids [5,61. The
major metabolic end point for glycogenolysis in
muscle is pyruvate, which is then metabolized to
lactate or decarboxylated to acetyl-CoA. Thus
the pyruvate dehydrogenase reaction is the flux
generating step in pyruvate oxidation; also,
because the lactate dehydrogenase reaction is
always near-equilibrium, the rate of pyruvate
oxidation assumes importance through its indirect effect on lactate production in exercise.
In rats, skeletal muscle contraction, induced by
sciatic nerve [71 or direct [81 stimulation, is
associated with activation of the enzyme and in
increases in lactate oxidation without an increase
in pyruvate concentration [ 71, indicating that
activation plays an important role in the regulation of energy metabolism in exercise.
The purpose of the present study was to
examine the part played by pyruvate dehydrogenase activation in the control of energy
metabolism in human skeletal muscle. We have
measured the total activity of the pyruvate
dehydrogenase complex and the extent of activation of the enzyme, in samples of muscle obtained
by needle biopsy in normal subjects before and
immediately after three types of exhaustive
exercise associated with different metabolic
responses (aerobic, intermittent anaerobic and
isometric anaerobic).
biopsies [ 101 were taken immediately before and
within 5 s of stopping exercise.
Progressive incremental aerobic exercise
Five subjects exercised on a cycle ergometer,
with an initial power output of 17 W, and the
workload was increased by 33 W every 2 min,
the subjects exercising until exhaustion. During
the study inspired ventilation was measured by a
dry gas meter (Parkinson-Cowan CD-4) and
mixed expired gas was continuously analysed for
CO, (Godart Capnograph) and 0, (Godart
Rapox). Oxygen intake and CO, output were
calculated [91. The total duration of the studies
was 20 k 3 min (mean k SD).
Intermittent maximal exercise
Six subjects exercised intermittently for 1 min
with periods of 3 min rest between each exercise
bout. The power output was calculated to be
140% of Vo, max. The study was continued until
the subject could no longer complete 1 min of
exercise. The average number of exercise bouts
was nine, the total duration being 35 2 6 min.
Maximal isometric contraction
In seven subjects maximal voluntary isometric
contraction of the quadriceps muscle group was
measured [ 111 by using an isokinetic dynamometer (Cybex 2, Lumex Isokinetic Systems);
50% of the maximal voluntary contraction was
used for the experiments. The time to exhaustion
at this constant isometric power output was
65-73 S.
Materials and methods
Supplementary studies
The subjects were 14 healthy males, age 19-25
years, height 179 k 6 cm, weight 79 k 9 kg. The
subjects gave their informed consent after a
complete description of the studies and the
attendant risks, and the project was approved by
the university's Ethics Committee.
One week before each study a progressive
incremental exercise test was carried out on a
cycle ergometer [9]. The oxygen intake measured
during the highest work load that could be
sustained for 1 min was taken to be the maximal
oxygen intake ( V o , rnax.).
Three series of experiments were carried out.
In each, the subjects came to the laboratory after
a 12-16 h fast. Two small skin incisions were
made over one vastus lateralis muscle, 4 cm
apart, under local anaesthesia. Needle muscle
To amplify the findings of the main studies,
two additional studies were carried out. In the
first, three subjects pedalled the cycle ergometer
with one leg for 10 min at a power output of
133 W; needle biopsies were taken immediately
afterwards from the vastus lateralis muscle of
both legs. In the second study, vastus lateralis
muscle biopsies were taken at rest in four elite
athletes [two oarsmen and. two distance runners
4 ml
who had high values for Vo, max. (71
min-' kg-I)] and in four subjects of similar age
and size, who were untrained (VO, max. 47 k 3
ml min-I kg-I).
The biopsy samples were frozen in liquid N,
within 2 s and stored in liquid N, for up to 1 week
before analysis. The muscle biopsy was dissected
free of fat, blood and connective tissue in a
Activation of human muscle pyruvate dehydrogenase
-2OOC cold room. The biopsy was weighed and
homogenized for 40 s (Polytron PT10) in 30 vol.
of ice-cold extraction solution containing potassium phosphate (10 mmol/l), dithiothreitol (1
mmol/l), sodium EDTA (1 mmol/l) and 1 g of
crystalline bovine serum albumin/l, at pH 7.4.
After homogenization, pyruvate dehydrogenase
activity was measured immediately. Separate
experiments indicated that both active and total
pyruvate dehydrogenase remained stable for
several weeks when frozen in liquid N, before
homogenization and for up to 12 h at O°C after
homogenization.
The enzyme was assayed by a modification of
the method described by Taylor & Jungas [121.
The assay solution for the active enzyme contained potassium phosphate (1 1 mmol/l), sodium
EDTA (2.5 mmol/l), magnesium chloride (2-5
mmol/l), coenzyme A (0.5 mmol/l), dithiothreitol
(2.5 mmol/l), NAD+ (30 mmol/l, thiamine
pyrophosphate (1 5 mmol/l), sodium pyruvate
(0.6 mmol/l), sodium [ l-I4Clpyruvate (4 pCi/ml)
and 2 g of crystalline bovine serum albumin/l, at
pH 7.6. The assay was carried out in rubbersealed 10 ml Erlenmeyer flasks with a centre well.
Before the assay 0.24 ml of incubation buffer was
added to the flask, 0.2 ml of Hyamine hydroxide
(1 mol/l) was placed in the centre well, and the
flask was stored on ice. For the assay the flasks
were warmed to 37OC for 3 rnin and the reaction
was started by addition of 0 - 2 ml of ice-cold
muscle homogenate. The reaction was carried out
at 37°C for 2 rnin in a Dubnoff incubator, and
was stopped by the addition of 0.8 ml of citric
acid (80 mmol/l)/phosphoric acid (40 mmol/l) at
pH 3.0 under sealed conditions. The flasks were
further incubated at 37OC for 60 rnin to allow
release of 14C0, and trapping by the Hyamine
hydroxide. After incubation, the Hyamine hydroxide was removed and I4C measured by scintillation counting. Counting efficiency was determined by the channels-ratio method. Enzyme
dependent 14C02 production was measured as
above, but with the extraction buffer solution and
muscle extract only.
The assay for total pyruvate dehydrogenase
activity was identical with that outlined above,
except that 0.3 mi of homogenate was incubated
with magnesium chloride (10 mmol/l) and calcium chloride (10 mmol/l) for 6 rnin at 37"C,
before addition of homogenate to the incubation
flask. A sample (0.2ml) was used for the assay
of total pyruvate dehydrogenase activity by the
procedure above.
A number of preliminary experiments were
carried out to establish the validity of the assay
procedures. As pyruvate dehydrogenase is pre-
89
sent in mitochondria, a variety of procedures has
been used to liberate the enzyme from mitochondria; studies with rat mitochondria and
muscle homogenates showed that freezing and
thawing, ultrasonication and Triton X-100 treatment yielded equally improved enzyme activity
over untreated preparations Il31. Under the
incubation conditions we used, repeated analyses
established that enzyme activity reached a maximum after 2 min, which was maintained for 15
min.
Of the initial biopsy, at least 30 mg was used
for enzyme analysis. The remainder was used for
the analysis of lactate, pyruvate, ATP, ADP,
phosphocreatine, creatine, glycogen and glucose
by standard enzymic fluorimetric techniques [ 141.
Results
Progressive incremental aerobic work
After an average of 20 min exercise, total
pyruvate dehydrogenase activity was unchanged
compared with rest (Fig. 1). At rest, the activity
of the active pyruvate dehydrogenase was 41 &
SEM 3.0% of the total activity; however, at
exhaustion the activity of the active pyruvate
dehydrogenase accounted for 88 ? 2.4%. This
type of exercise was associated with a decrease in
muscle glycogen to 43% of the resting value
(Table l), and an increase in lactate from 1.29 to
9.41 pmol/g. There was a small increase in
pyruvate concentration and the lactate/pyruvate
ratio increased from 18.4 to 47.0. There was a
small decrease in ATP, a small increase in ADP
and a fall in the ATP/ADP ratio. Phosphocreatine concentration decreased markedly.
=
4001
300
M
C
'Z
-.-
2*
v
x
.->
.-
9
100
0
R
E
Aerobic
R
E
Anaerobic
R
E
Isometric
Fig. 1. Pyruvate dehydrogenase activity in three types
of exercise, showing total and active (cross-hatched)
forms. R, Rest; E, exercise. Bars are f SEM.
G . R . Ward et al.
90
TABLE
1. Effect of various types of exercise on tissue metabolite concentrations in human skeletal muscle at rest and after
exhaustive exercise
Results are means f SEM. The subjects in each type of work were exercised to exhaustion as outlined in the Materials and
methods section. The values are in pmol/g wet weight of muscle.
Progressive exercise
Rest
Time (min) . . .
Glycogen
Lactate
Pyruvate
ATP
ADP
Phosphocreatine
Creatine
Exercise
Intermittent exercise
Rest
Exercise
36.4 f 2.56
9.4 f 0.30
0 . 2 0 f 0.03
4.29f0.18
1.38 f 0.09
3.3 ? 0.88
18.3 f 1.55
5
5
No. of observations
Intermittent maximal exercise
After 35 & 6 min, the activity of active
pyruvate dehydrogenase was increased from 38
f 3% at rest to 60 t 3.0% at exhaustion, but the
total pyruvate dehydrogenase activity remained
unchanged. The exercise was associated with
greater glycogen depletion, compared with the
aerobic work study, glycogen falling to 30% of
resting values. This was associated with a greater
increase in lactate, but similar changes in
pyruvate, ATP, ADP and phosphocreatine
concentrations (Table 1).
Rest
35 ? 6
20 f 3
84.1 k 12.39
1.3 f 0.06
0.07 f 0.01
5.23k0.16
0.92 k 0.07
15.6 f 1.30
10.6 f 0.50
Maximal isometric exercise
80.3 f 3.34
1 . 2 f 0.16
0.07 f 0.003
4.99 f 0.24
0 . 5 3 f 0.02
16.1 f 0 . 7 9
9.7f0.48
Exercise
1.2 f 3
2 4 . 2 k 5.33
1 3 . 1 f 0.48
0.19 f 0.02
3.51 ? 0.18
0.61 ? 0.03
5 . 1 k 1.01
21.5-t 1 . 1 1
71.9 f 8.88
1.8 f 0.39
0.08 k 0.02
5.06 f 0.19
0.57 f 0 . 0 4
17.13 f 1.00
9 . 2 0.42
+
44.3 f 13.55
17.6 f 0.99
0.54 ? 0.04
2.90 f 0 . 1 3
0.75 f 0.03
2.85 f 0.31
20.3 f 0.81
6
6
6
6
leg. Active pyruvate dehydrogenase was twice as
high in the active leg (225 t 6 nmol min-' g-I) as
the inactive leg (1 12 f 2 nmol rnin-' g-I).
The total pyruvate dehydrogenase activity in
the resting muscle of four athletes was similar to
that in four untrained subjects (309 f 7 compared with 304 f 4 nmol min-' g-I respectively)
but 70% of the enzyme was in the active form in
the athletes compared with 41% in the untrained
subjects (215 k 15 compared with 124 t 9 nmol
min-I g-' respectively) (P< 0.001).
Discussion
Maximal isometric exercise
After 65 ? 3 s of this type of exercise, the
activity of active pyruvate dehydrogenase was 39
& 1.2%, a value not significantly different from
that at rest (37 f 1.0%). This type of exercise
was associated with a fall in glycogen concentration to 61% of the resting value (Table l), a
marked increase in lactate and pyruvate
concentrations, marked falls in phosphocreatine
concentration and a significant fall in ATP and
the ATP/ADP ratio.
The proportion of the total pyruvate dehydrogenase activity accounted for by the active form
was significantly greater with progressive aerobic
exercise than with intermittent exercise and both
were significantly greater than values after
isometric exercise (P< 0.00 I).
Supplementary studies
In a comparison between the active and
inactive leg after one-legged exercise in three
subjects, the pyruvate dehydrogenase activity
was the same, 305 f 4 nmol min-' gg' in the
active and 302 5 nmol min-' g-' in the inactive
Heavy aerobic exercise greatly increases skeletal
muscle 0, consumption and CO, production,
mainly owing to increased oxidation of carbohydrate derived from muscle glycogen and blood
glucose 1151. In relatively light exercise of long
duration, on the other hand, there is increase in
lipolysis and increased skeletal muscle oxidation
of non-esterified fatty acids [161, with a relatively
small increase in carbohydrate oxidation. The
increase in free fatty acid oxidation in this type of
exercise exerts a sparing effect on glycogen,
which is probably mediated in part by an
inhibition of phosphofructokinase by citrate [ 171,
and of pyruvate dehydrogenase by changes in the
acetyl-CoA/CoA and NADH/NAD ratios [ 18,
191. Studies in rat heart have shown a strong
inhibitory effect of palmitoylcarnitine in the
presence of Ca2+ [20], which may depend on a
specific Ca2+ transporting system into the mitochondrion 1211. Thus changes in Ca2+may play a
part in the activation of pyruvate dehydrogenase
which occurs in exercising muscle. The results of
the present study suggest that almost all the
pyruvate dehydrogenase is in the active form
during aerobic exercise and that much less is in
Activation of human muscle pyruvate dehydrogenase
the active form during very heavy short term or
isometric exercise, in which lactate production is
much greater. The reason for this finding is not
clear, but may indicate that H+ may influence the
enzymes controlling the interconversion between
the two forms of pyruvate dehydrogenase. The
finding cannot be explained by delay in the
activation processes beyond the duration (1 min)
of the exercise; in stimulated rat skeletal muscle
activation reaches maximum values within 20 s,
which are then maintained for several minutes 18,
131.
The ratio of active to total pyruvate dehydrogenase activity found in the present study in
human resting vastus lateralis muscle is higher
than previously found in rat muscle [81. Studies
in our laboratory, with methods similar to those
used in the present work [131, showed total
pyruvate dehydrogenase activity to be 831 +_ 28
nmol min-' g-' with 31 f 3% in the active form
in resting rat gastrocnemius muscle (n = 20);
corresponding values for rat heart were 5210 f
480 nmol min-' g-' and 21 f 3% (n = 8). These
results emphasize the variation in pyruvate
dehydrogenase activity in different species and
different muscles of the same species.
High levels of muscle 0, consumption and
CO, production were reached in the incremental
aerobic exercise study (means of 3.6 l/min and
4.1 l/min respectively). This in our subjects, 150200 mmol of O,/min was being used, equivalent
to a flux of 50-65 mmol of pyruvate/min into the
tricarboxylic acid cycle. Assuming the active
muscle mass to be about 25 kg, these values
imply a flux of at least 2000 nmol min-' g-'
through the pyruvate dehydrogenase reaction.
Although considerable caution is required in
applying enzyme activity measurements in the
reaction flask or cuvette to the activity in
exercising muscle in vivo I221, it is of interest to
calculate the extent to which our measurements
might account for the flux into the tricarboxylic
acid cycle in exercise. The highest value for active
pyruvate dehydrogenase activity was about 300
nmol of pyruvate min-' g-l. However, assays
were carried out at a pyruvate concentration of
20 pmolll; as the K, for pyruvate is 0.1 pmol/ml
and the pyruvate concentration in muscle after
heavy exercise was 0.2 pmol/g we may calculate,
using the Michaelis-Menten relationships, that
the flux could be increased to about lo00 nmol
min-I g-'. Although this is only half the
calculated flux in heavy exercise, we lack precise
information regarding active muscle mass, mitochondrial NADH/NAD+ and acetyl-CoA/CoA
ratios, and of other factors known to modulate
the activity of the active enzyme [2, 20, 211.
91
Changes in these factors and in muscle pH
presumably account for the differences seen
between the three types of exercise we employed.
The lack of change in pyruvate dehydrogenase
activity in inactive muscle supports the dominant
role of intramuscular factors in the control of the
interconversion between the two forms of the
enzyme, although in adipose tissue hormonal
control may be important [231. The increase in
muscle pyruvate concentration in all forms of
exercise, particularly the maximal isometric
activity, indicates that the enzyme is rate limiting,
particularly in view of the fact that the increase in
pyruvate concentration bore an inverse relationship to the degree of pyruvate dehydrogenase
activation (Table 1).
Conversion of the inactive into the active form
of the enzyme may be of obvious importance in
athletic events in which a rapid increase in
aerobic processes is required, and anaerobic
metabolism is kept to a minimum. Whipp &
Wasserman [241 studied the kinetics of oxygen
uptake at the onset of exercise, and suggested
that well trained athletes increase oxygen uptake
more rapidly at the onset of exercise than do
untrained individuals. Although they suggested
that this difference is due to a more rapid
adaptation of oxygen delivery mechanisms at the
onset of exercise, it is also possible that highly
trained individuals are capable of increasing the
flux through the pyruvate dehydrogenase reaction, and thus oxygen utilization, more rapidly
than untrained subjects. Our demonstration of a
difference between athletes and untrained subjects is consistent with an adaptive change
occurring as a result of training. The production
of lactate by muscle at the onset of exercise, or in
heavy 'anaerobic' exercise, may not be due
entirely to the classical concept of 'oxygen debt'
but to an imbalance between the rate of
glycolysis, controlled by phosphorylase and
phosphofructokinase, and the rate of pyruvate
incorporation into the tricarboxylic acid cycle,
controlled by active pyruvate dehydrogenase.
Acknowledgments
Excellent technical assistance was provided by
Rheal Leveille and Elizabeth Head. We thank Dr
Robert Jungas for his invaluable advice in setting
up the experimental technique.
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