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Carbohydrate Ingestion Prior to Exercise Augments the Exercise‐Induced

Activation of the Pyruvate Dehydrogenase Complex in Human Skeletal Muscle

Article  in  Experimental Physiology · September 2000

DOI: 10.1111/j.1469-445X.2000.02043.x · Source: PubMed

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 Carbohydrate ingestion prior to exercise augments the exercise-
induced activation of the pyruvate dehydrogenase complex in
human skeletal muscle
K. Tsintzas*†, C. Williams†, D. Constantin-Teodosiu‡, E. Hultman§, L. Boobis¨
and P. Greenhaff‡
†Human Muscle Metabolism Research Group, Loughborough University, UK, ‡School of
Biomedical Sciences, Nottingham University, UK, §Department of Medical Laboratory Science
and Technology, Huddinge University Hospital, Sweden and ¨Sunderland Royal
General Hospital, UK
(Manuscript received 31 March 2000; accepted 15 August 2000)

This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase
complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and
phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state
exercise. Seven male subjects performed two 10 min treadmill runs at 70% maximum oxygen uptake (ýOµ,max),
1 week apart. Each subject ingested 8 ml (kg body mass (BM))¢ of either a placebo solution (CON trial) or a
5.5% CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the
vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise
in the CHO trial compared with the CON trial (1.78 ± 0.18 and 1.27 ± 0.16 mmol min¢ (kg wet matter
(WM))¢, respectively; P < 0.05) and this was accompanied by lower acetylcarnitine (7.1 ± 1.2 and
9.1 ± 1.1 mmol kg¢ (dry matter (DM))¢ in CHO and CON, respectively; P < 0.05) and citrate concentrations
(0.73 ± 0.05 and 0.91 ± 0.10 mmol (kg DM)¢ in CHO and CON, respectively; P < 0.05). No difference was
observed between trials in the rates of muscle glycogen and PCr breakdown and lactate accumulation. This is
the first study to demonstrate that CHO ingestion prior to exercise augments the exercise-induced activation of
muscle PDC and reduces acetylcarnitine accumulation during the transition from rest to steady-state exercise.
However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis.
Experimental Physiology (2000) 85.5, 581—586.

During the transition from rest to steady-state exercise, a
significant part of the energy necessary to sustain force
generation is derived from PCr hydrolysis and glycogenolysis.
Classically, the extent of this anaerobic ATP production
through substrate level phosphorylation at the onset of exercise
has been attributed to a lag in blood flow and oxygen delivery
to the contracting muscle (Margaria et al. 1963). More
recently, however, it has been demonstrated that the activity of
PDC and the availability of acetyl groups to the tricarboxylic
acid (TCA) cycle are important determinants of the metabolic
responses during the onset of exercise (Timmons et al. 1997,
1998).
The PDC controls the rate-limiting step in CHO oxidation, the
oxidative decarboxylation of pyruvate to acetyl-CoA. The
activity of PDC increases during exercise (Constantin-Teodosiu
et al. 1992), resulting in an increase in pyruvate flux, the
formation of acetyl-CoA and a concomitant increase in CHO
oxidation. When the rate of acetyl-CoA formation by the PDC
exceeds its rate of oxidation by the TCA cycle, the excess
acetyl-CoA is buffered by carnitine, resulting in the formation
of acetylcarnitine (Constantin-Teodosiu et al. 1992). However,
at the onset of exercise there is a delay in the activation of
PDC and provision of acetyl groups to the TCA cycle which
seems to be responsible for the increased contribution of
substrate level phosphorylation to energy metabolism (Timmons
et al. 1997; Howlett et al. 1999).
Both hyperglycaemia and hyperinsulinaemia increase the
activity of PDC in resting human skeletal muscle (Mandarino
et al. 1987, 1993). Therefore, CHO-mediated increases in
blood glucose and insulin concentrations may also result in a
faster rate of PDC activation, which in turn would increase
the provision of substrate (i.e. acetyl groups) for use by the
TCA cycle, at the onset of exercise. This could then lead to a
faster onset of oxidative ATP production and a reduction in

Publication of The Physiological Society * Corresponding author:kostas.tsintzas@nottingham.ac.uk

2043
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582 K. Tsintzas and others
the requirement for ATP resynthesis by substrate level
phosphorylation.
Therefore, the purpose of this study was to examine the effect
of an orally ingested CHO load on PDC activity and acetyl
group availability in human skeletal muscle during the
transition from rest to steady-state running and its influence
on the contribution of substrate level phosphorylation to ATP
resynthesis.
METHODS
Subjects
Seven male recreational runners gave their informed consent and
volunteered to participate in this study, which was approved by the
University of Loughborough Ethical Advisory Committee. The mean
age, height, body mass, ýOµ,max and maximum heart rate (HRmax)
of the subjects were 27.6 ± 2.2 years, 178 ± 1 cm, 76.1 ± 2.1 kg,
57.5 ± 1.7 ml kg¢ min¢ and 191 ± 5 beats min¢, respectively.
Preliminary measurements
Following familiarisation with treadmill running and experimental
procedures, the subjects undertook two preliminary tests in order to
determine (i) the relationship between running speed and ýOµ using a
16 min incremental submaximal running test, and (ii) the ýOµ,max
using an uphill incremental treadmill running test to exhaustion
(Taylor et al. 1955). Expired air samples were collected using the
Douglas bag method and analysed for ýOµ, carbon dioxide production
(ýCOµ), and respiratory exchange ratio (RER), as previously
described (Tsintzas et al. 1995). The heart rate value obtained at the
end of the ýOµ,max test was taken as the HRmax value of the individual.
Experimental design
All subjects completed two 10 min runs at 70% ýOµ,max on a
motorised treadmill 7 days apart. On each occasion, subjects
ingested 8 ml (kg BM)¢ of either a placebo solution containing
artificial sweetener (CON trial) or a 5.5% CHO solution (CHO trial)
10 min before each run. The CHO solution was a 5.5% solution
which contained glucose (1.7%), fructose (1.1%), maltose (0.6%),
higher saccharides (1.9%) and electrolytes (sodium, 26.5 mmol l¢;
potassium, 2.6 mmol l¢). The osmolality of the CHO solution was
280 mosmol kg¢. The experiment was conducted using a single-
blind design, with the CON trial occurring first. A random order of
trials was not employed because this study was part of a bigger
project where the main aim was to investigate the metabolic
environment within the muscle at that point in time during the CHO
trial that coincided with exhaustion in the CON trial. Therefore,
because we had recruited experienced subjects who were familiar
with the experimental procedures employed in this study and made
no performance measurements, the employment of a single-blind
design was considered sufficient to examine the main hypothesis of
the present study, as described in the Introduction.
The subjects were asked to refrain from heavy exercise, alcohol,
caffeine and tobacco consumption for 2 days before each trial and to
weigh and record their food intake for 3 days preceding the first
trial. They were then required to replicate exactly the same diet for
the same period of time before the second trial.
Protocol
On the day of the experiment each subject arrived in the laboratory
following a 12 h overnight fast and his nude body mass was
obtained. Following this, an indwelling cannula (venflon, 18 G,
Ohmeda, Hatfield, Herts, UK) was inserted in an ante-cubital vein
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Exp. Physiol. 85.5
while the subject was lying on an examination couch. The cannula
was kept patent by infusion with sterile saline. A resting muscle
sample was then obtained from the vastus lateralis muscle. A 10 ml
resting venous blood sample was also obtained after which 5 min of
warm-up exercise was performed at 60% ýOµ,max. The treadmill was
then stopped for 10 min and the subject consumed 8 ml (kg BM)¢
of the assigned fluid while standing on the treadmill. In the CHO
trial, this fluid ingestion resulted in the consumption of 33.6 ± 1.1 g
of CHO. At the end of this 10 min resting period a second 10 ml
venous blood sample was collected (0 min sample). Immediately
after this 10 min period, the treadmill speed was increased and each
subject ran for 10 min at a speed equivalent to 70% ýOµ,max. A
1 min expired gas sample and a 10 ml venous blood sample were
obtained during the final minute of the run. Upon the completion of
the run, the treadmill was stopped and the subject was quickly
transferred to an adjacent couch where a second muscle sample was
obtained from the vastus lateralis. Dry bulb temperatures within the
laboratory were 18.7 ± 0.3 and 19.0 ± 0.1 °C during the CON and
CHO trials, respectively.
Blood sample collection and analysis
Serum insulin, plasma free fatty acids (FFAs), whole blood lactate
and glucose concentrations, and percentage changes in plasma
volume (% PVC) were obtained by methods previously described
(Tsintzas et al. 1995). The resting blood sample was used as the
baseline reference for the calculation of % PVC.
Muscle sample collection and analysis
All muscle samples were obtained from the vastus lateralis muscle
using the needle biopsy technique (Bergstrom, 1962). Each sample
was taken through a separate skin incision (3—5 mm long). All
incisions were made under local anaesthesia (2—3 ml of 1% lidocaine
(lignocaine)) using a surgical blade before the start of exercise while
the subject was lying on an examination couch. After removing the
biopsy needle from the leg, the biopsy needle was immediately
immersed in liquid nitrogen. The time delay between the end of
exercise and the freezing of the muscle sample in liquid nitrogen was
on average between 10 and 12 s, and there was no difference
between trials. Each frozen sample was divided into two parts while
under liquid nitrogen. One part was kept in liquid nitrogen until it
was used to determine the active form of PDC (PDCa) by a method
previously described (Constantin-Teodosiu et al. 1991).
The remaining part of the snap-frozen muscle sample was freeze
dried, after which it was stored at −70°C. At a later date, the
freeze-dried muscle was dissected free of visible blood and
connective tissue, powdered and washed twice with 40% petroleum
ether to remove fat. Muscle metabolites (PCr, glucose-6-phosphate
(G-6-P), pyruvate and lactate) were extracted and determined
enzymatically (Lowry & Passonneau, 1972; Harris et al. 1974).
The neutralised muscle extract was also used for the determination
of free carnitine and acetylcarnitine by enzymatic assays using
radioisotopic substrates as previously described (Cederblad et al.
1990) and for the determination of citrate (Bergmeyer, 1974). A
portion of the freeze-dried muscle powder was also used for the
determination of muscle glycogen. The muscle powder was digested
in 0.5 mol l¢ NaOH and neutralised with HCl—citrate buffer
(pH = 4.9). The glycogen present in the supernatant was hydrolysed
with á_amyloglucosidase and analysed for glucosyl units by an
enzymatic method (Harris et al. 1974).
Statistical analysis
The normal distribution of data and their homogeneity were
checked before applying appropriate statistical analysis. Analysis
Exp. Physiol. 85.5 Carbohydrate feeding and the pyruvate dehydrogenase complex 583

of variance (ANOVA) for repeated measures on two factors –––––––––––––––––––––––––––––

(experimental treatment and sampling time) was used to assess Table 1. Cardiovascular and respiratory responses to CON and
overall differences between physiological and metabolic responses to CHO trials
both trials. When a significant difference was obtained, the –––––––––––––––––––––––––––––
Neuman-Keul post hoc test was used to locate any differences. CON CHO
Statistical significance was accepted at a 5% level. Results are –––––––––––––––––––––––––––––
presented as means ± s.e.m. % ýOµ,max 68.8 ± 1.9 67.8 ± 1.4
RER 0.95 ± 0.01 0.97 ± 0.01
RESULTS CHO oxidation (g min¢) 3.0 ± 0.2 3.1 ± 0.1
Cardiovascular and respiratory responses Fat oxidation (g min¢) 0.3 ± 0.1 0.2 ± 0.1
% PVC −2.8 ± 1.3 —1.5 ± 1.5
The average percentage ýOµ,max values sustained during the –––––––––––––––––––––––––––––
CON and CHO trials were 68.8 ± 1.9 and 67.8 ± 1.4%, Values are means ± s.e.m. given in (g min¢) for oxidation rates;
respectively (range 64—75%). Based on the ýOµ and blood n = 7. % PVC, percentage change in plasma volume.
lactate response to exercise and the ratings of perceived –––––––––––––––––––––––––––––
exertion, the exercise intensity employed in this study was
submaximal for all subjects. There were no differences Blood metabolites
between trials in RER and CHO and fat oxidation rates, as Carbohydrate ingestion increased blood glucose and serum
calculated from ýOµ and RER values (Table 1). The percentage insulin concentrations (Fig. 1) immediately before and at
change in plasma volume was also similar between trials. 10 min of exercise (P < 0.01). Plasma FFA concentrations

Figure 1
Blood glucose, serum insulin and plasma FFA concentrations during the CON and CHO trials. Values are
means ± s.e.m.; n = 7. * P < 0.01 from CON; ** P < 0.05 from CON.

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584 K. Tsintzas and others Exp. Physiol. 85.5

––––––––––––––––––––––––––––––––––––––––––––––
Table 2. Muscle metabolites before and after the 10 min run in the CON and CHO trials
––––––––––––––––––––––––––––––––––––––––––––––
CON CHO
––––––––––––– ––––––––––––
Rest 10 min run Rest 10 min run
––––––––––––––––––––––––––––––––––––––––––––––
Glycogen 369 ± 31 302 ± 35* 365 ± 34 299 ± 30*
PCr 74.6 ± 1.0 65.9 ± 2.3** 75.5 ± 2.6 67.4 ± 2.3 **
G-6-P 1.2 ± 0.2 3.0 ± 0.6 1.2 ± 0.3 2.2 ± 0.5
Pyruvate 0.53 ± 0.03 0.69 ± 0.06** 0.55 ± 0.02 0.63 ± 0.04
Lactate 5.3 ± 0.5 8.9 ± 1.2** 6.3 ± 0.4 9.1 ± 0.4**
Citrate 0.64 ± 0.06 0.91 ± 0.10** 0.55 ± 0.07 0.73 ± 0.05†
––––––––––––––––––––––––––––––––––––––––––––––
Values are means ± s.e.m.; units are mmol glucosyl units (kg DM)¢ for glycogen and mmol (kg DM)¢ for
other metabolites; n = 7; * P < 0.01 from rest; ** P < 0.05 from rest; † P < 0.05 from CON.
––––––––––––––––––––––––––––––––––––––––––––––
were not different at rest and 0 min. At the end of the run the Muscle metabolites
plasma FFA concentration was lower (P < 0.05) in the CHO As Table 2 shows, CHO ingestion resulted in similar muscle
trial compared with the CON trial (Fig. 1). There was no PCr, glycogen, G-6-P, pyruvate and lactate concentrations at
difference between trials in blood lactate concentration at the the end of exercise when compared with placebo ingestion.
end of exercise (0.9 ± 0.1 mmol l¢). There was no difference between trials in PDC activity at rest
(Fig. 2). In both trials, exercise stimulated PDC activity
(P < 0.01). However, at the end of the run PDCa was higher
in the CHO trial than in the CON trial (Fig. 2). The activity of
PDC increased in all subjects in response to CHO feeding.
The average increase was 51 ± 23%. The PDC data is
presented for only six subjects because the determination of
PDC was precluded in one subject due to lack of material.
In the CHO trial, muscle citrate (Table 2) and acetylcarnitine
(Fig. 2) accumulation were lower during exercise when
compared with the CON trial (P < 0.05). No difference was
observed in total muscle carnitine concentration (defined as
the sum of acetylcarnitine and free carnitine concentrations)
between the CON and CHO trials at rest (22.8 ± 1.5 vs.
23.1 ± 2.0 mmol (kg DM)¢) and at 10 min of exercise
(23.4 ± 2.1 vs. 22.4 ± 1.8 mmol (kg DM)¢).
DISCUSSION
This is the first study to demonstrate that CHO ingestion prior
to exercise augments the exercise-induced activation of muscle
PDC and reduces acetylcarnitine accumulation during the
transition from rest to steady-state exercise. However, those
changes do not appear to affect the contribution of substrate
level phosphorylation (PCr hydrolysis and glycogenolysis) to
ATP resynthesis.
At the onset of exercise there is a delay in the activation of
PDC and formation of acetyl-CoA which appears to be
associated with an increase in the contribution of substrate
level phosphorylation to energy metabolism (Timmons et al.
1997; Howlett et al. 1999). When this delay in PDC activation
Figure 2 was circumvented by maximally activating PDC using
Muscle PDC activity and acetylcarnitine concentrations at rest dichloroacetate (DCA) prior to contraction (Timmons et al.
and after 10 min of exercise in the CON and CHO trials. 1997, 1998; Howlett et al. 1999), a reduction in muscle PCr
Values are means ± s.e.m.; n = 6 for PDC and n = 7 for hydrolysis and lactate formation was observed within the first
acetylcarnitine. ** P < 0.05 from CON. few minutes of exercise. This finding is in contrast with the
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Exp. Physiol. 85.5 Carbohydrate feeding and the pyruvate dehydrogenase complex
results from the present study which showed that a CHO-
induced increase in PDC activation during running does not
reduce the requirement for ATP resynthesis by substrate level
phosphorylation. There are a number of possible explanations
for this apparent discrepancy.
Firstly, in the present study a relatively small amount of PCr
(•10% of resting values) was used during the first 10 min of
exercise. Previous human studies using submaximal cycling
(Howlett et al. 1999) and submaximal single leg knee
extension (Timmons et al. 1998) reported a much higher rate
of PCr degradation within the first 8—10 min of exercise, i.e.
•40—50% of resting values. The extent of PCr breakdown
during exercise inversely reflects the magnitude of substrate
oxidation. Thus, the modest muscle PCr hydrolysis and lactate
accumulation observed at the onset of submaximal running
suggest that the rate of acetyl group provision to the TCA cycle
might not be a limiting factor in oxidative phosphorylation at
the onset of this type and intensity of exercise. Alternatively,
the modest changes in PCr hydrolysis and lactate accumulation
observed in the present study could be attributed, at least in
part, to a stockpile of acetyl groups as a result of the warm-up
exercise performed prior to the main trials. Secondly, the 2- to
3-fold lower activation of PDC observed in the present study,
when compared with the values recorded in DCA studies,
might have resulted in lower provision of acetyl groups to the
TCA cycle and thus might have precluded any measurable
changes in substrate level phosphorylation. Thirdly, it is also
possible that any such changes might have been confined to a
specific fibre type and hence, under the conditions of this
study, conclusions drawn from analysis of homogenates of
muscle samples might not offer a clear insight into the
metabolic responses during the onset of exercise. The latter
suggestion is prompted by the results of a more recent study in
which we reported that during the first 10 min of submaximal
running a significant hydrolysis of PCr occurs in type II
fibres only (Tsintzas et al. 1999). Furthermore, most of the
acetylcarnitine accumulation also occurs in type II fibres
(Constantin-Teodosiu et al. 2000). Therefore, it is not
unreasonable to conclude that any CHO-induced changes in
substrate utilisation during the transition from rest to exercise
might be confined to type II fibres. Finally, it is also possible
that the CHO-induced changes in the transformation of PDC
to its active form, as measured in the present study, did not
alter the flux through the PDC reaction, since the latter is also
determined by pyruvate availability.
Hyperglycaemia and hyperinsulinaemia have been shown to
increase PDC activation in resting skeletal muscle (Mandarino
et al. 1987, 1993). Hyperglycaemia is thought to stimulate
PDC through an increase in pyruvate availability as a result of
increases in glucose uptake and glycolysis (Mandarino et al.
1993). However, in the present study, there were no differences
between trials in G-6-P and pyruvate concentrations and
hence the rate of glycolysis. The rates of glycogen breakdown
were also similar between trials. It is possible, therefore, that
the increase in PDC activation observed in the CHO trial was
due to a CHO-induced increase in serum insulin concentration,
which has been shown to activate the PDC phosphatase, the
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585
regulatory enzyme responsible for the dephosphorylation and
hence activation of PDC (Mandarino et al. 1987; Patel &
Roche, 1990). Whatever the mechanism underlying the
activation of PDC in the present study, the possibility cannot
be excluded that the pre-exercise CHO feeding may have
activated PDC at rest, producing a readily available pool of
CHO-derived acetyl groups for use by the TCA cycle at the
onset of exercise. However, this is unlikely to explain the
results from the present study since the time which elapsed
between the end of the CHO ingestion and the onset of
exercise (•5 min) was probably too short to fully account for
the increase in PDC activation. Nevertheless, further studies
are required to confirm this hypothesis.
Alternatively, the increase in PDC activity in the CHO trial
could also be attributed to a reduction in the formation of
acetyl groups from fat oxidation following the insulin-induced
reduction in plasma FFA concentrations. A decrease in acetyl-
CoA has been shown to decrease the activity of PDC kinase,
thereby favouring the transformation of PDC to its active form
(Cooper et al. 1975). This, however, is unlikely to explain the
results from the present study since the contribution of fat
oxidation to energy metabolism at the onset of exercise was
small (as indicated by the high RER values observed in both
trials) and was unaffected by CHO ingestion.
When a mismatch between the rate of acetyl-CoA formation
by the PDC and its rate of oxidation by the TCA cycle occurs
during exercise, then the excess of acetyl groups are buffered by
carnitine (a reaction catalysed by carnitine acetyltransferase),
resulting in muscle acetylcarnitine accumulation (Constantin-
Teodosiu et al. 1992). Thus, it is possible that the reduced
acetylcarnitine accumulation observed in the CHO trial was
attributable to a better match between CHO-derived acetyl
group formation and utilisation by the TCA cycle. In the
absence of an increase in whole body CHO oxidation rate, this
conclusion could not be confirmed. However, one might argue
that the use of indirect calorimetry to estimate whole body
substrate oxidation rates might have precluded the detection of
small changes in those rates across the exercising muscles,
especially if such changes were confined to a specific muscle
fibre type.
Recently, it has been shown that pharmacological activation of
PDC using DCA decreases the concentrations of the TCA
cycle intermediates (TCAI) at rest, possibly by directing
pyruvate to acetyl-CoA formation and, thus, limiting the
available pool of pyruvate for anaplerosis (Constantin-Teodosiu
et al. 1999; Gibala & Saltin, 1999). During exercise, however,
the total concentration of TCAI was not affected by the
activation of PDC prior to muscle contraction (Gibala & Saltin,
1999). It should be noted that in the latter study, despite the
marked difference in PDC activity at rest, no difference in
PDC activity was observed at the onset of exercise. However,
in the present study the PDC activity was higher at 10 min of
exercise in the CHO trial compared with the CON trial and
was accompanied by a decrease in citrate accumulation. The
strong negative correlation observed between PDC activity
and muscle citrate concentration (CON: r = −0.89, P < 0.05;
 586 K. Tsintzas and others
CHO: r = −0.80, P = 0.05) provides some support to the
hypothesis that an increase in PDC activity at the onset of
exercise may result in a greater fraction of pyruvate being
directed towards acetyl-CoA formation rather than towards
anaplerotic pathways.
In summary, pre-exercise CHO ingestion augmented the
exercise-induced activation of muscle PDC, and reduced
acetylcarnitine accumulation, during the transition from rest
to steady-state exercise. However, those changes did not appear
to affect the rates of muscle PCr and glycogen degradation and
lactate accumulation. The type and intensity of exercise
employed in this study might explain the failure of CHO
ingestion to induce any measurable changes in the contribution
of substrate level phosphorylation to energy metabolism. In
addition, the lower degree of PDC activation observed in this
study compared with previous studies in which pharmacological
interventions were used to activate PDC might also explain the
absence of CHO-induced effects on muscle glycogenolysis and
PCr hydrolysis. Further studies are required to elucidate the
precise mechanism underlying the CHO-induced augmentation
of PDC activation at the onset of exercise.
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