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Skeletal muscle and exercise |
1 Canadian Centre for Activity and Aging
2 School of Kinesiology, Faculty of Health Sciences
3 Department of Physiology and Pharmacology
4 Departments of Clinical Neurological Sciences and Rehabilitation Medicine, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5B9
5 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
6 Faculty of Applied Health Sciences, Brock University, St Catharines, Ontario, Canada L2S 3A1
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Abstract |
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during the transition to moderate-intensity exercise (Mod) is faster following a prior bout of heavy-intensity exercise. In the present study we examined the activation of pyruvate dehydrogenase (PDHa) during Mod both with and without prior heavy-intensity exercise. Subjects (n
= 9) performed a Mod1–heavy-intensity–Mod2 exercise protocol preceded by 20 W baseline. Breath-by-breath 
kinetics and near-infrared spectroscopy-derived muscle oxygenation were measured continuously, and muscle biopsy samples were taken at specific times during the transition to Mod. In Mod1, PDHa increased from baseline (1.08 ± 0.2 mmol min–1 (kg wet wt)–1) to 30 s (2.05 ± 0.2 mmol min–1 (kg wet wt)–1), with no additional change at 6 min exercise (2.07 ± 0.3 mmol min–1 (kg wet wt)–1). In Mod2, PDHa was already elevated at baseline (1.88 ± 0.3 mmol min–1 (kg wet wt)–1) and was greater than in Mod1, and did not change at 30 s (1.96 ± 0.2 mmol min–1 (kg wet wt)–1) but increased at 6 min exercise (2.70 ± 0.3 mmol min–1 (kg wet wt)–1). The time constant of 
was lower in Mod2 (19 ± 2 s) than Mod1 (24 ± 3 s). Phosphocreatine (PCr) breakdown from baseline to 30 s was greater (P < 0.05) in Mod1 (13.6 ± 6.7 mmol (kg dry wt)–1) than Mod2 (6.5 ± 6.2 mmol (kg dry wt)–1) but total PCr breakdown was similar between conditions (Mod1, 14.8 ± 7.4 mmol (kg dry wt)–1; Mod2, 20.1 ± 8.0 mmol (kg dry wt)–1). Both oxyhaemoglobin and total haemoglobin were elevated prior to and throughout Mod2 compared with Mod1. In conclusion, the greater PDHa at baseline prior to Mod2 compared with Mod1 may have contributed in part to the faster 
kinetics in Mod2. That oxyhaemoglobin and total haemoglobin were elevated prior to Mod2 suggests that greater muscle perfusion may also have contributed to the observed faster 
kinetics. These findings are consistent with metabolic inertia, via delayed activation of PDH, in part limiting the adaptation of pulmonary 
and muscle O2 consumption during the normal transition to exercise.
(Received 2 May 2006;
accepted after revision 21 September 2006;
first published online 21 September 2006)
Corresponding author J. M. Kowalchuk: Canadian Centre for Activity and Aging, School of Kinesiology, Faculty of Health Sciences, HSB 411C, The University of Western Ontario, London, Ontario, Canada N6A 5B9. Email: jkowalch{at}uwo.ca
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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during the transition to moderate-intensity exercise resides in the ability to provide adequate substrate (i.e. O2, ADP, Pi, NADH and H+) for mitochondrial electron transport and oxidative phosphorylation (Tschakovsky & Hughson, 1999). In an attempt to isolate the mechanism(s), studies have tried to remove a potential O2 delivery limitation through manipulation of convective and/or diffusive O2 delivery (Grassi, 2001; Hughson et al. 2001), or have tried to overcome a metabolic inertia by removing a nitric oxide (NO)-induced inhibition of cytochrome oxidase and oxidative phosphorylation by inhibition of NO synthase using L-NAME (Kindig et al. 2002; Jones et al. 2003; Grassi et al. 2005) or through prior activation of muscle enzymes, mainly the mitochondrial pyruvate dehydrogenase complex (PDH; Howlett et al. 1999; Greenhaff et al. 2002; Rossiter et al. 2003; Howlett & Hogan, 2003). PDH has been proposed as a possible site of metabolic inertia as it controls the entry of carbohydrate-derived substrate into the tricarboxylic acid (TCA) cycle, and thus the provision of reducing equivalents (in the form of NADH and FADH2) to the electron transport chain.
Recently we demonstrated that the adaptation of 
at the onset of moderate-intensity exercise (i.e. an exercise intensity not associated with significant muscle lactate accumulation) was faster when the exercise was preceded by a bout of heavy-intensity exercise (Gurd et al. 2005). Both muscle perfusion (and O2 delivery) (Fukuba et al. 2004; Endo et al. 2005; Paterson et al. 2005) and mitochondrial PDH activity (Putman et al. 1995; Parolin et al. 1999) probably remain elevated in the immediate postexercise recovery from a bout of heavy-intensity exercise (i.e. an intensity associated with significant lactate accumulation and metabolic acidosis). In the study of Gurd et al. (2005), heart rate and local muscle oxygenation (determined using near-infrared spectroscopy) both remained elevated after the bout of heavy-intensity exercise and immediately prior to the onset of moderate-intensity exercise, consistent with greater muscle perfusion and O2 delivery. However, the relationship between PDH activity and 
kinetics at the onset of moderate-intensity exercise with and without prior heavy-intensity exercise was not established.
A speeding of 
kinetics during the transition to moderate intensity has not always been reported following interventions that attempt to manipulate either muscle blood flow and O2 delivery (i.e. perfusion limitation) or muscle enzyme activity and substrate provision (i.e. metabolic limitation). It is possible that an intervention designed to overcome only a single limiting factor to 
will only expose another limitation which still prevents a rapid acceleration of 
from being observed. With a model of prior ‘heavy-intensity exercise’ it might be possible to overcome limitations at a number of muscle sites, and thereby remove many of the inhibitions imposed on 
adaptation. Therefore, prior heavy exercise would be expected to: (i) increase the activity of PDH and possibly other mitochondrial enzymes, and thus elevate the concentration of substrates (e.g. acetyl CoA, NADH) for the tricarboxylic acid (TCA) cycle and electron transport chain; (ii) elevate muscle (presumably cytosolic) lactate concentration, and thereby provide a readily available source of substrate (via reversal of the LDH reaction and formation of pyruvate and NADH) for the PDH reaction and for the mitochondrial electron shuttle systems; and (iii) elevate muscle perfusion (relative to muscle O2 consumption), and thereby raise microvascular 
and the driving pressure for O2 diffusion into the mitochondria. Also, the use of multiple repetitions of the exercise protocol to reduce the variability and increase the signal-to-noise of the 
response provides an improved confidence in our parameter estimation of the time course of the 
response for individual subjects, thereby allowing small differences between both individual and group values to be discerned.
Therefore, the purpose of the present study was to examine the effect of a prior bout of heavy-intensity exercise on the adaptation of pulmonary 
, muscle PDH activation and metabolism, and muscle oxygenation, to better understand the underlying limitations to muscle O2 consumption during moderate-intensity exercise. We hypothesized that prior heavy-intensity exercise would be associated with (1) a faster adaptation of pulmonary 
during the transition to a subsequent bout of moderate-intensity exercise, (2) a higher baseline active form of PDH (PDHa) activation, (3) faster PDH activation to steady-state levels, and (4) elevated baseline and exercise muscle oxygenation (as determined by near-infrared spectroscopy).
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Methods |
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Nine young healthy males adults (age, 24 ± 4 years; 
, 47 ± 2 ml kg–1 min–1) volunteered and gave written informed consent to participate in the study. All subjects were recreationally active but not involved in a specific training programme at the time of the study. The study was approved by The University of Western Ontario Research Ethics Board for Health Sciences Research Involving Human Subjects and conformed with the Declaration of Helsinki.
Exercise protocol
Subjects reported to the laboratory on six separate occasions at approximately the same time of day, approximately 2 h after consuming a small meal high in carbohydrate and low in fat. Subjects performed an incremental ramp exercise test (25 W min–1) to the limit of tolerance on an electronically braked cycle ergometer (model H-300-R; Lode) on the first day of testing for determination of the estimated lactate threshold (
L) and 
. The L was defined as the 
at which CO2 output 
began to increase out of proportion relative to 
, combined with a systematic rise in the ventilatory equivalent for 
and end-tidal 
with no concomitant rise in the ventilatory equivalent for 
or end-tidal 
. 
was calculated as the average 
over the final 30 s of the ramp exercise test. From the results of this ramp test, work rates (WRs) were identified that elicited a 
corresponding to 
90%
L (i.e. moderate-intensity exercise), and 
50% (50% of the difference between the 
at L and 
, i.e. heavy-intensity exercise).
During four of the subsequent five visits to the laboratory, subjects performed two step-transitions in WR of moderate intensity (Mod1 and Mod2) separated by a step increase in WR of heavy intensity, as previously described (Scheuermann et al. 2002; Gurd et al. 2005). Exercise was performed continuously; the duration of each step-transition was 6 min, and each transition was preceded by 6 min baseline cycling at 20 W. Changes in WR were initiated as a step function without warning to the subject. This continuous protocol was performed four times, resulting in four repetitions for each subject and condition. In one other visit, placed randomly amongst the final 2–5 visits, subjects repeated the protocol but with the cycling being interrupted and muscle biopsy samples being taken during baseline (20 W) cycling and at 30 and 360 s of the transition to each of the two moderate-intensity exercise bouts (see below).
measurement
Gas exchange was measured as previously described (Babcock et al. 1994; Scheuermann et al. 2002). Briefly, inspired and expired flow rates were measured with a low dead-space (90 ml) bi-directional turbine (Alpha technologies VMM 110), which was calibrated before each test with a syringe of known volume (3 l). Inspired and expired gases were sampled continuously at the mouth and analysed for concentrations of O2, CO2 and N2 by mass spectrometry (AMIS 2000) after calibration with precision-analysed gas mixtures. Changes in gas concentration were aligned with gas volumes by measuring the time delay for a square-wave bolus of gas passing the turbine to the resulting changes in fractional gas concentrations as measured by the mass spectrometer. Data collected every 20 ms were transferred to a computer, which aligned concentrations with the volume data to build a profile of each breath. Breath-by-breath alveolar gas exchange was calculated by the algorithms of Beaver et al. (1981).
Near-infrared spectroscopy
Near-infrared spectroscopy (NIRS; Hamamatsu NIRO 300; Hamamatsu Photonics KK, Japan) was used to measure, continuously, changes in concentration of local muscle oxyhaemoglobin (O2Hb), deoxyhaemoglobin (HHb) and total haemoglobin-myoglobin (HbTOT) of the vastus lateralis muscle of the right leg. Optodes were placed on the belly of the muscle midway between the lateral epicondyle and the greater trochanter of the femur. The optodes were housed in an optically dense plastic holder to ensure that their separation remained constant. The optode assembly was secured on the skin surface with tape, covered with an optically dense black vinyl sheet to minimize the intrusion of extraneous light and loss of NIR-transmitted light from the field of interrogation, and wrapped with an elastic bandage to minimize movement of the optodes, while still permitting freedom of movement for cycling. This preparation essentially prevented any optode movement relative to the skin surface.
Use of NIRS to monitor changes in local muscle oxy- and deoxygenation status during exercise was previously described by DeLorey et al. (2004). The NIRS unit uses four different wavelength laser diodes (775, 810, 850 and 910 nm) pulsed in rapid succession, with the reflected light detected by the photomultiplier tube. The intensity of incident and transmitted light was recorded continuously at 1 Hz and, along with the relevant specific extinction coefficients and optical path length (assuming a differential path length factor of 3.83; Delorey et al. 2004), used for online estimation and display of the relative concentration changes from the ‘zero’ set during the resting baseline of O2Hb, HHb and HbTOT. The raw attenuation signals (in optical density units) were transferred to computer and stored for further analysis.
The NIRS-derived HHb signal is a reliable estimator of changes in intramuscular deoxygenation and represents the balance between local muscle O2 delivery and O2 utilization, which reflects microvascular O2 and muscle O2 extraction within the NIRS field of interrogation (De Blasi et al. 1994; Ferrari et al. 1997).
Muscle sampling
During one of the visits to the laboratory, a total of six muscle biopsy samples were obtained from each subject, with three biopsy samples taken from a single leg during each of the two moderate-intensity exercise transitions. Biopsy samples were obtained from the vastus lateralis muscle using the needle biopsy technique (Bergstrom, 1975). Initially three biopsy sites were prepared by making incisions through the skin to the deep fascia under local anaesthesia (2% lidocaine (lignocaine) without adrenaline). The subject then was moved to the cycle ergometer and began baseline cycling at 20 W. A biopsy sample was taken after 5 min baseline cycling, and two exercise samples were taken at 30 s and 6 min of the transition to Mod1. After approximately 1 h resting recovery, during which time three biopsy sites were prepared on the other leg, the subject returned to the cycle ergometer and exercised for 6 min at the 20 W baseline, followed by 6 min of heavy-intensity exercise, 6 min baseline exercise (at 20 W) and 6 min moderate-intensity exercise (i.e. Mod2). Muscle biopsy samples were taken 1 min before (i.e. 5 min recovery from heavy-intensity exercise) and at 30 s and 6 min of Mod2. Muscle biopsy samples were immediately frozen in liquid N2, removed from the needle while frozen and stored in liquid N2 until later analysis.
Modelling of the 
and NIRS responses
The breath-by-breath 
data obtained during each step increase in WR were filtered and linearly interpolated to 1 s intervals. Each transition was time-aligned, ensemble-averaged to yield a single profile, and time-averaged into 10 s bins to give a single response for each subject. The on-transient responses to Mod1 and Mod2 were modelled as a monoexponential of the form:
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is the time constant (i.e. the time taken to reach 63% of the steady-state response) and TD is the time delay. 
data were fitted from the phase 1 to phase 2 transition to the end of exercise, as previously described (Rossiter et al. 2001; Gurd et al. 2005).
The time delay before an increase in HHb after exercise onset (HHb-TD) was determined by second-by-second data and corresponded to the time to the first point demonstrating a consistent increase above the nadir of the signal. The estimation of the HHb-TD was performed on each individual trial and reported as the average of the four trials for each subject. The NIRS-derived HHb, O2Hb and HbTOT data were time-aligned, ensemble-averaged and time-averaged into 5 s bins to yield a single response for each subject. As the HHb response between the HHb-TD and 90 s of the exercise transition increases in an exponential-like manner, these data were modelled using an exponential function of the form given in eqn (1) to determine the time course of the muscle HHb response (HHb). The mean response time (MRT = HHb-TD +
HHb) was calculated to provide a description of the overall time course for muscle HHb. The O2Hb and HbTOT signals do not approximate an exponential response, and thus the analysis of these data was limited to determining the steady-state baseline and end-exercise values.
Muscle analysis
A small piece of frozen muscle (10–15 mg) was chipped from each muscle sample under liquid N2 and used for determination of the active form of PDH (PDHa) as previously described (Constantin-Teodosiu et al. 1991; Putman et al. 1993). The remaining muscle sample was freeze-dried, powdered and dissected free of all visible blood and connective tissue, and extracted with 0.5 M perchloric acid containing 1 mM EDTA, and neutralized with 2.2 M KHCO3, for determination of muscle metabolite concentrations. Creatine, phosphocreatine (PCr), ATP, lactate and pyruvate were analysed by spectrophotometric assays (Bergmeyer, 1974; Harris et al. 1974), while acetyl-CoA was determined radioisotopically (Cederblad et al. 1990). All muscle measurements were normalized to the highest total creatine measured amongst the six biopsy samples from each subject.
Calculations
Muscle contents of free ADP (ADPf) and AMP (AMPf) were calculated by assuming equilibrium of the creatine kinase and adenylate kinase reactions, respectively (Dudley et al. 1987). ADPf was calculated by using the measured ATP, creatine, PCr, estimated H+ concentration and the creatine kinase equilibrium constant of 1.66 x 106; H+ concentration was calculated from the measured pyruvate and lactate contents as described by Sahlin et al. (1976). AMPf was calculated with the estimated ADPf and measured ATP content using the adenylate kinase equilibrium constant of 1.05 (Dudley et al. 1987). Free inorganic phosphate (Pif) was calculated by adding the estimated free phosphate content of 10.8 mmol (kg dry wt)–1 to the difference in PCr content relative to the baseline value.
Statistical analysis
Parameter estimates for 
and NIRS-derived HHb responses for the two moderate-intensity exercise bouts were compared using a one-way ANOVA for repeated measures. PDHa and muscle metabolite contents were compared using a two-way ANOVA for repeated measures with main effects of exercise bout and time. Statistical significance was accepted at P < 0.05. Data are presented as means ±
S.D.
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Results |
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kinetics
In the present study the moderate-intensity exercise represented 89%
L (±4) (44%
(±3) at a power output of 100 ± 15 W) while the heavy-intensity exercise represented 50% (78%
(±4) at a power output of 228 ± 25 W). A summary of the parameter estimates for the on-transient 
response to Mod1 and Mod2 are presented in Table 1, and the response for an individual subject with exponential model fit (and residuals) is shown in Fig. 1. The baseline and end-exercise 
were higher (P < 0.05) and the 
amplitude was lower (P < 0.05) in Mod2 compared with Mod1. Heavy-intensity exercise was associated with a speeding of 
kinetics in eight of nine subjects; the phase 2 
time constant 
was reduced (P < 0.05) in Mod2 (19 ± 2 s) compared with Mod1 (24 ± 3 s).
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During Mod1, the activity of PDHa increased (P < 0.05) from baseline (1.08 ± 0.20 mmol min–1 (kg wet wt)–1) to 30 s exercise (2.05 ± 0.21 mmol min–1 (kg wet wt)–1), and was not different at 6 min exercise (2.07 ± 0.34 mmol min–1 (kg wet wt)–1; Fig. 2). After heavy-intensity exercise, baseline PDHa activity was elevated (P < 0.05) (1.88 ± 0.30 mmol min–1 (kg wet wt)–1) compared with Mod1 and did not change significantly during the first 30 s exercise in Mod2 (1.96 ± 0.20 mmol min–1 (kg wet wt)–1). At 6 min exercise in Mod2, PDHa activity (2.70 ± 0.30 mmol min–1 (kg wet wt)–1) was greater (P < 0.05) than at 30 s of Mod2 and at end-exercise in Mod1 (Fig. 2).
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Muscle pyruvate content did not change during moderate-intensity exercise and was not affected by prior heavy-intensity exercise (Table 2). The muscle lactate content did not change during Mod1 (Table 2). After heavy-intensity exercise, the muscle lactate content was elevated (P < 0.05) at baseline (28.8 ± 19.3 mmol min–1 (kg wet wt)–1), and although the muscle lactate content decreased (P < 0.05) throughout Mod2, it remained elevated (P < 0.05) at end-exercise (Table 2, Fig. 3). Calculated muscle [H+] was increased at 30 s and 6 min compared to baseline in Mod1 and was elevated prior to Mod2 compared with Mod1 baseline and did not change throughout Mod2 (Table 2).
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Muscle creatine content increased (P < 0.05) from baseline to 30 s in Mod1 and remained elevated at 6 min (Table 3). After heavy-intensity exercise, creatine content was elevated compared with the Mod1 baseline, remained unchanged after 30 s exercise, and increased (P < 0.05) by end-exercise. During Mod1, PCr content decreased (P
= 0.06) from baseline to 30 s exercise, with no further change at end-exercise (Table 3). Baseline PCr content was lower (P < 0.05) prior to Mod2 than Mod1 (by 6.5 mmol (kg dry wt)–1); PCr content was not significantly changed at 30 s exercise in Mod2, but after 6 min, PCr content was lower (P < 0.05) than both the Mod2 baseline and the Mod1 end-exercise value (Fig. 3). During the first 30 s exercise PCr breakdown was greater (P < 0.05) in Mod1 (13.6 ± 6.7 mmol (kg dry wt)–1) than Mod2 (6.5 ± 6.2 mmol (kg dry wt)–1), but during the subsequent 5.5 min exercise PCr breakdown was greater (P < 0.05) in Mod2 (13.6 ± 9.1 mmol (kg dry wt)–1) than Mod1 (1.1 ± 8.9 mmol (kg dry wt)–1), which resulted in a similar (P > 0.05) total PCr breakdown between conditions (Mod1, 14.8 ± 7.4 mmol (kg dry wt)–1; Mod2, 20.1 ± 8.0 mmol (kg dry wt)–1). ATP and ADPf were unchanged compared with baseline during both Mod1 and Mod2 (Table 3).
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A summary of the parameter estimates for HHb, O2Hb and HbTOT is presented in Table 4. The baseline and end-exercise O2Hb and HbTOT were elevated (P < 0.05) in Mod2 compared with Mod1. The baseline for HHb was not different between Mod1 and Mod2, while the amplitude of the HHb response was greater (P < 0.05) in Mod2 compared with Mod1 (Table 4). The time course of HHb adaptation was described by a shorter (P < 0.05) HHb-TD and greater (P < 0.05) HHb in Mod2 compared to Mod1; the HHb mean response time was not different between the two transitions.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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, PDH activity, muscle metabolite contents and muscle deoxygenation during the transition to moderate-intensity exercise in healthy young adults. After heavy-intensity exercise phase 2 pulmonary 
kinetics (a reflection of the time course of muscle O2 consumption) was faster (by 20%) and baseline and end-exercise O2Hb and HbTOT (consistent with greater muscle perfusion and O2 delivery) were higher than in the transition to exercise without the prior heavy exercise, as shown previously (Gurd et al. 2005). The major new finding of this study was that the faster 
kinetics during the transition to moderate-intensity exercise after heavy-intensity exercise (i.e. Mod2) was associated with an elevated baseline PDHa activity (an activity similar to that seen during the exercise steady-state of Mod1) and no further activation of PDHa during the early transition to Mod2, suggesting a role for PDHa activation in limiting 
kinetics during exercise. Also, the faster 
kinetics and elevated muscle PDHa activity in Mod2 were accompanied by a reduced substrate-level phosphorylation as determined by lower PCr breakdown during the initial 30 s of exercise.
kinetics, PDH activity and substrate-level phosphorylation
The lower 
in Mod2 compared with Mod1 agrees with our previous findings in young (Gurd et al. 2005) and older adults (Scheuermann et al. 2002; DeLorey et al. 2004). However, a speeding of 
kinetics in moderate exercise in young adults is not always seen after a priming bout of heavy-intensity exercise (Gerbino et al. 1996; Burnley et al. 2000; Scheuermann et al. 2002; DeLorey et al. 2004). In the present study we demonstrated that the reduction in 
between Mod1 and Mod2 was directly related to the 
in Mod1 (i.e. without prior heavy exercise; Fig. 4) (see also Scheuermann et al. 2002; Gurd et al. 2005), and thus the relatively slower 
kinetics in the present study 
compared with others (16–20 s; Burnley et al. 2000; Scheuermann et al. 2002) may explain why these studies were unable to observe a measurable and significant speeding in the 
response during moderate-intensity exercise.
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1.0 to 2.0 mmol acetyl CoA min–1 (kg wet wt)–1 at 30 and 360 s exercise (at 44%
). In agreement, Howlett et al. (1998) reported steady-state exercise values for PDHa activity of 1.5 and 3.0 mmol acetyl CoA min–1 (kg wet wt)–1 for exercise at 35% and 65%
, respectively. The higher PDHa activity at baseline prior to Mod2 (to 1.9 mmol acetyl CoA min–1 (kg wet wt)–1) reflects the slower rate of transformation of PDH activity back to its inactive form when light exercise (i.e. 20 W) rather than a resting recovery is performed after the ‘priming’ heavy-intensity exercise (Putman et al. 1995; Parolin et al. 1999).
Faster activation of mitochondrial respiration and muscle O2 consumption (as reflected by faster pulmonary 
kinetics) is expected to reduce substrate-level phosphorylation and lower muscle PCr degradation and lactate accumulation in exercise (Timmons et al. 1998; Howlett et al. 1999; Parolin et al. 2000; Campbell-O'Sullivan et al. 2002; Roberts et al. 2002). In the present study, baseline PCr content was lower immediately prior to the onset of Mod2 and probably is related to an elevated muscle [H+] and its effect on the creatine kinase equilibrium, while PCr breakdown was reduced in the immediate transition to Mod2 compared with Mod1. Muscle lactate content remained low and did not change throughout Mod1, as expected with exercise performed below the estimated lactate threshold (90%
L), suggesting that substrate-level phosphorylation from glycolysis did not contribute significantly to ATP production during this moderate exercise bout. Muscle lactate content was elevated as a consequence of the heavy-intensity exercise and the progressive decrease in muscle lactate observed throughout Mod2 reflects a greater lactate oxidation by reversal of the cytosolic and/or mitochondrial (via an intramuscular lactate shuttle system) LDH reaction to form pyruvate, with subsequent oxidative decarboxylation via the PDH reaction (see Brooks, 2000 and Gladden, 2004 for reviews on the topic). Therefore, the faster adaptation of pulmonary 
kinetics in Mod2, together with prior exercise-induced activation of PDH activity, and reduced PCr breakdown in the initial moments of Mod2, are consistent with faster activation of mitochondrial oxidative phosphorylation during the transition to moderate-intensity exercise after heavy-intensity exercise (Timmons et al. 1998; Howlett et al. 1999). Additionally, there was a positive relationship (albeit weak; r
= 0.46) between the difference in baseline PDHa activity in Mod2 and Mod1 and the reduction in the phase 2 
which is consistent with PDH contributing, at least in part, to the speeding of 
kinetics seen after the heavy-intensity exercise bout in this study. These findings support the contention that metabolic inertia (i.e. delayed activation of enzymes (e.g. PDH) and provision of oxidative substrates other than O2), in part, limits the adaptation of pulmonary 
and muscle O2 consumption during the normal transition to exercise (i.e. Mod1).
Alternatively, the lower baseline [PCr] and decreased PCr breakdown early during the transition to Mod2 may have contributed to the faster 
kinetics observed in this transition. PCr may serve as an energy buffer within muscle cells to limit the increase in cytosolic and mitochondrial [ADP] and therefore attenuate the drive for mitochondrial respiration. In recent studies utilizing iodoacetamide-induced creatine kinase (CK) inhibition (Harrison et al. 2003; Kindig et al. 2005) and CK knockout mice (Gustafson & Van Beek, 2002), muscle oxidative metabolism was shown to activate faster than in control CK-active muscles where the rise in [ADP] would be limited by the transfer of high-energy phosphate between PCr and ADP. The lower baseline PCr seen prior to Mod2 in the present study might shift the equilibrium nature of the CK reaction towards a higher ADP-to-ATP ratio during the exercise transition, contributing to a greater drive to activate mitochondrial respiration, with a consequent speeding of 
kinetics.
It also has been suggested that acetyl group availability (in the form of acetyl CoA and acetylcarnitine) is limiting for the activation of oxidative phosphorylation in the transition to exercise (Roberts et al. 2002), although the only demonstration of this has been in an ischaemic canine hindlimb preparation (Roberts et al. 2002). Acetylcarnitine was not measured in the present study, but changes in acetylcarnitine content during exercise are expected to be reflected by changes in acetyl CoA content (Howlett et al. 1998; Roberts et al. 2002). In the present study, a decrease in acetyl CoA content was not seen in the immediate transition to Mod1 (i.e. at 30 s), but rather, there was a tendency for acetyl CoA to increase throughout Mod1 (and also Mod2). However, acetyl CoA (and presumably acetylcarnitine) was elevated prior to and throughout Mod2 compared with Mod1, reflecting a greater PDH activity at the start of Mod2, and may have contributed to greater substrate provision to the TCA cycle and thereby faster activation of oxidative phosphorylation.
Although a reduced substrate-level phosphorylation (Timmons et al. 1998; Howlett et al. 1999; Parolin et al. 2000; Roberts et al. 2002) and faster fall in intracellular 
(Howlett & Hogan, 2003) have been seen following dichloroacetate (DCA)-induced activation of PDH activity, faster pulmonary 
and muscle O2 consumption kinetics have not been demonstrated (Bangsbo et al. 2002; Grassi et al. 2002; Rossiter et al. 2003; Jones et al. 2004; Koppo et al. 2004). However, these discrepancies may reflect a possible DCA-mediated increase in metabolic efficiency (Grassi, 2005) as demonstrated by lower amplitudes for 
and PCr for a given work rate during knee-extension exercise in humans (Rossiter et al. 2003) and similar 
amplitude but lower PCr degradation and lower muscle fatigue in an electrically stimulated canine gastrocnemius muscle preparation (Grassi et al. 2002). Alternatively, these differences may, in part, be related to the fact that activation of muscle O2 consumption may be limited at reaction steps other than, or in addition to, PDH (i.e. possibly other dehydrogenases located in the TCA cycle), and thus these additional enzyme-catalysed steps may need to be activated before a faster acceleration of muscle O2 consumption can occur. Therefore, unlike in previous studies where only a single variable may have been altered to affect either metabolic inertia or muscle blood flow and O2 delivery, in the present study PDH activity, and possibly other mitochondrial dehydrogenases, were activated as a consequence of the heavy-intensity exercise. Support for this can be found in the studies of Hogan (2001) and Behnke et al. (2002) where a greater reduction in the time delay preceding a fall in intracellular and microvascular 
(suggestive of faster onset of oxidative phosphorylation) was demonstrated in the second bout of a series of electrically induced contractions (with an overall activation of muscle enzymes) than was observed with DCA administration alone (with only PDH activated prior to onset of contractions) (Howlett & Hogan, 2003). Also, the upregulation of muscle metabolism probably occurred in combination with greater muscle perfusion and O2 delivery. Higher HbTOT and O2Hb levels (present study) and heart rate before and throughout Mod2 (Scheuermann et al. 2002; DeLorey et al. 2004; Gurd et al. 2005) are consistent with better perfusion in this condition. Also, femoral arterial blood flow has been shown to be higher during 6 min baseline recovery from a bout of heavy-intensity knee-extension exercise (Fukuba et al. 2004; Endo et al. 2005; Paterson et al. 2005). Better perfusion (relative to muscle O2 consumption) would result in greater convective O2 delivery and, presumably, a higher microvascular 
(and thus diffusive O2 delivery) prior to and during the transition to Mod2 compared with Mod1.
While it has been suggested that O2 delivery does not limit 
kinetics during moderate-intensity exercise (Grassi, 2001), it has been shown that mitochondrial oxidative phosphorylation is O2 dependent, whereby the respiratory rate can be maintained despite alterations in O2 levels, by adjustments to the cellular redox and/or phosphorylation potential (Hogan et al. 1992; Wilson, 1994; Haseler et al. 1998). It is possible that in studies where metabolic activation was accelerated without a corresponding increase in O2 delivery, a condition is created where O2 availability is unable to sustain the higher rate of metabolic substrate delivery to the TCA cycle and electron transport chain and thus O2 utilization cannot be accelerated. Thus the faster 
kinetics seen in the current study is likely to be a result of a combination of both increased NADH availability (via elevated PDH activation) and elevated mitochondrial 
(via increased bulk and local blood flow).
Surprisingly, while PCr breakdown was lower in the first 30 s of Mod2, net PCr breakdown continued to end-exercise, a finding not consistent with the lower PCr degradation in the first 30 s of Mod2, or with the steady-state conditions in the latter part of Mod2, evidenced by the constant pulmonary 
during the final 5 min exercise 
. However, total PCr degradation was similar in Mod2 and Mod1, resulting in a lower end-exercise PCr content in Mod2, a finding consistent with previous studies where PCr degradation was attenuated early in the transition to exercise but was not different by the end of exercise (Timmons et al. 1998, 2004; Howlett et al. 1999; Campbell-O'Sullivan et al. 2002; Roberts et al. 2005). While this observation is of interest, the results of the present (and previous) research do not provide an explanation as to why this occurred. Further research is required to better understand the interaction between the faster 
kinetics and reduced PCr breakdown early in exercise but similar overall PCr breakdown during the course of the entire exercise period.
Interestingly, in the present study, although a decrease in 
was observed in Mod2 compared with Mod1, a significant limitation to the activation of 
persisted, in spite of local conditions which would be expected to overcome any limitation imposed by O2 delivery or enzyme activation (PDHa; TCA cycle enzymes) and substrate provision (acetyl CoA; NADH; pyruvate (through conversion from lactate)). It is possible that this limitation may reflect an inherent delay imposed by provision of adequate levels of ADP (and Pi) to the mitochondria during the transition to exercise.
Muscle deoxygenation and 
kinetics
The NIRS-derived deoxygenation (i.e. HHb) response reflects the balance between local muscle O2 consumption and muscle blood flow. In the present study, after heavy-intensity exercise a shorter HHb-TD was observed before the increase in HHb (and thus O2 extraction), similar to the shorter time delay preceding the fall in microvascular 
(Behnke et al. 2002) and intracellular 
(Hogan, 2001) in the second of two electrically stimulated contraction bouts using animal preparations. The shorter HHb-TD in Mod2 represents an earlier ‘imbalance’ between muscle O2 utilization and muscle perfusion and thus an earlier requirement for O2 extraction from haemoglobin, in spite of possibly greater local blood flow in Mod2, and is consistent with faster 
kinetics in Mod2 compared with Mod1. The greater HHb in Mod2 implies that with time after the onset of Mod2, a further increase in local blood flow exceeded O2 demand, thereby slowing the rate of O2 extraction in this transition. Together the HHb-TD and HHb support the findings of faster 
kinetics and greater muscle perfusion during the transition to Mod2. The greater HHb amplitude in Mod2 is in agreement with the greater end-exercise 
in Mod2.
Conclusion
Therefore, this study demonstrated faster 
kinetics during the transition to moderate-intensity exercise when the exercise was preceded by a bout of heavy-intensity exercise. An elevated PDH activity and greater muscle perfusion and local muscle oxygenation (as determined by a greater O2Hb and HbTOT) prior to the start of Mod2 was accompanied by a speeding of 
kinetics and shorter HHb-TD during the transition to Mod2 and a decreased reliance on substrate-level phosphorylation (as determined by a lower PCr degradation) in the first 30 s exercise in Mod2. These results suggest that the speeding of moderate-intensity 
kinetics after a bout of heavy-intensity exercise occurs as a consequence of prior activation of mitochondrial enzyme activity (i.e. PDH and possibly other dehydrogenases) and substrate provision in combination with elevated muscle perfusion and O2 delivery, thereby ensuring adequate provision of all substrates needed to support oxidative phosphorylation.
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during the on-transition to Mod1 and Mod2
response of a representative subject (
, and grey line of best fit and residuals) with 
(0–100% end-exercise) with line of best fit inset.
P < 0.05 versus baseline. 
P < 0.05 versus 30 s.
time constant ( 
) between Mod1 and Mod2.