B. J. Gurd 1,2,S.J.Peters 6, G. J. F. Heigenhauser 5, P. J. LeBlanc 6,T.J.Doherty 4, D. H. Paterson 1,2 and J. M. Kowalchuk 1,2,3

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1 J Physiol (2006) pp Prior heavy exercise elevates pyruvate dehydrogenase activity and speeds O 2 uptake kinetics during subsequent moderate-intensity exercise in healthy young adults B. J. Gurd 1,2,S.J.Peters 6, G. J. F. Heigenhauser 5, P. J. LeBlanc 6,T.J.Doherty 4, D. H. Paterson 1,2 and J. M. Kowalchuk 1,2,3 1 Canadian Centre for Activity and Aging, 2 School of Kinesiology, Faculty of Health Sciences, 3 Department of Physiology and Pharmacology and 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 The adaptation of pulmonary oxygen uptake ( V O2 ) 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 Mod 1 heavyintensity Mod 2 exercise protocol preceded by 20 W baseline. Breath-by-breath V O2 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 Mod 1, 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 Mod 2, PDHa was already elevated at baseline (1.88 ± 0.3 mmol min 1 (kg wet wt) 1 ) and was greater than in Mod 1, 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 V O2 was lower in Mod 2 (19 ± 2 s) than Mod 1 (24 ± 3 s). Phosphocreatine (PCr) breakdown from baseline to 30 s was greater (P < 0.05) in Mod 1 (13.6 ± 6.7 mmol (kg dry wt) 1 ) than Mod 2 (6.5 ± 6.2 mmol (kg dry wt) 1 ) but total PCr breakdown was similar between conditions (Mod 1, 14.8 ± 7.4 mmol (kg dry wt) 1 ; Mod 2, 20.1 ± 8.0 mmol (kg dry wt) 1 ). Both oxyhaemoglobin and total haemoglobin were elevated prior to and throughout Mod 2 compared with Mod 1. In conclusion, the greater PDHa at baseline prior to Mod 2 compared with Mod 1 may have contributed in part to the faster V O2 kinetics in Mod 2. That oxyhaemoglobin and total haemoglobin were elevated prior to Mod 2 suggests that greater muscle perfusion may also have contributed to the observed faster V O2 kinetics. These findings are consistent with metabolic inertia, via delayed activation of PDH, in part limiting the adaptation of pulmonary V O2 and muscle O 2 consumption during the normal transition to exercise. (Resubmitted 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. jkowalch@uwo.ca The mechanism(s) controlling the rate of muscle O 2 consumption and pulmonary O 2 uptake ( V O2 ) during the transition to moderate-intensity exercise resides in the ability to provide adequate substrate (i.e. O 2, ADP, P i, 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 O 2 delivery limitation through manipulation of convective and/or diffusive O 2 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 DOI: /jphysiol

2 986 B. J. Gurd and others J Physiol 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 FADH 2 ) to the electron transport chain. Recently we demonstrated that the adaptation of V O2 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 O 2 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 O 2 delivery. However, the relationship between PDH activity and V O2 kinetics at the onset of moderate-intensity exercise with and without prior heavy-intensity exercise was not established. A speeding of V O2 kinetics during the transition to moderate intensity has not always been reported following interventions that attempt to manipulate either muscle blood flow and O 2 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 V O2 will only expose another limitation which still prevents a rapid acceleration of V O2 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 V O2 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 O 2 consumption), and thereby raise microvascular P O2 and the driving pressure for O 2 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 V O2 response provides an improved confidence in our parameter estimation of the time course of the V O2 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 V O2, muscle PDH activation and metabolism, and muscle oxygenation, to better understand the underlying limitations to muscle O 2 consumption during moderate-intensity exercise. We hypothesized that prior heavy-intensity exercise would be associated with (1) a faster adaptation of pulmonary V O2 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). Methods Subjects Nine young healthy males adults (age, 24 ± 4 years; V O2 peak, 47 ± 2mlkg 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 (θ L ) and V O2 peak.theθ L was defined as the V O2 at which CO 2 output ( V CO2 ) began to increase out of proportion relative to V O2, combined with a systematic rise in the ventilatory equivalent for V O2 and end-tidal P O2 with no concomitant rise in the ventilatory equivalent for V CO2 or end-tidal P CO2. V O2 peak was calculated as the average V O2 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 V O2 corresponding to 90% θ L (i.e. moderate-intensity exercise), and 50% ( 50% of the difference between the V O2 at θ L and V O2 peak, i.e. heavy-intensity exercise).

3 J Physiol Pyruvate dehydrogenase activity and V o2 kinetics in moderate exercise 987 During four of the subsequent five visits to the laboratory, subjects performed two step-transitions in WR of moderate intensity (Mod 1 and Mod 2 ) 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). V O2 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 O 2,CO 2 and N 2 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 (O 2 Hb), deoxyhaemoglobin (HHb) and total haemoglobin-myoglobin (Hb TOT ) 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 O 2 Hb, HHb and Hb TOT. 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 O 2 delivery and O 2 utilization, which reflects microvascular O 2 and muscle O 2 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 Mod 1. 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. Mod 2 ). 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 Mod 2. Muscle biopsy samples were immediately frozen in liquid N 2, removed from the needle while frozen and stored in liquid N 2 until later analysis.

4 988 B. J. Gurd and others J Physiol Modelling of the V O2 and NIRS responses The breath-by-breath V O2 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 Mod 1 and Mod 2 were modelled as a monoexponential of the form: Y (t) = Y (BSL) + Amp [ 1 e [ (t TD)/τ ]] (1) where Y (t ) represents the variable at any time (t), Y (BSL) is the baseline value of Y before the step increase in WR, Amp is the amplitude (i.e. steady-state increase in Y above baseline), τ is the time constant (i.e. the time taken to reach 63% of the steady-state response) and TD is the time delay. V O2 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, O 2 Hb and Hb TOT 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 (τ HHb). The mean response time (MRT = HHb-TD + τ HHb) was calculated to provide a description of the overall time course for muscle HHb. The O 2 Hb and Hb TOT 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 ( mg) was chipped from each muscle sample under liquid N 2 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 KHCO 3, 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 (ADP f ) and AMP (AMP f ) were calculated by assuming equilibrium of the creatine kinase and adenylate kinase reactions, respectively (Dudley et al. 1987). ADP f was calculated by using the measured ATP, creatine, PCr, estimated H + concentration and the creatine kinase equilibrium constant of ; H + concentration was calculated from the measured pyruvate and lactate contents as described by Sahlin et al. (1976). AMP f was calculated with the estimated ADP f and measured ATP content using the adenylate kinase equilibrium constant of 1.05 (Dudley et al. 1987). Free inorganic phosphate (P if ) 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 V O2 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 < Data are presented as means ± s.d. Results V O2 kinetics In the present study the moderate-intensity exercise represented 89% θ L (±4) (44% V O2 peak (±3) at a power output of 100 ± 15 W) while the heavy-intensity exercise represented 50% (78% V O2 peak (±4) at a power output of 228 ± 25 W). A summary of the parameter estimates for the on-transient V O2 response to Mod 1 and Mod 2 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 V O2 were higher (P < 0.05) and the V O2 amplitude was lower (P < 0.05) in Mod 2 compared with Mod 1. Heavy-intensity exercise was associated with a speeding of V O2 kinetics in eight of nine subjects; the phase 2 V O2 time constant (τ V O2 ) was reduced (P < 0.05) in Mod 2 (19 ± 2 s) compared with Mod 1 (24 ± 3 s).

5 J Physiol Pyruvate dehydrogenase activity and V o2 kinetics in moderate exercise 989 Table 1. Summary of parameter estimates for V O2 during the on-transition to Mod 1 and Mod 2 Mod 1 Mod 2 Amplitude (l min 1 ) 0.71 ± ± 0.05 Baseline (l min 1 ) 0.97 ± ± 0.04 End-exercise (l min 1 ) 1.68 ± ± 0.08 τ V O2 (s) 24 ± 3 19± 2 τ V O2,O 2 uptake time constant. Values are means ± S.D. (n = 9). Significant difference (P < 0.05) between Mod 1 and Mod 2. Muscle PDH activity During Mod 1, 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 Mod 1 and did not change significantly during the first 30 s exercise in Mod 2 (1.96 ± 0.20 mmol min 1 (kg wet wt) 1 ). At 6 min exercise in Mod 2, PDHa activity (2.70 ± 0.30 mmol min 1 (kg wet wt) 1 ) was greater (P < 0.05) than at 30 s of Mod 2 and at end-exercise in Mod 1 (Fig. 2). Muscle metabolite content 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 Mod 1 (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 Mod 2, 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 Mod 1 and was elevated prior to Mod 2 compared with Mod 1 baseline and did not change throughout Mod 2 (Table 2). Acetyl-CoA content increased progressively throughout Mod 1 and Mod 2 reaching significance (P < 0.05) at 6 min relative to both baseline and 30 s (Table 2). After heavy-intensity exercise, acetyl-coa was greater (P < 0.05) at all times compared with Mod 1 (Table 2). Muscle creatine content increased (P < 0.05) from baseline to 30 s in Mod 1 and remained elevated at 6 min (Table 3). After heavy-intensity exercise, creatine content was elevated compared with the Mod 1 baseline, remained unchanged after 30 s exercise, and increased (P < 0.05) by end-exercise. During Mod 1, 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 Mod 2 than Mod 1 (by 6.5 mmol (kg dry wt) 1 ); PCr content was not significantly changed at 30 s exercise in Mod 2, but after 6 min, PCr content was lower (P < 0.05) than both the Mod 2 baseline and the Mod 1 end-exercise value (Fig. 3). During the first 30 s exercise PCr breakdown was greater (P < 0.05) in Mod 1 (13.6 ± 6.7 mmol (kg dry wt) 1 ) than Mod 2 (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 Mod 2 (13.6 ± 9.1 mmol (kg dry wt) 1 ) than Mod 1 (1.1 ± 8.9 mmol (kg dry wt) 1 ), which resulted in a similar (P > 0.05) total PCr breakdown between conditions (Mod 1, 14.8 ± 7.4 mmol (kg dry wt) 1 ;Mod 2, 20.1 ± 8.0 mmol (kg dry wt) 1 ). ATP and ADP f were Figure 1. Absolute V O2 response of a representative subject (τ Mod 1 = 30 s; τ Mod 2 = 21 s) Line of best fit and residuals for Mod 1 (, and black line of best fit and residuals) and Mod 2 (, and grey line of best fit and residuals) with V O2 (0 100% end-exercise) with line of best fit inset. VO 2 (l min 1 ) Time (s)

6 990 B. J. Gurd and others J Physiol Table 2. Muscle contents of pyruvate, lactate, H + and acetyl-coa prior to and during Mod 1 and Mod 2 Moderate exercise Condition Baseline 30 s 360 s Pyruvate Mod ± ± ± 0.22 Mod ± ± ± 0.15 Lactate Mod ± ± ± 5.5 Mod ± ± ± 10.7 H + Mod ± ± ± 4.9 Mod ± ± ± 10.7 Acetyl-CoA Mod ± ± ± 1.1 Mod ± ± ± 2.0 Values are means ± S.D. (n = 9) in mmol (kg dry wt) 1 except [H + ] which is in 10 9 mol l 1. Significant difference (P < 0.05) between Mod 1 and Mod 2 at specific time. Significant difference (P < 0.05) from baseline for that condition. unchanged compared with baseline during both Mod 1 and Mod 2 (Table 3). NIRS-derived muscle oxygenation A summary of the parameter estimates for HHb, O 2 Hb and Hb TOT is presented in Table 4. The baseline and end-exercise O 2 Hb and Hb TOT were elevated (P < 0.05) in Mod 2 compared with Mod 1.The baseline for HHb was not different between Mod 1 and Mod 2, while the amplitude of the HHb response was greater (P < 0.05) in Mod 2 compared with Mod 1 (Table 4). The time course of HHb adaptation was described by a shorter (P < 0.05) HHb-TD and greater (P < 0.05) τ HHb in Mod 2 compared to Mod 1 ; the HHb mean response time was not different between the two transitions. PDHa (mmol actyl CoA min 1 (kg wet wt) 1 ) * * Time(s) Figure 2. Pyruvate dehydrogenase activity of the active form (PDHa) at rest and at 30 s and 6 min of moderate-intensity exercise during Mod 1 ( ) and Mod 2 ( ) Values are means ± S.D. P < 0.05 versus Mod 1. P < 0.05 versus baseline. P < 0.05 versus 30 s. Discussion This is the first study to examine the effect of prior heavy-intensity exercise on the adaptation of pulmonary V O2, 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 V O2 kinetics (a reflection of the time course of muscle O 2 consumption) was faster (by 20%) and baseline and end-exercise O 2 Hb and Hb TOT (consistent with greater muscle perfusion and O 2 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 V O2 kinetics during the transition to moderate-intensity exercise after heavy-intensity exercise (i.e. Mod 2 ) was associated with an elevated baseline PDHa activity (an activity similar to that seen during the exercise steady-state of Mod 1 ) and no further activation of PDHa during the early transition to Mod 2, suggesting a role for PDHa activation in limiting V O2 kinetics during exercise. Also, the faster V O2 kinetics and elevated muscle PDHa activity in Mod 2 were accompanied by a reduced substrate-level phosphorylation as determined by lower PCr breakdown during the initial 30 s of exercise. V O2 kinetics, PDH activity and substrate-level phosphorylation The lower τ V O2 in Mod 2 compared with Mod 1 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 V O2 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 τ V O2 between Mod 1 and Mod 2 was directly related to

7 J Physiol Pyruvate dehydrogenase activity and V o2 kinetics in moderate exercise 991 Table 3. Mod 2 Muscle content of creatine, PCr, ATP and ADP f prior to and during Mod 1 and Moderate exercise Condition Baseline 30 s 360 s Creatine Mod ± ± ± 4.6 Mod ± ± ± 8.6 PCr Mod ± ± ± 13.1 Mod ± ± ± 17.8 ATP Mod ± ± ± 5.6 Mod ± ± ± 8.1 ADP f Mod ± ± ± 28.5 Mod ± ± ± 35.6 Values are means ± S.D. (n = 9) in mmol (kg dry wt) 1 except ADP f (free ADP) which is in μmol (kg dry wt) 1. Significant difference (P < 0.05) between Mod 1 and Mod 2 at specific time. Significant difference (P < 0.05) from baseline. Significant difference (P < 0.05) from 30 s. the τ V O2 in Mod 1 (i.e. without prior heavy exercise; Fig. 4) (see also Scheuermann et al. 2002; Gurd et al. 2005), and thus the relatively slower V O2 kinetics in the present study (τ V O2 25 s) compared with others ( 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 V O2 response during moderate-intensity exercise. During Mod 1, PDHa activity increased from 1.0 to 2.0 mmol acetyl CoA min 1 (kg wet wt) 1 at 30 and 360 s exercise (at 44% V O2 peak). 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% V O2 peak, respectively. The higher PDHa activity at baseline prior to Mod 2 (to 1.9 mmol acetyl CoA min 1 (kg wet wt) 1 ) 100 PCr (mmol (kg dry wt) 1 ) * * * Figure 3. Muscle lactate and phosphocreatine at baseline and at 30 s and 6 min of moderateintensity exercise during Mod 1 ( ) and Mod 2 ( ) Values are means ± S.D. (n = 9) in mmol/kg dry wt. P < 0.05 versus Mod 1. P < 0.05 versus baseline. Lactate (mmol (kg dry wt) 1 ) * * Time(s)

8 992 B. J. Gurd and others J Physiol Table 4. Summary of parameter estimates for NIRS-derived ΔO 2 Hb, ΔHb TOT and ΔHHb during Mod 1 and Mod 2 Mod 1 Mod 2 O 2 Hb Baseline (μm) 0.9 ± ± 8.7 End-exercise (μm) 1.4 ± ± 6.0 Hb TOT Baseline (μm) 7.4 ± ± 8.5 End-exercise (μm) 2.7 ± ± 8.0 HHb Baseline (μm) 8.4 ± ± 7.3 Amplitude (μm) 7.4 ± ± 5.6 End-exercise (μm) 0.9 ± ± 5.3 HHb-TD (s) 12.9 ± ± 2.0 τhhb (s) 10.3 ± ± 6.6 MRT (s) 23.2 ± ± 6.3 MRT, mean response time; O 2 Hb, change in oxyhaemoglobin; Hb TOT, change in total haemoglobin; HHb, change in deoxyhaemoglobin; HHb-TD, time delay before an increase in HHb after exercise onset; τhhb, HHb time constant. Values are means ± S.D. (n = 9). Significant difference (P < 0.05) between Mod 1 and Mod 2. Significant difference (P < 0.05) from baseline. 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 O 2 consumption (as reflected by faster pulmonary V O2 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 Mod 2 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 Mod 2 compared with Mod 1. Muscle lactate content remained low and did not change throughout Mod 1, as expected with exercise performed below the estimated lactate threshold ( 90% θ 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 Mod 2 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 V O2 kinetics in Mod 2, together with prior exercise-induced activation of PDH activity, and reduced PCr breakdown in the initial moments of Mod 2, 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 Mod 2 and Mod 1 and the reduction in the phase 2 τ V O2 which is consistent with PDH contributing, at least in part, to the speeding of V O2 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 O 2 ), in part, ΔτVO 2 (Mod 1 - Mod 2 )(s) slope = 0.47 intercept = 6.36 r = 0.84 (P < 0.05) τvo 2 Mod 1 (s) Figure 4. Relationship between τ Mod 1 and the change (Δ) in V O2 time constant (τ V O2 ) between Mod 1 and Mod 2.

9 J Physiol Pyruvate dehydrogenase activity and V o2 kinetics in moderate exercise 993 limits the adaptation of pulmonary V O2 and muscle O 2 consumption during the normal transition to exercise (i.e. Mod 1 ). Alternatively, the lower baseline [PCr] and decreased PCr breakdown early during the transition to Mod 2 may have contributed to the faster V O2 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 Mod 2 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 V O2 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 Mod 1 (i.e. at 30 s), but rather, there was a tendency for acetyl CoA to increase throughout Mod 1 (and also Mod 2 ). However, acetyl CoA (and presumably acetylcarnitine) was elevated prior to and throughout Mod 2 compared with Mod 1, reflecting a greater PDH activity at the start of Mod 2, 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 P O2 (Howlett & Hogan, 2003) have been seen following dichloroacetate (DCA)-induced activation of PDH activity, faster pulmonary V O2 and muscle O 2 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 V O2 and PCr for a given work rate during knee-extension exercise in humans (Rossiter et al. 2003) and similar V O2 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 O 2 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 O 2 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 O 2 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 P O2 (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 O 2 delivery. Higher Hb TOT and O 2 Hb levels (present study) and heart rate before and throughout Mod 2 (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 O 2 consumption) would result in greater convective O 2 delivery and, presumably, a higher microvascular P O2 (and thus diffusive O 2 delivery) prior to and during the transition to Mod 2 compared with Mod 1. While it has been suggested that O 2 delivery does not limit V O2 kinetics during moderate-intensity exercise (Grassi, 2001), it has been shown that mitochondrial oxidative phosphorylation is O 2 dependent, whereby the respiratory rate can be maintained despite alterations in O 2 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 O 2 delivery, a condition is created where O 2 availability is unable to sustain the higher rate of metabolic substrate delivery to the TCA cycle and electron transport chain and thus O 2 utilization cannot be accelerated. Thus the faster V O2 kinetics seen in the current study is likely to be a result of a combination of both increased NADH availability (via elevated PDH

10 994 B. J. Gurd and others J Physiol activation) and elevated mitochondrial P O2 (via increased bulk and local blood flow). Surprisingly, while PCr breakdown was lower in the first 30 s of Mod 2, net PCr breakdown continued to end-exercise, a finding not consistent with the lower PCr degradation in the first 30 s of Mod 2, or with the steady-state conditions in the latter part of Mod 2, evidenced by the constant pulmonary V O2 during the final 5 min exercise (τ V O2 19 s). However, total PCr degradation was similar in Mod 2 and Mod 1, resulting in a lower end-exercise PCr content in Mod 2, 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 V O2 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 τ V O2 was observed in Mod 2 compared with Mod 1,a significant limitation to the activation of V O2 persisted, in spite of local conditions which would be expected to overcome any limitation imposed by O 2 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 P i ) to the mitochondria during the transition to exercise. Muscle deoxygenation and V O2 kinetics The NIRS-derived deoxygenation (i.e. HHb) response reflects the balance between local muscle O 2 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 O 2 extraction), similar to the shorter time delay preceding the fall in microvascular P O2 (Behnke et al. 2002) and intracellular P O2 (Hogan, 2001) in the second of two electrically stimulated contraction bouts using animal preparations. The shorter HHb-TD in Mod 2 represents an earlier imbalance between muscle O 2 utilization and muscle perfusion and thus an earlier requirement for O 2 extraction from haemoglobin, in spite of possibly greater local blood flowinmod 2, and is consistent with faster V O2 kinetics in Mod 2 compared with Mod 1. The greater τ HHb in Mod 2 implies that with time after the onset of Mod 2,a further increase in local blood flow exceeded O 2 demand, thereby slowing the rate of O 2 extraction in this transition. Together the HHb-TD and τ HHb support the findings of faster V O2 kinetics and greater muscle perfusion during the transition to Mod 2. The greater HHb amplitude in Mod 2 is in agreement with the greater end-exercise V O2 in Mod 2. Conclusion Therefore, this study demonstrated faster V O2 kinetics during the transition to moderate-intensity exercise when the exercise was preceded by a bout of heavy-intensity exercise. 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