277:890-900, 1999. Am J Physiol Endocrinol MetabJones and George J. F. Heigenhauser Michelle L. Parolin, Alan Chesley, Mark P. Matsos, Lawrence L. Spriet, Norman L.

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Regulation of skeletal muscle glycogen phosphorylaseand PDH during maximal intermittent exercise

MICHELLE L. PAROLIN,1 ALAN CHESLEY,2 MARK P. MATSOS,1 LAWRENCE L. SPRIET,2NORMAN L. JONES,1 AND GEORGE J. F. HEIGENHAUSER1

1Department of Medicine, McMaster University, Hamilton, Ontario L8N 3Z5;and 2Department of Human Biology and Nutritional Sciences,University of Guelph, Guelph, Ontario, Canada N1G 2W1

Parolin, Michelle L., Alan Chesley, Mark P. Matsos,Lawrence L. Spriet, Norman L. Jones, and George J. F.Heigenhauser. Regulation of skeletal muscle glycogen phos-phorylase and PDH during maximal intermittent exercise.Am. J. Physiol. 277 (Endocrinol. Metab. 40): E890–E900,1999.—The time course for the activation of glycogen phos-phorylase (Phos) and pyruvate dehydrogenase (PDH) andtheir allosteric regulators was determined in human skeletalmuscle during repeated bouts of maximal exercise. Six sub-jects completed three 30-s bouts of maximal isokinetic cyclingseparated by 4-min recovery periods. Muscle biopsies weretaken at rest and at 6, 15, and 30 s of exercise during bouts 1and 3. Phos was rapidly activated within the first 6 s of bout 1from 12% at rest to 47% at 6 s. The activation of PDHincreased from 14% at rest to 48% at 6 s and 95% at 15 s ofbout 1. Phos reverted back to basal values at the end of thefirst bout, whereas PDH remained fully activated. In con-trast, in the third bout, PDH was 42% at rest and wasactivated more rapidly and was nearly completely activatedby 6 s, whereas Phos remained at basal levels (range 14–20%). Lactate accumulation was marked in the first bout andincreased progressively from 2.7 to 76.1 mmol/kg dry wt withno further increase in bout 3. Glycogen utilization was alsomarked in the first bout and was negligible in bout 3. Therapid activation of Phos and slower activation of PDH in bout1 was probably due to Ca21 release from the sarcoplasmicreticulum. Lactate accumulation appeared to be due to animbalance of the relative activities of Phos and PDH. Theincrease in H1 concentration may have served to reducepyruvate production by inhibiting Phos transformation andmay have simultaneously activated PDH in the third boutsuch that there was a better matching between pyruvateproduction and oxidation and minimal lactate accumulation.As each bout progressed and with successive bouts, there wasa decreasing ability to stimulate substrate phosphorylationthrough phosphocreatine hydrolysis and glycolysis and ashift toward greater reliance on oxidative phosphorylation.

pyruvate dehydrogenase; lactate metabolism; acetylcarni-tine; glycolysis; oxidative phosphorylation; phosphocreatine

FOR MORE THAN A DECADE, we have been examining theregulation of energy provision during repeated bouts ofmaximal isokinetic cycling (26, 35, 38, 45, 50). Othergroups have also performed similar studies with theuse of different sprint models (2, 6, 18, 20). Duringsprint exercise, power outputs are elicited in excess oftwo to three times that at which maximal oxygen

consumption (VO2max) is achieved. To meet the maximaldemand for ATP, the majority of energy for short-termmaximal exercise is derived via substrate phosphoryla-tion from phosphocreatine (PCr) hydrolysis and glycoly-sis (26, 27, 33). However, studies using intermittentexercise consisting of two to four bouts of 30 s ofmaximal cycling separated by 4 min of rest havedemonstrated that there is a progressive shift to oxida-tive metabolism in later bouts (6, 38, 45, 50). Inaddition, significant lactate accumulation occurred inthe first bout compared with the third and fourth bouts,where lactate accumulation was negligible (35, 38, 45).Reduced flux through the glycolytic pathway and agreater degree of activation of pyruvate dehydrogenase(PDH) before the third bout was accompanied by anincrease in oxygen uptake (VO2), which contributed toreduced accumulation of lactate and increased oxida-tion of pyruvate (38). Associated with a reduction inlactate accumulation was an increase in oxidativephosphorylation in the third bout compared with thefirst.

During maximal sprint exercise, glycogen is theprimary substrate, with extracellular glucose providing,2–3% of the total (29, 38). Therefore, glycogen phos-phorylase (Phos) catalyzes the rate-limiting step inglycogenolysis and sets the upper limit for the rates ofglycolysis. PDH catalyzes the oxidative decarboxyl-ation of pyruvate to acetyl-CoA and thus regulates therate of entry of pyruvate into the tricarboxylic acid(TCA) cycle. When the rate of pyruvate productionthrough glycolysis exceeds its rate of oxidation throughPDH, it is converted to lactate by the near-equilibriumenzyme lactate dehydrogenase whose equilibrium fa-vors lactate formation. The balance between the activi-ties of Phos and PDH determines the accumulation oflactate. During sprint exercise, when the demand forATP is maximal, there will be a large imbalancebetween flux through Phos and PDH. Thus, withrepeated exercise, the progressive reduction in lactateaccumulation may be due to a reduction in Phosactivation, an increase in PDH, or a combination of thetwo.

Although many studies have examined the metabolicresponses to sprinting before and after 30-s bouts ofsprint exercise (6, 18, 20, 26, 35, 38, 45), few haveexamined these responses during the exercise bout.Therefore, to examine these factors more closely, thisstudy specifically examined the time course of theactivation of Phos and PDH during the first and thirdbouts of maximal intermittent cycling. Muscle biopsieswere obtained at rest and at 6, 15, and 30 s of the first

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

0193-1849/99 $5.00 Copyright r 1999 the American Physiological SocietyE890

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and third bouts of exercise to construct a time course ofPhos and PDH activation and to ascertain the regula-tory factors responsible for Phos and PDH activity. Thisprotocol also allowed us to calculate the relative contri-butions to ATP regeneration from substrate level phos-phorylation by PCr hydrolysis and glycolysis and fromoxidative phosphorylation within the two bouts ofexercise.

METHODS

Subjects

Six male subjects (mean 6 SE, age 28.5 6 1.83 yr, height178.8 6 2.1 cm, and weight 85.8 6 3.1 kg) participated in thisstudy. Informed consent was obtained from each subject aftera verbal and written explanation of the experimental protocoland its attendant risks. The study was approved by theMcMaster University Ethics Committee.

Experimental Protocol

The exercise protocol for this study consisted of three 30-sbouts of maximal isokinetic cycling at 100 rpm separated by4 min of rest. The isokinetic cycle ergometer used in thisstudy has been described previously (34). Briefly, the cyclewas fitted with a 3-hp direct current motor that set the upperlimit of crank velocity at 100 rpm. The forces exerted by thesubjects were recorded by strain gauges attached to the pedalcranks. Electrical signals were transferred to a computer, andwork was calculated for each pedal stroke as the product ofimpulse and angular velocity. The total work for each 30-speriod was calculated as the sum of 50 pedal strokes for twolegs. The total work and power output accomplished duringeach time interval (i.e., 0–6, 6–15, and 15–30 s) werecalculated from the sum of the pedal strokes for two legs overeach interval.

Biopsies of the vastus lateralis were taken at rest and at 6,15, and 30 s of bouts 1 and 3. Because it was not possible toobtain all of the biopsies on the same day, the sampling wasdistributed over three trials each separated by 1 wk (Fig. 1).The study was designed such that 1) the predeterminedexercise intervals (i.e., 6, 15, or 30 s) were completed uninter-rupted from rest before each biopsy was taken and 2) theresting and 30-s biopsies were obtained on the same day for

bouts 1 and 3, respectively, so that muscle glycogenolysiscould be determined within each bout from the differencebetween resting glycogen content and glycogen content at 30 sof exercise. During trial 1, two biopsies were taken at rest,one at 30 s of bout 1, and another at 6 s of bout 3. During trial2, one biopsy was taken at 15 s of bout 1, two biopsies weretaken at rest before bout 3, and one biopsy was taken at 30 s ofbout 3. During trial 3, biopsies were taken at 6 s of bout 1 and15 s of bout 3. A total of 10 biopsies were taken. The order ofthe trials was randomized for each subject and took place atthe same time each day. Subjects were asked to consume ahigh-carbohydrate diet 48 h before trial 1 and to replicate thisdiet before the remaining two trials. The subjects wereinstructed to abstain from the consumption of caffeine and torefrain from intense exercise for 48 h before each trial.

Muscle Sampling

Before each trial, one thigh was prepared for needlebiopsies of the vastus lateralis. Two or four incisions weremade through the skin to the deep fascia under local anesthe-sia (2% lidocaine without epinephrine). Alternate legs wereused on each visit so that one leg received four biopsies andthe contralateral leg received six biopsies in total. Biopsies ofthe vastus lateralis were obtained as described by Bergstrom(5). All muscle biopsies were taken while the subject was onthe bike and were immediately plunged in liquid N2. At theappropriate time, the exercise was interrupted and the clockwas stopped. After the biopsy, cycling was resumed for thetime remaining in the bout so that subjects completed a totalof 30 s of maximal cycling for each bout. The time delaybetween interrupting and resuming cycling was 6–9 s. Thetime elapsed between the start of the biopsy to completefreezing was ,10 s. One of the resting biopsies before bouts 1and 3 was frozen in liquid N2 after a 30-s delay to obtainresting measurements of the active form of Phos (Phos a; seeRef. 41). The biopsies were removed from the needle whilefrozen and were stored in liquid N2.

Analyses

A 5- to 15-mg piece of muscle was chipped from each biopsyunder liquid N2 and was dissected free of blood and connec-tive tissue for the determination of the active form of PDH(PDHa). On two larger biopsies, another piece of wet musclewas used for the determination of total PDH activity (PDHt).

Fig. 1. Experimental design. Muscle biopsies weretaken at the times indicated by the arrows.

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PDHa and PDHt were measured as described by Constantin-Teodosiu et al. (13) and modified by Putman et al. (39). Theremaining muscle was freeze-dried, dissected free of blood,connective tissue, and fat, and stored dry at 270°C forsubsequent analysis.

One aliquot of powdered muscle was used for the determi-nation of Phos activity, as described by Young et al. (52).Briefly, 3–4 mg of powdered muscle were homogenized at225°C in 200 µl of buffer containing 100 mM Tris, 60%glycerol, 50 mM KF, and 10 mM EDTA (pH 7.5). Homogenateswere then diluted with 800 µl of the above buffer withoutglycerol and were homogenized further at 0°C. Total (a 1 b)Phos activity (in the presence of 3 mM AMP) and Phos aactivity (in the absence of AMP) were measured by followingthe production of glucose 1-phosphate spectrophotometricallyat 30°C. Maximal velocity (Vmax) and the mole fraction of Phosa and a 1 b was calculated from the measured activities asdescribed by Chasiotis et al. (11).

Asecond aliquot of powdered muscle was assayed enzymati-cally for glycogen, as described by Harris et al. (21). Theremaining dry powdered muscle was extracted in a solution of0.5 M perchloric acid (PCA), and 1 mM EDTA and wasneutralized with 2.2 M KHCO3. The neutralized PCA extractswere assayed for ATP, PCr, creatine, pyruvate, lactate, glu-cose, glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P), and glycerol 3-phosphate (Gly-3-P) as described by Berg-meyer (4) and acetyl-CoA, the reduced form of coenzyme A(CoASH), and acetylcarnitine as described by Cederblad et al.(8). Muscle metabolites and enzyme activities were correctedto the highest total creatine content for each subject to correctfor nonmuscle contamination.

Calculations

Intracellular H1 concentration ([H1]) was calculated frommuscle lactate and pyruvate concentrations according toSahlin et al. (44) and was validated by Spriet et al. (45) for thetype of exercise used in the present study. The concentrationsof free ADP and free AMP were calculated from the near-equilibrium reactions of creatine kinase and adenylate ki-nase, respectively (15); however, these calculations do notprovide information as to the exact location and local concen-trations of these metabolites within the cell.

The concentration of free Pi was calculated as the differ-ence between the concentrations of resting and exercise PCr,less the accumulation of G-6-P, F-6-P, and Gly-3-P plus theassumed resting concentration of 10.8 mmol/kg dry wt (15).The concentrations of the monoprotonated (HPO4

22) anddiprotonated (H2PO4

12) forms of Pi were calculated from thetotal concentration of Pi, and the calculated pH, using a pKa of6.78.

The rate of glycogenolysis, expressed as millimole glucosylunits per kilogram dry weight per second, was estimatedaccording to Spriet et al. (47) from the accumulation ofglycolytic intermediates plus the flux of pyruvate throughPDHa during each time interval. Because previous studieshave shown that flux through PDH is equivalent to its level ofactivity (19, 22, 38), PDHa was converted to units of millimoleper kilogram dry weight per second, assuming a wet-to-drymuscle ratio of 4:1 at rest and 4.5:1 during exercise (37)

glycogenolysis

5 (D([G-6-P] 1 [F-6-P]) 1 D([Gly-3-P] 1 [lactate] 1 [pyruvate])/2)

4 time 1 PDHa/2

where D indicates change and brackets indicate concentra-tion.

The rate of pyruvate production, expressed as millimolepyruvate units per kilogram dry weight per second, wascalculated from the accumulation of pyruvate and lactateplus the flux of pyruvate through PDHa during each timeinterval. From a previous study using the same protocol (31),lactate efflux from the muscle was found to be very small andwas thus not included in the calculation of pyruvate produc-tion

pyruvate production 5 D([lactate] 1 [pyruvate]) 4 time 1 PDHa

The rate of pyruvate oxidation was estimated from the fluxthrough PDHa, which was converted to units of millimole perkilogram dry weight per second. The rate of lactate accumula-tion was calculated from the differences in concentrationbetween two time points, divided by the elapsed time.

The rate of ATP turnover from PCr was calculated from thebreakdown of PCr, whereas the rate of ATP turnover fromglycolysis was calculated from the accumulation of lactateand the flux of pyruvate through PDHa. The rate of ATPturnover from oxidative phosphorylation was calculated fromtotal acetyl-CoA production as the area under the PDHacurves for each bout; 1 mmol of acetyl-CoA from glycogenoly-sis was equal to 15 mmol of ATP. The contribution of fat fuels(25) and exogenous glucose (29) was assumed to be negligible.

Statistical Analysis

Data were analyzed by a two-way ANOVA with repeatedmeasures over time. When a significant F ratio was found, theNewman-Keuls post hoc test was used to compare the means.Net changes of metabolites during bouts 1 and 3 werecompared using a paired dependent-samples t-test. Resultswere considered significant at P , 0.05.

RESULTS

Total Work and Average Power Output

Total work performed during the 30-s bouts of iso-kinetic cycling was 18.7 6 0.8 and 13.8 6 1.0 kJ in bouts1 and 3, respectively, and was significantly greater inbout 1. The total power output generated during eachbout decreased significantly from 622 6 27 W during

Fig. 2. Average power generated during each time interval of maxi-mal isokinetic cycling at 100 rpm in the first bout (open bars) andthird bout (filled bars). Data are means 6 SE. *Significantly differentfrom first 6 s of same bout. †Significant difference between bouts 1and 3. ‡Significantly different from 7 to 15 s of same bout.

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the first bout to 459 6 32 W in the third bout. Theaverage power declined progressively during each bout,and this decline was more pronounced in bout 1 (Fig. 2).

Phos

The maximal total Phos activities were similar be-tween bouts and across time (Table 1). In contrast, thecalculated maximal Phosa activity increased from restduring bout 1, returned to basal levels at the end ofbout 1, and remained unchanged throughout bout 3(Table 1). Consequently, during bout 1, the percentageof Phos in the a form increased rapidly from a restingvalue of 11.8 6 2.7 to 46.8 6 5.3% at 6 s, remainedelevated at 15 s, and reverted back to basal levels at30 s (Fig. 3). In bout 3, the resting Phosa mole fractionwas similar to the resting value before bout 1 andremained unchanged throughout exercise (Fig. 3).

PDH

Before bout 1, resting PDHa was 0.53 6 0.02mmol·min21 ·kg wet wt21 and increased in a linearfashion to 1.81 6 0.16 mmol·min21 ·kg wet wt21 at 6 sand to 3.56 6 0.50 mmol·min21 ·kg wet wt21 at 15 s(Table 1). Because PDHt was 3.74 6 0.59 mmol·min21 ·kgwet wt21, this represented complete activation of PDH(95.2 6 13.2%), which was maintained for the remain-der of bout 1 (Fig. 3). Resting PDHa was approximatelythreefold higher before bout 3 vs. bout 1. In bout 3, PDHwas nearly completely activated to PDHa by 6 s and wasfully activated for the remainder of exercise (Table 1and Fig. 3).

Muscle Metabolites

Muscle glycogen utilization was 89.2 6 31.3 mmol/kgdry wt during bout 1 but was negligible in bout 3(4.2 6 28.5 mmol/kg dry wt; Fig. 4). The rate of muscle

glycogenolysis during the initial 6 s of bout 1 was 4.04mmol glucosyl units ·kg dry wt21 ·s21, remained ele-vated from 6 to 15 s, and dropped to 0.58 mmol glucosylunits·kg dry wt21 ·s21 in the final 15 s (Fig. 5). Glycogen-olytic rates were depressed throughout bout 3 to levelssimilar to those in the last 15 s of bout 1.

The concentrations of G-6-P, F-6-P, and Gly-3-Pincreased significantly from rest to 15 s of exercise inbout 1 and remained elevated (Table 2). In contrast, theconcentrations of G-6-P, F-6-P, and Gly-3-P did notincrease above resting levels during bout 3. The concen-tration of free glucose did not change within either boutbut was significantly greater in bout 3 (Table 2).

During bout 1, the concentrations of lactate, pyru-vate, and H1 increased progressively from rest until 15s and remained elevated at 30 s (Fig. 4 and Table 2).Before bout 3, the concentrations of lactate, pyruvate,and H1 were similar to those found at the end of bout 1and did not increase further during bout 3.

The rates of total pyruvate production and oxidationand lactate accumulation are shown in Fig. 6. The rateof pyruvate production was highest in the first 15 s ofbout 1 but was reduced to ,1⁄3 to 1⁄6 in the last 15 s ofbout 1 and throughout bout 3. In contrast, the rate ofpyruvate oxidation increased more than threefold dur-

Table 1. Muscle Phos activities and PDHa at rest andduring maximal intermittent isokinetic cycling

Phos

PDHaVmax for Phos a 1 b Vmax for Phos a

Rest

Bout 1 1.9760.22 0.2460.08 0.5360.02Bout 3 2.5660.43 0.3560.13 1.5660.25†

6 s

Bout 1 2.7360.30 1.2860.22* 1.8160.16*Bout 3 3.0860.55 0.3960.05† 3.0860.55*†

15 s

Bout 1 2.3860.25 1.0960.17* 3.5660.50*‡Bout 3 2.6060.33 0.5460.10† 3.3160.40*

30 s

Bout 1 2.3060.29 0.5260.19‡ 3.7160.38*Bout 3 2.2160.35 0.2760.14 3.4660.42*

Data are means 6 SE. Vmax, for phosphorylase (Phos) a 1 b,calculated maximal total Phos activity; Vmax, for Phos a, calculatedmaximal Phos a activity in mmol·kg dry wt21 ·s21. Active form ofpyruvate dehydrogenase (PDHa) is expressed in mmol ·kg wetwt21 ·min21. *Different from rest of same bout. †Difference betweenbouts 1 and 3. ‡Different from previous time point in same bout.

Fig. 3. Muscle pyruvate dehydrogenase (PDH) in the active (a) formand phosphorylase (Phos) a mole fractions at rest and duringmaximal isokinetic cycling in the first bout (s) and third bout (r).Data are means 6 SE. *Significantly different from rest of same bout.†Significant difference between bouts 1 and 3. ‡Significantly differ-ent from previous time point in same bout.

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ing the last 15 s of bout 1. Lactate accumulated at a ratethat mirrored the rate of pyruvate production.

High energy phosphates. The concentration of PCrdecreased from a resting value of 88.2 6 4.7 to 7.6 6 3.2mmol/kg dry wt at 30 s of exercise in bout 1 (Table 3).PCr resynthesis was incomplete before bout 3 and was79% of the amount available before bout 1. Total PCrhydrolysis was greater in bout 1 compared with bout 3(80.7 vs. 59.9 mmol/kg dry wt).

The concentration of ATP was unchanged duringexercise and between bouts (Table 3). The calculatedconcentrations of free ADP and AMP increased in atime-dependent fashion and were significantly greaterat 30 s of exercise in bout 1 compared with bout 3(Table 3).

The concentration of total free Pi increased rapidlyduring the first 6 s of exercise and was significantlyelevated throughout bout 3 compared with bout 1, dueto increased binding to accumulated hexose monophos-phates in bout 1 (Table 3). Due to a greater change in[H1] in the first bout compared with the third, differ-ences were shown between the two bouts when totalfree Pi was partitioned into its two molecular species.

The diprotonated form (H2PO412) followed a similar

pattern of change as total Pi, whereas the monoproto-nated form (HPO4

22) increased as a function of time onlywith no differences between bouts 1 and 3 (Table 3).

Acetyl group accumulation. The concentrations oftotal and free CoA remained constant throughout bothbouts of cycling (Table 2). The concentration of acetyl-CoA increased from resting values during each boutand was significantly elevated throughout bout 3 com-pared with bout 1 (Table 2). The concentration ofacetylcarnitine remained constant throughout eachexercise bout but was greater in bout 3 compared withbout 1 (Table 2).

DISCUSSION

In the present study, we examined the regulation ofmetabolic changes in muscle during the first and last ofthree bouts of maximal exercise for 30 s separated by 4min of rest. During bout 1, the challenge of the extremeATP demand was met through substrate phosphoryla-tion by rapid PCr breakdown and the rapid onset ofglycogenolysis. Glycogenolysis and pyruvate produc-tion were maximal during the first 15 s and farexceeded the rate of oxidation through PDH, resultingin significant lactate accumulation. However, duringthe last 15 s, Phos had returned to resting levels withlow rates of glycogenolysis and pyruvate production,whereas PDH was fully active by 15 s and pyruvateoxidation was more closely matched to its rate ofproduction, resulting in minimal further lactate accu-mulation. In bout 3, Phos b-to-a transformation wasinhibited such that glycogenolysis and pyruvate produc-tion were low. PDH activity was elevated before bout 3,and full activation was rapidly achieved during thethird bout such that pyruvate oxidation was moreclosely matched to pyruvate production with minimallactate accumulation. As each bout progressed and with

Fig. 4. Muscle glycogen, lactate, and pyruvate at rest and duringmaximal isokinetic cycling in the first bout (s) and third bout (r). dw,Dry wt. *Significantly different from rest of same bout. †Significantdifference between bouts 1 and 3. ‡Significantly different fromprevious time point in same bout.

Fig. 5. Estimated rates of muscle glycogenolysis during maximalisokinetic cycling in the first bout (open bars) and third bout (filledbars).

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each progressive bout, there was decreased substratephosphorylation by PCr hydrolysis and glycolysis and agreater reliance on oxidative phosphorylation.

Contribution of Substrate Phosphorylation andOxidative Phosphorylation to ATP Production

In response to the high demand for ATP turnoverduring the initial 6 s of maximal exercise, substratelevel phosphorylation by rapid PCr hydrolysis providedan immediate source of ATP at a rate of 7.0 mmol·kgdry wt21 ·s21 (Fig. 7). The activation of Phos and glyco-genolysis allowed substrate level phosphorylation byglycolysis at a rate of 6.2 mmol·kg dry wt21 ·s21,whereas the rate ofATP turnover by oxidative phosphor-ylation was 1.32 mmol·kg dry wt21 ·s21 in the first 6 s ofexercise. Thus a total maximal ATP turnover rate of14.5 mmol·kg dry wt21 ·s21 was obtained in the first 6 sof bout 1 and was in agreement with values obtained inother studies using similarly strenuous protocols (24).In the last 15 s of bout 1, when the PCr stores had been

depleted and glycogenolysis was inhibited, substratelevel phosphorylation accounted for a decreasing propor-tion of the total ATP turnover rate. In bout 3, PCr wasstill an important source of ATP regeneration, but, dueto the inhibition of Phos, substrate level phosphoryla-tion by glycolysis contributed very little. As each boutprogressed and with each progressive bout, the decreas-ing ability to maintain substrate phosphorylation byPCr hydrolysis and glycolysis was supplemented byoxidative phosphorylation. As each bout progressed,and the average power output was reduced, the ATPturnover rate was reduced accordingly (Figs. 2 and 7).

Regulation of Phos

Glycogen supplies the majority of the energy re-quired for rapid ATP provision during short-term maxi-mal exercise (7, 18, 26, 35, 46). During the initial 6 s ofthe first bout, the glycogenolytic rate was estimated tobe 4.04 mmol glucosyl units ·kg dry wt21 ·s21, whichremained elevated during the next 9 s, and declined to

Table 2. Muscle metabolite content in the vastus lateralis at rest and during maximalintermittent isokinetic cycling

Measure

Bout 1 Bout 3

Rest 6 s 15 s 30 s Rest 6 s 15 s 30 s

Glucose, mmol/kg dry wt 1.360.2 4.062.2 2.060.3 4.361.6 12.863.4† 12.962.8† 9.760.9† 10.261.4†G-6-P, mmol/kg dry wt 0.960.2 9.062.1* 18.462.6*‡ 16.861.6* 8.562.5† 7.061.4 11.861.6† 10.962.6†F-6-P, mmol/kg dry wt 0.260.1 1.560.4* 3.460.5*‡ 3.961.3* 1.660.4† 1.860.5 2.460.4 2.060.6†Gly-3-P, mmol/kg dry wt 0.560.1 4.060.7* 7.460.3*‡ 6.561.4* 11.161.7† 10.861.4† 11.760.6† 11.661.6†H1, nM 62.360.3 110.365.4 157.469.8* 182.9612.4* 216.4648.0† 254.5645.8† 231.4613.0 254.5636.1Acetyl-CoA, µmol/kg dry wt 8.061.4 9.660.7 11.061.1* 14.464.3*‡ 14.361.5† 18.961.1*† 18.161.4*† 21.261.8*†‡Free CoASH, µmol/kg dry wt 58.469.6 67.2613.2 66.667.6 60.9621.6 64.0612.0 62.868.8 66.1610.6 58.968.4Total CoASH, µmol/kg dry wt 66.469.9 76.8613.6 77.768.1 75.2623.4 78.3613.2 81.769.8 84.1612.0 80.069.7Acetylcarnitine, mmol/kg dry wt 4.661.6 2.060.4 3.361.0 5.461.8 11.860.9† 11.762.0† 10.360.7† 11.161.1†

Data are means 6 SE. G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; Gly-3-P, glycerol 3-phosphate. *Different from rest of samebout. †Difference between bouts 1 and 3. ‡Different from previous time point in same bout.

Fig. 6. Comparison of the rates of pyru-vate production, pyruvate oxidation, andlactate accumulation during each time in-terval in the first (A) and third (B) bouts.

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0.58 mmol glucosyl units ·kg dry wt21 ·s21 in the final 15s (Fig. 5). The initial glycogenolytic rates obtained inthe present study are similar to those that have previ-ously been reported for maximal short-term exercise (7,18, 26). Additionally, the decline in glycogenolytic rateobserved over the first bout is in agreement with aprevious study from this laboratory (26) and a studyfrom Boobis et al. (7) in which glycogenolysis occurred,for the most part, within the first 6–10 s of a 30-ssprint. Furthermore, there was a decrease in glycogenutilization in the third bout relative to the first bout.During the first bout, glycogen utilization over the 30 swas 89.2 6 31.3 mmol glycosyl units/kg dry wt but only4.2 6 28.5 mmol glycosyl units/kg dry wt in the thirdbout. This decline in glycogen utilization with repeatedbouts of maximal exercise is consistent with previousobservations (6, 18, 45).

The breakdown of glycogen is regulated by the rate-limiting enzyme Phos. Phos is regulated by reversibleenzymatic phosphorylation and exists in two intercon-vertible forms, a less active b form and a more active aform. Phos b is transformed to its a form by Phoskinase, whereas dephosphorylation back to the b formis catalyzed by Phos phosphatase. The transformationof Phos is regulated at the hormonal level by cAMP andat the contractile level by Ca21 release from the sarco-plasmic reticulum (9, 43). In the present study, thetransformation of Phos to its more active a form wasrapid and reached a peak (46.8 6 5.3%) within theinitial 6 s of bout 1 and remained elevated until 15 s.This rapid transformation was probably due to Ca21

release from the sarcoplasmic reticulum at the onset ofcontraction.

Posttransformational regulation of Phos activity andglycogenolysis occurs via the availability of substrate(Pi and glycogen) and the presence of positive (free AMPand IMP) and negative (G-6-P) allosteric modulators.The Michaelis constant (Km) values of Phos a and a 1 bfor Pi are 26.2 and 6.8 mM, respectively (11). Further-more, the presence of 0.01 mM free AMP reduces the Kmof Phos a for Pi to 11.8 mM (42). Phos b is also active inthe presence of high levels of free AMP (.0.02 mM) andIMP (1). In response to the high demand for ATPturnover during the initial 6 s of maximal exercise, therapid hydrolysis of PCr resulted in significant accumu-lation of free Pi, which falls within the range of the Kmfor Phos.

The high glycogenolytic rate attained during the first6 s of bout 1 and maintained during the next 9 s wouldrequire a Phos activity of 4.04 mmol glucosyl units ·kgdry wt21 ·s21. The calculated Vmax of Phos a at 6 s wasnot sufficient to accommodate such a high glycogeno-lytic rate. This suggests that complete activation ofPhos a 1 b would be required to achieve this flux. Dueto the rapid reversal of Phos a to b (41), the 10-s delayfrom muscle sampling to complete freezing in our studymay have led to an underestimation of Phos a activityand hence Phos activation.

Previous studies using submaximal exercise intensi-ties ranging from 30 to 90% VO2max have shown thatactivation of Phos is in excess of the estimated fluxthrough the enzyme and that glycogenolytic flux through

Fig. 7. ATP turnover rates from PCrhydrolysis, glycolysis, and oxidativephosphorylation during each time inter-val in the first (A) and third (B) bouts ofmaximal isokinetic cycling.

Table 3. Muscle high energy phosphate content in the vastus lateralis at restand during maximal intermittent isokinetic cycling

Measure

Bout 1 Bout 3

Rest 6 s 15 s 30 s Rest 6 s 15 s 30 s

ATP, mmol/kg dry wt 20.861.6 22.460.5 20.462.0 16.264.9 17.661.3 16.462.0 18.160.9 17.861.4Free ADP, µmol/kg dry wt 101.068.7 208.8621.6 274.8617.3* 651.2613.8*‡ 49.266.4 168.9638.5 295.4658.0* 455.4695.9*†‡Free AMP, µmol/kg dry wt 0.560.1 1.960.4 3.860.6 20.060.3*‡ 0.160.0 1.860.6 5.161.6 15.664.1*†‡PCr, mmol/kg dry wt 88.264.7 46.264.0* 28.262.6*‡ 7.663.2* 69.764.3† 25.763.9*† 14.964.5* 9.861.8*Total Pi, mmol/kg dry wt 10.8 36.763.4* 45.569.4* 47.866.5* 18.8610.4 55.766.5*† 62.167.4*† 63.768.6*†HPO4

22, mmol/kg dry wt 7.860.0 22.161.6* 24.165.3* 22.162.9* 7.764.2 20.862.0* 25.462.9* 24.363.5*H2PO4

12, mmol/kg dry wt 3.060.0 14.661.8* 21.464.2*‡ 25.763.6* 11.266.5† 35.065.5*† 36.764.6*† 39.465.8*†

Data are means 6 SE. PCr, phosphocreatine. *Different from rest of same bout. †Different between bouts 1 and 3. ‡Different from previoustime point in same bout.

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Phos is mediated primarily through posttransforma-tional regulation (substrate and allosteric; see Refs. 12,16, 22). In the present study, the estimated glycogeno-lytic flux was equal to the total Phos activity, and thepeak glycogenolytic rates were ,20-fold higher thanrates of 0.17–0.25 mmol glucosyl units · kg drywt21 ·min21 observed at 80–90% VO2max (12, 16, 22).The power outputs generated during the first 15 s ofexercise in bout 1 were 3.5-fold greater than thatobtained in the study by Howlett et al. (22) in which theaverage power output was 229 6 19 W at 90% VO2max.Thus it appears that, during the first 15 s of maximalexercise, total Phos activation is required to maintainglycogenolytic flux. This involves upregulation of Phosactivity by both transformational and posttransforma-tional mechanisms.

Compared with the first 15 s of bout 1, glycogenolysiswas reduced by 86–95% in the latter one-half of bout 1and throughout bout 3. This large reduction in glycogen-olysis coincided with Phos a to b reversion and theinability to reactivate Phos transformation in bout 3. Inaddition, the predicted flux through Phos a during thelast 15 s of bout 1 and throughout bout 3 correspondedmore closely to the observed glycogenolytic fluxes. Atthe end of the first bout, Vmax for Phos a correspondedmore closely to the glycogenolytic rate estimated overthe last 15 s of bout 1 (Table 1 and Fig. 5). Similarly,during the third bout, the estimated glycogenolyticrates were in the range of the calculated Vmax for Phos aduring each time interval (Fig. 5 and Table 1). Incontrast, the maximal Phos a 1 b activity remainedstable during the entire exercise protocol (Table 1).These observations imply that Phos b was minimallyactivated during this time despite elevations in freeAMP. It is conceivable that the accumulation of G-6-P, aknown allosteric inhibitor of Phos b (17), could haveoverridden positive modulation by free AMP.

The reduction in Phos transformation observed atthe end of the first bout and throughout bout 3 suggeststhat the mechanism of activation by Ca21 was beingoverridden. Krebs et al. (30) have shown in vitro thatelevated [H1] inhibited Phos kinase, thus reducing thetransformation of Phos to its a form. During the last15 s of the first bout, [H1] increased significantly to182.9 6 12.4 nM (pH 6.74) and up to 254.5 6 45.8 nM(pH 6.59) at 6 s in the third bout (Table 2). Chasiotis etal. (10) also observed in human skeletal muscle thatthere was reduced Phos b to a transformation at a pH of6.6 to 6.7.

Increases in [H1] may also have a dual inhibitoryeffect by influencing the concentration of the substratefor Phos. An increase in [H1] increases the prevalenceof the diprotonated Pi, H2PO4

12, over the monoproto-nated form, HPO4

22. The latter is considered to be theonly active substrate of Phos (28). The fall in pH from6.74 at the end of bout 1 to 6.61 at the end of the thirdbout implies that the proportion of Pi in the diproto-nated form increases from 25.7 mmol/kg dry wt at theend of bout 1 to 39.4 mmol/kg dry wt at the end of bout 3in association with the increase in the concentration oftotal Pi, nullifying the change in the monoprotonated

form. Thus, although the total Pi was higher in thethird bout, the availability of the monoprotonated formwas similar in both bouts (Table 3). The availability ofsubstrate for Phos does not appear to be the factor thatdownregulates glycogenolysis in the latter part of bout 1and in bout 3.

The reduced rate of glycogenolysis during the last15 s of the first bout and throughout the third boutappears to be complex as it is modulated by thecumulative effects of a multiplicity of factors. In bout 1,the reduction in glycogenolysis appears to be primarilydue to reversion of Phos a back to the b form andinhibition of Phos b activity at the end of the bout. Inbout 3, the inability to transform Phos b to a could bedue to increased activity of the Phos phosphatase ordecreased activity of the Phos kinase due to elevated[H1]. Although substrate was available, the inhibitoryeffect of G-6-P appears to override the positive alloste-ric effects of free AMP on flux through Phos.

Regulation of PDH

PDH is the rate-limiting enzyme that regulates theentry of glycolytically generated pyruvate into oxida-tive metabolism. The PDH complex is a mitochondrialenzyme that is also regulated by reversible phosphory-lation. PDH kinase catalyzes the phosphorylation ofPDH with concomitant inactivation, whereas PDHphosphatase dephosphorylates the enzyme, therebyactivating PDH. The activity of PDH is determined bythe proportion of the complex in the active form. PDHkinase is inhibited by pyruvate and elevated ratios ofCoASH to acetyl-CoA and NAD to NADH and, con-versely, is stimulated by an elevated ratio of ATP toADP. PDH phosphatase is stimulated by Ca21 (see Refs.3, 40, and 51 for review).

Ca21 released from the sarcoplasmic reticulum at theonset of exercise was probably the primary stimulus forthe activation of PDH. The Ca21 released activatesPDH phosphatase (23) and may simultaneously inacti-vate PDH kinase (40), thus increasing the proportion ofPDHa. Increases in Ca21 concentration in rat heartmitochondria have previously been shown in vitro toresult in 60% activation of PDH (14), which is similar towhat we observed in the first 6 s of contraction. Theremaining 40% of the activation of PDH may have beendue to other allosteric activators of PDH. Although theeffects of Ca21 on PDH activation were studied incardiac muscle (14), it is generally believed that Ca21

is also important in PDH activation in skeletal muscle(51).

Pyruvate is a known activator of PDH and inacti-vates PDH kinase (23). Pyruvate increased linearlyduring the first 15 s, served as substrate for thereaction, and may also have functioned as an allostericregulator that stimulated activation of PDH. The levelsof pyruvate appear to be within the estimated range ofthe inhibitory constant of PDH kinase for pyruvate of0.5–2.0 mM (51). Increases in [H1] followed the sametime course as pyruvate and may have played a role inPDH activation. Again, the only available evidence hasbeen demonstrated in cardiac muscle. A study in per-

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fused rat heart reported that acidosis increased PDHactivation (36). Hucho et al. (23) also reported pHoptima of 7.0–7.2 for bovine kidney and rat heart PDHkinases and 6.7–7.1 for the PDH phosphatases, suggest-ing that an increase in [H1] would favor the activity ofthe phosphatase over the kinase and increase thetransformation of PDH to its active form. Althoughacetyl-CoA is known to inhibit activation of PDH byinhibition of PDH phosphatase and activation of PDHkinase, significant increases in acetyl-CoA in the last15 s of exercise did not appear to exert an inhibitoryeffect over the activation of PDH. It appears that theincreases in Ca21 with contraction and increases in[pyruvate] and [H1] override the acetyl-CoA inhibition.

As in a previous study (38), before the initiation ofexercise in the third bout, the activity of PDHa wassignificantly elevated above resting values before bout1. The increased activation was probably due to ele-vated levels of pyruvate and H1, which continued tostimulate activation to PDHa, overriding the elevatedlevels of the negative allosteric modulator acetyl-CoA.Before the onset of contraction in the third bout, themole fraction of PDHa was 41.8 6 6.8%, and with theonset of contraction PDH became totally activatedwithin 6 s, likely due to the release of Ca21 duringcontraction. Therefore, compared with the first bout,the higher levels of PDHa in bout 3 would provideincreased levels of acetyl-CoA for increased oxidativephosphorylation during the initial 6 s.

Timmons et al. (48, 49) have demonstrated that, atthe onset of submaximal exercise, oxidative phosphory-lation was limited by the availability of acetyl-CoA forthe TCAcycle. They showed that dichloroacetate admin-istration before exercise increased PDHa and acetylcar-nitine levels in human skeletal muscle and reducedPCr hydrolysis, glycogenolysis, and lactate productionduring the rest to exercise transition. In the presentstudy, there was a tendency for acetylcarnitine todecrease (P 5 0.11) at the onset of exercise in the firstbout, and the major contribution of energy provisionwas from substrate level phosphorylation (Fig. 7),which suggests that the availability of acetyl groupsmay be limiting to oxidative phosphorylation. Beforethe third bout of exercise, we observed a similar degreeof activation of PDH and accumulation of acetyl groupsas in the studies of Timmons et al. The increasedavailability of the acetyl groups did not prevent thelarge and rapid rate of PCr hydrolysis in the first 6 s butdid result in a shift to oxidative phosphorylation withminimal utilization of glycogenolysis, resulting in lac-tate accumulation. In a previous study, we observed a10% increase in VO2 in the third bout compared withbout 1 (38), which was mirrored by an overall increaseof 10% in the rate of ATP provision from oxidativephosphorylation in the present study. Other investiga-tors have observed an 18% increase in VO2 in the secondbout relative to the first bout (6) and an increase in thetransient rise in VO2 observed in a second exercise boutwhen it was preceded by high-intensity exercise (32).

Lactate Production

During intense exercise, Phos provides substrate tothe glycolytic pathway. Glycolysis in turn providessubstrate for the TCA cycle in the form of pyruvate viadecarboxylation by the rate-limiting enzyme PDH.Thus the accumulation of pyruvate is determined by itsrate of production by glycolysis and its rate of oxidationby PDH. Glycolysis produced pyruvate at a rate of 4.31,3.89, and 1.36 mmol·kg dry wt21 ·s21 from 0 to 6, 6 to15, and 15 to 30 s in bout 1, respectively (Fig. 6). Thecorresponding rates at which PDHa could oxidize pyru-vate were 0.09, 0.20, and 0.27 mmol·kg dry wt21 ·s21

(Fig. 6). Thus the glycolytic rate was 48- and 19-foldgreater than PDHa during the first 15 s of bout 1 butonly 5-fold greater in the last 15 s of bout 1. The largedisparity between the rates of glycolytic pyruvate pro-duction and oxidation resulted in the net accumulationof pyruvate and lactate in the first 15 s of exercise (Fig.6). As bout 1 progressed, there was a better matchbetween pyruvate production and oxidation due todecreased glycogenolysis and increased PDH activa-tion, which was evidenced by the reduced accumulationof pyruvate and thus lactate toward the end of the firstbout (Fig. 6). Throughout the third bout, the rates ofpyruvate production were considerably reduced to ,0.67mmol·kg dry wt21 ·s21, whereas the rates of pyruvateoxidation through PDH were maintained at 0.17–0.25mmol·kg dry wt21 ·s21, representing less than a four-fold difference. The better matching between the ratesof pyruvate production and pyruvate oxidation in thethird bout resulted in no further accumulation ofpyruvate or lactate.

During the initial seconds of maximal exercise, largerates of glycogenolysis and glycolysis are required toprovide a rapid and immediate source of ATP, and Ca21

appears to be the primary mechanism responsible forPhos and PDH activation. The rapid hydrolysis of ATPand PCr resulted in an accumulation of free Pi and AMPthat increased Phos activity and substrate for glycoly-sis. The increased glycolytic rate translated into en-hanced pyruvate production, which may have exerted apositive allosteric effect on the activation of PDH. Theincrease in [H1] toward the end of the first bout mayhave inhibited Phos b to a transformation and glycogen-olysis, while simultaneously activating PDH. The ele-vated [H1] at the beginning of and throughout the thirdbout may have completely inhibited the transformationof Phos and glycogenolytic flux and may have simulta-neously maintained a high level of PDH activity. Thismechanism of regulation may have served to spare theglycogen stores, inhibit further accumulation of lactate,and limit further increases in [H1]. Thus the increasein [H1] that accompanies intense exercise may be theprimary mechanism responsible for limiting furtherincreases in H1 by inhibiting Phos and the accumula-tion of lactate while increasing PDH activity and theoxidation of lactate. The result was a tighter matchbetween flux through Phos and PDH and a shift towardgreater reliance on oxidative phosphorylation and less

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on substrate phosphorylation by PCr hydrolysis andglycolysis.

We thank Tina Bragg and George Obminski for technical assis-tance.

This study was supported by operating grants from the MedicalResearch and Natural Sciences and Engineering Research Councilsof Canada. M. L. Parolin was supported by a Natural Sciences andEngineering Research Council scholarship and by the Ontario Tho-racic Society. G. J. F. Heigenhauser is a Career Investigator of theHeart and Stroke Foundation of Ontario (I-2576).

Address for reprint requests and other correspondence: G. J. F.Heigenhauser, Dept. of Medicine, McMaster Univ. Medical Centre,1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail:heigeng@fhs.csu.mcmaster.ca).

Received 14 December 1998; accepted in final form 23 June 1999.

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