Anaerobic metabolism in human skeletal muscle ...

Anaerobic metabolism in human skeletal muscle during short-term, intense activity' LAWRENCE k. SPRIET School of Humn Biology, Universifp of Guelph, Guelpbt, Ont. , Camda Nl G 2 WJ

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Hunan Normal University on 06/05/13 For personal use only.

Received November 5, 19W SPWIET,L. L. 1992. Anaerobic metabolism in human skeletal muscle during short-term, intense activity. Can. J. Physiol. Pharmcol. TO: 157 - 165. The ability of human skeletal muscle to provide anaerobically derived ATP during short-term, intense activity is examined. The paper emphasizes the information obtained from direct measurements of substrates, intermediates, and products of the pathways in muscle that provide anaerobically derived ATP. The capacity of muscle to provide ATP via anaerobic pathways is 370 mmollkg dry muscle (dm) during dynamic exercise lasting 3 min. Anaerobic glycolysis provided 80%, phosphocreatine (PCr) degradation 1696, and depletion of the ATP store -4% of the total ATP provided. When the blood flow to the working muscles is reduceel or occluded, the anaerobic capacity decreases to 300 mmollkg dm. This reduction is due to a lower glycolytic capacity associated with an inability to remove lactate from the muscles. Directly measured maximal rates of anaerobicdy derived ATP provision from PCr degradation and glycolysis during intense muscular activity are each 9- 10 mmol . kg-' dm s-'. Evidence suggests that both of these pathways are activated instantaneously at the onset of maximal activity. Spring training does little to the capacity or rates of the pathways, although a 10-20% increase in glycolytic ATP provision has been reported. The only study comparing direct and indirect estimates of the anaerobic capacity in humans suggests that 0, deficit measured at the mouth accurately predicts the anaerobic capacity s f a single muscle group and that 0, debt dms not. There are many unresolved issues regarding the capacity of the PCr and glycogeno%ytic-glycolytic systems to provide ATP during short-term intense muscular activity in humans. Considerable effort is now being directed to understanding the in viva regulation of the regulatory and flux-generating glycogenolytic enzyme, phosphorylase. Key words: glycogenolysis, glycolysis, phosphocreatine, ATP, sprinting.

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SPWIET,L. L. 1992. Anaerobic metabolism in human skeletal muscle during short-term. intense activity. Can. I. PhysioB. Pharmacol. 196D : 157-165. On examine la capacite du muscle squelettique h u m i n de fournir de I'ATP derivk anakrobiquement duraat une activitk intense de courte dur&. L'article met I'accent sur l'information obtenue de mesures directes de substrats, intermediaires et produits des voies musculaires qui foumissent de 1 I T P dCrivCe anakrobiquement. La capacitC du muscle de fournir de I'ATP par les voies anakrobies correspond 370 mmollkg de muscle sec (ms) durant un exercice dynamique d9unedurke de 3 min. La glycolyse anakrobie a fourni 80%, la degradation de la phosphocreatine (PCr) 16% et la depletion de la reserve d9ATP -4% du total d'ATP fournie. Lorsque le dCbit sangblin aux muscles actifs est rCduit ou bloquC, la capacite anaerobic diminue 2 -300 mmol/kg ms. Cette reduction est due 2 une plus faible capacitk glycolytique assmike une incapaeitk d'eliminer le lactate des muscles. Les taux maximaux d'apport en ATP d6rivCe anakrobiquement, mesures directement pour ce qui est de la glycolyse et de la dkgradation de h PCr durant une activitk musculaire intense, correspondent chacun B -9- 10 mmol - kg-' ms . s-'. Les faits sugg$rent que ces deux voies sont activks instantankment au dCbut de I'activitC maximale. L'entrainement au sprint modifie peu leur capacite ou leur taux, bien qu'une augmentation de 18-2Q% dd'apport en ATP glycolytique ait kt6 rapportke. L'unique etude comparant les Cvaluations directes et indirectes de Ba capacitk anakrobie chez l'humain suggkre que le dCficit en O,, mesure au niveau de la bouche, pr&lit prCcisCment h capacitC anairobie pour I nous reste encore beaucoup i apprendre sur la capacitC des un g r o u p de muscles seuls, ce que ne fait pas la dette d'8,. B syst&mesglycogenolytiques -glycolytiques et de la PCr de fornmir de I'ATP durant une activitk musculaire intense de csurte d u r k chez les humains. Un effort considirable est maintenant dirigC vers la compr6hension de la regulation in vivs de l'emyme glycog~nolytiquephosphorylase. Mots clefs : glycogCnolyse, glycolyse, phosphmrCatine, ATP, sprint. [Traduit par la rkdaction]

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Introduction Engaging in intense muscular activity leading to exhaustion in 3 rnin increases the activity of the enzymes that hydrolyze adenosine 5'-triphosphate (ATP) to extreme levels. These include the myosin, Ca2+ and Na+ -K ATPases. Consequently, ATP must be resynthesized at an extremely high rate. This occurs at a time when oxygen (Q2) is in short supply as the cardiovascular system increases 0,delivery to the working muscles in an attempt to match the metabolic demand. Even when the muscles attain their maximal rate of aerobic ATP provision, the demand for ATP is in excess sf the aerobic capability. In this situation, anaerobically derived ATP provi-

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'This paper was presented at the Skeletal Muscle Metabolism Symposium, held during the meeting of the 1998 Canadian Association of Sports Sciences, in Minaki, Ont., Canada, September 29, 1990, and has undergone the Journal's usual peer review. Printed in Canada I Imprime au Canada

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sion is paramount and may provide up to 80 -90% of the tstd ATP required, depending on the duration of the activity. There is a Imge body sf literature that has indirectly examined the ability of skeletal muscle to provide anaerobic ATP using measurements of Q2 deficit and O2 debt. A more direct method is to estimate the provision of anaerobic ATP from measurements s f the substrates, intermediates, and products sf the anaerobic pathways in skeletal muscle. Simultaneous estimations of anaerobically derived ATP provision using indirect and direct methods are required to demonstrate whether the indirect and non-invasive approach is valid. Owing to the invasiveness of the required measurements, few studies have attempted the direct approach in humans. The direct measurements that have been made demonstrate that significant amounts of anaerobicdly derived ATP can be provided from phosphocreatine (PCr) degradation and the glycolytic pathway (Fig. 1). The endogenous ATP store can

CAN. J . PHYSHOL. PWARMACBL. VOL. 70, 1992

ANAEROBIC ENERGY SOLIRCES PCY+ADP+H+~ATP+C~

ATP r e s e r v e


AT P

FIG. 1. A schematic representation of the sources sf anercpbic energy.

also contribute a small amount of energy as the ATP concentration in muscle may fdl by up to 50% when the demand for ATP is extreme. Anaerobically derived ATP can be provided at much higher rates than aerobic pathways, but the capacity for ATP provision is extremely limited. This is consistent with the ability of humans to attain extremely high power outputs, but only maintain them for short perids of time. The purpose of this paper is to review the current howledge regarding anaerobic metabolism during intense short-term muscular activity. The pathways of anaerobic metabolism in skeletal muscle are examined in terms of their capacity to provide ATP, the rates at which ATP can be resynthesized and how quickly they are activated. The paper idso examines selected issues regarding the regulation of these pathways, including the responsiveness to sprint training. Adult human data are emphasized and supplemented with data from animal models where necessary. Data obtained from direct measurements on human skeletal muscle are given preference over data obtained indirectly, although comparisons between direct and indirect approaches are made when appropriate. For overviews of indirect approaches to studying anaerobically derived ATP provision the reader should consult the following papers: Hermansen 1969, Medbo et al. 1988, Saltin 1990. The exercise or contraction tasks examined in this paper produced exhaustion or required extremely high rates of ATP provision for a short period of time.

The capacity sf skeletal muscle to provide anaerobic ATP To accurately assess the anaerobic capacity of a given muscle or muscle group, measurements of ATP, Per, and lactate content are required from muscle biopsies obtained before and after an exhausting task. The amount of lactate that has escaped the muscle must also be estimated using arterial and venous catheters and blood Wow measurements and then normalized to the mass of working muscle. The invasive nature sf this approach has limited the number of complete studies in this area. However, several studies reported intramuscular changes following intense exercise lasting 38- 150 s (Table 1). While measurements of escaped lactate were not always made, estimates of anaerobic ATP provision per kilogram dry muscle (dm) were made using the following equation: -

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ATP provision = A PCr

-+ 1.5 A lactate + (2 A ATP - A ADP)

This assumes that the majority of carbohydrate substrate originates from muscle glycogen and that decreases in muscle ATP concentration are accounted for by increases in inosine 5 '-monophosphate (IMP). The data of Karlsson and Saltin (1970) indicate a total anaerobic ATP provision of 160 mmollkg dm

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during intense cycling (Table I). Glycolysis provided 60 % of the ATP, Per breakdown contributed 33 % and depletion of the ATP store provided only 7 % . During an isometric contraction to exhaustion, the estimated anaerobic ATP was considerably greater at -225 mmol/kg dm (Karlsson et al. 1975). The increase was due to a greater glycolytic contribution that probably resulted from lower leg blood flow and less lactate escape during the isometric contraction. Jones et al. (1985) also reported a high ATP contribution from glycolysis (75 %) during isokinetic cycling at extreme power outputs. In this case blood Wow was undoubtedly- high, but there was little time (30 s) for lactate to escape the muscle. In a different approach, Spriet et al. (1987) occluded the circulation to one leg and electrically stimulated the quadriceps muscles to fatigue. With a closed circulation no significant glucose or O2 uptake occurred and lactate did not escape the muscles. The electrical stimulation protocol was 1.6 s on, 1.6 s off at 20 Hz for 200 s. During the final 50 s of continuing stimulation, peak tetanic tension decreased to less than 15% sf initial tension and no appreciable P e r degradation, glycolytic activity, or further reductions in ATP concentration were measured, indicating that the muscles were fatigued. It is also interesting to note that the muscles did not enter into a state of rigor with continued electrical stimulation. Total contributions from Per, ATP, and glycolysis were 75, 20, and 210 mmol ATB/kg dm, respectively, for a total of 305 mmol/kg dm (Table 1). Bangsbo et al. (1990) quantified the anaerobic ATP provision of quadriceps muscles during intense dynamic exercise to exhaustion with an open circulation. The task required subjects to extend the lower leg about the knee once per second against a resistance corresponding to a power output of 130% of the leg's peak aerobic power output. The mean time to exhaustion was 192 s. The criteria u s d to determine exhaustion were a decrease in the force magnitude or a drop in the rate of extensions. The glycolytic anaerobic contribution in this study was estimated from muscle lactate and glycolytic intermediate accumulations and the lactate released from a known amount of muscle. The anaerobic ATP provision from Per, ATP, and glycolysis was -60, 10, and 300 mmol/kg dm, respectively, for a total of 370 rnmol/kg dm (Table 1). The high glycolytic contribution could be partitioned into 67% from the intramuscular accumulation of lactate and glycolytic intermediates and 33 % from escaped lactate. The anaerobic ATP contribution from glycolysis during exhausting contractions increased by -33% when the circulation was open as compared with occluded (Spriet et al. 1987). The escape of lactate and associated changes in ion movements appeared to delay the onset of muscular fatigue when the circulation was open.

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Comparison of direct and indirect estimates of anaerobic capacity The main purpose of the study by Bangsbo et al. (1990) was to compare direct and indirect estimates of the capacity to provide anaerobic energy. Arterial and venous catheterization and blood flow measurements permitted the estimation of leg O2 uptake. A graded exercise test for the knee extensors provided the relationship between O2 uptake and submaximal power outputs. This relationship was used to predict the energy d e m d of power outputs above that which elicited leg Vo2rnax. The O2 deficit during intense work was then estimated as the difference between the energy demand and the aerobic energy

SPRIET


TABLE 1. Direct measurements of the capacity of human vastus lateralis muscle to provide anaerobic ATP from PCr degradation, glycolysis, and the ATP store

Reference

Activity

Karlsson and Saltin (1970)

Cycling (385 W) Isometric (50% MVC) Isok. cycling (750 W) El. stim (20 Hz) Isok. leg extension

Karlsson et al. (1975) Jones et al. (1985) Spriet et al. (1987) Bangsbo et aH. (1990)

Time (s)

Anaerobic ATB provision (mmollkg dm) Duty cycle

PCr

GHycolysis

ATB

Total

143

- 111

55

95 3-

10

1663+

-90

-

70

153+

3

30

- 111

45

1N+

10

245+

205

111

75

210

20

305

192

- 111

60

3W

10

370

(193

+ 95 + 12)

226

(65 W) ABBREVIATIONS: W, watts; MVC, maximal voluntary contraction; El. stim, electrical stimulation; Isok. cycling, isokinetie cycling. +, indicates no attempt to include the lactate that escaped from muscle. Bracketed values under glycolysis from Bangsbo et al. (1990) indicates ATP contributions from accumulated muscle lactate, escaped lactate, and accumulated glycolytic intermediates, respectively. Exercise was continued to fatigue in all studies except Jones et a!. (1985).

provision. A comparison of direct and indirect (02 deficit) methods for quantifying the anaerobic ATP capacity of the knee extensor muscles gave estimates of 9 1.2 and 9 1.6 mmollkg wet muscle, respectively. In addition, the calculated O2 deficits across the leg and at the lung were 0.46 and 0.44 L O,, respectively, indicating that the O2 deficit determined at the lung represents the anaerobic ATP capacity of this single group of working muscles. These findings suggest that an accurately obtained O2 deficit at the lung is a vdid indirect method for quantifying a person's anaerobic capacity during this type of exhausting task. The O2 deficit technique has been in use to estimate anaerobic capacity in several laboratories (Hermansen 1969; Karlsson and Saltin 1970; M d b o et al. 2988; Pate et al. 1983). Frequent determinations of O2 uptake and O2 deficit during exhausting exercise permits an accurate partitioning of the proportions of energy arising from anaerobic and aerobic sources. In the study by Bangsbo et d. (1990), contributions from anaerobic and aerobic sources were 80 %/20% in the initial 30 s945'36155% from 60 to 90 s, and 30%/70% from 120 to 192 s. The average over the 3 min of exercise was 45 % anaerobic and 55 % aerobic.

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Rates sf anaerobic ATP provision The rates that anaerobic pathways provide ATP are critical to the development and maintenance of high power outputs. To assess these rates, serial sampling of muscle is required at frequent intervals, Table 2 presents a summary of studies where data permit an estimation of the rates of anaerobic ATP provision from K r degradation and glyco8ysis. When examining these values, it must be remembered that as muscular activity extends beyond 10 s, power output will decline and rates sf glycolytic and Per-derived ATP provision will not be maximal. In addition, most sf the glycolytic estimates do not account for escaped lactate and are therefore underestimations. The highest rates for PCr and glycolytic ATP provision during various contraction modes lasting up to I0 s are -6.0-9.8 and 6.0-9.3 mmol ATP . kg- "dm . s u l , respectively. The submaximal intensities of many of the contraction tasks and the contraction durations used to determine average

rates ( > 10 s) are responsible for the low rates reported in many of the studies.

How quickly are anaerobic pathways activated? Margaria et al. (1964, 2969) proposed that PCr degradation was the immediate and only substrate for ATP resynthesis during the early stages (< 10 s) of intense exercise. Upon depletion of this substrate, glycogenolysis was activated to provide a continued ATP supply through glycolysis. The experimental evidence suggests that PCr degradation does occur instantaneously at the onset sf exercise (Table 2). This is consistent with the extremely high maximal activities of creatine phosphokinase (CPK) measured in vitro and the near-equilibrium nature of the enzyme (Newskolme and Leech 1983). Therefore, the PCr store is a powerful energy buffer, as small changes in ATP and ADP concentrations ensure rapid ATP resynthesis. In fact, most investigations demonstrate that muscle ATP concentration is maintained in the face of intense contractile activity until PCr is is largely depleted (Boobis el al. 1982; Sahlin et al. 1975; Spriet et al. 1987). The earliest report of PCr degradation in human muscle during intense exercise measured a decrease from 70.0 to 8.0 mmollkg dm duringcycling to exhaustion in 77 s (Hultman et al. 19b7). The suggestion that PCr degradation and anaerobic glycolysis are "serially mobilized" in maximally contracting skeletal muscle is clearly not supported by experimental evidence. The data in Table 3 demonstrate that the glycolytic pathway is rapidly activated during intense muscular contractions. Saltin et d o(1971) proposed that anaerobic glycolysis commenced with the onset of muscular contraction. They observed a significant increase in muscle lactate concentration in two individuals after 10 s of cycling at 110% kro2max. B o ~ b i set al. (1982) also reported very significant increases in muscle lactate concentration following only 6 s of cycling at high power outputs in four males. However, Jacobs et d. (1982, 1983) were the first to systematically examine the hypothesis that anaerobic glycolysis commenced well before the PCr store was depleted. Their initial study demonstrated that lactate accumulated from 9.0 to 60.5 mmollkg dm and that PCr concentration decreased by 60% during 30 s of maximal cycling

CAN. J.

PHYSIOL. PHAWMACOL. VOL. 70,

1992

TABLE2. Rates of anaerobic ATP provision from phssphocreatine degradation and glycolysis during intense rnuscu1ar contraction

Reference


Hultman and SjshoHm (19836)

HuItrnan and Sjoholm ( 1 9 8 3 ~ )

WP of exercise

Duration (s)

PCP

Glyeolysis

El. stim (50 Hz) (20 Hz) El. stim (20 Hz)

Boobis et al. (1982)

Cycling

Jacobs et al. (1983)

(M)Cycling (F) CycHing (M) Cycling (F) Cycling Isok. cycling

Jones et al. (1985)

ATP provision (mmsl. kg-' d m . s-')

(m v m ) Spriet et al. (1987aj

Cheetham eb al. (1986) Jacobs et al. (1982) Nevill et al. (1989) Costill et al. (1883) Bultman et al. (1967) Kabrlssorn et aB. (1975) Karlsson and Saltin (1970) Bangsbo et al. (1990)

EB. stim Cycling Isok. cycling rpm) Running Cycling Running Rumaqing 125 $0 (G) Vo,rniax Running 4OCl rn Cycling Isometric (50% MVC) Cycling Isok. knee extensions

MOTE: References are arranged with the shortest contraction perids listed first. ABBREVIATIONS: PCr, phosphscreatine; El. stim, electrical stimulation: Hsok. cycling, isokinetis cycling; M,male; F, female; hpm, revolutions per minute; G , gastrocnemius; MVC, maximal vol~nhkycontraction. All muscle biopsies were from the vastus lateralis unless noted otherwise.

in females (Jacobs et al. 1982). A subsequent study demonstrated that lactate accumulated to 46.1 and 25.2 mrnol/kg dm following I 0 s of maximal cycling in males and females, respectively. Unfor%kanate8y9resting lactate and resting and 18 s PCr measurements were not made. Jones et al. (1985) also reported large increases in the muscle lactate concentration s f two males after only 10 s of maximal isokinetic cycling. The Per concentration decreased to -25-38% of the resting content in only 10 S. Convincing evidence that anaerobic gHycolysis is activated at the onset of muscular contraction arises from the electrical stimuIatisn work of Hultman and Sjokolm (1983a, 1983b). They stimulated the vastus lateralis (VL) muscle of males and females for 1.28 to I 0 s at 20-50 Hz and consistently measured increases in muscle lactate concentration (Table 4). In these experiments, the circuIation to the stimulated muscle was occluded during and following the short stirnulation periods

to minimize the chance of recovery before biopsies were taken. It should be remembered that these were electrical stimulation experiments and not dynamic exercise, but activation of all motor units during each contraction would be expected in both situations.

Effects of sprint training on the anaerobic capacity Can the anaerobic capacity be increased through sprint training? Surprisingly, only a few studies have examined this question, considering the impact that training-induced increases could have on the performance of athletic activities where anaerobic ATP provision is paramount. MacDougall et al. (1977) subjected nine males with no previous experience to a 5-month weight training program. Biopsies of the triceps brachii muscle before and after the training program demonstrated significant increases in P e r , A ' P , and glycogen contents following training (Table 4). Although the relationship between

TABLE3. Experimental evidence for the rapid onset of anaerobic glycolysis during intense, shortterm muscular activity Lactate (rnrnd/kg dm)

Time Sdtin et al. (1971)


(s)

Activity

Reference

Rest

n

Exercise

Cycling (I 10 % 'ooZmax) Cycling El. stim (28 Hz) El. stim (50 Hz) (20 Hz) Cycling (747 W) (539 W) Isok. cycling (758 W, 60 rgsrn) (900 W, I48 p m )

Boobis et d. (1982) Hultmn and Sjoholrn (1983a) Hultmn and Sjoholrn (19833) Jacobs et al. (1983) Jones et al. (2985)

NOTE:References are listed chrondogicdIy. Aee~svr~TnOprs: W,watts; M, m d e ; F, female; rpm, revolutisns/rnin.

TABLE 4. Effects of sprint training on resting BCr, ATP, and glycogen contents and anaerobic ATP provision during intense short-term exercise -

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Reference

Training

MasDougall et al. (1977) Boobis et aI. (1983)

4 -5 months weight train. 8 weeks, 5/week cycling 36) s cycling 8 weeks, 4/wwk running 30 s running 2 min run. (1 10 % Vo,max)

Nevi11 ee al. (1989)

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Anaerobic ATB provision

Resting concn. PCr

ATP

Glycogen

-5 %

- 18%

-32 %

ATP

Per

Glycslysis

$%*

9%

-

36%

-

-

-

11%"

10%"

-

-

20 9%

-

6%*

10%

-

-

-

NOTE:Resting Kr and AT$, mmollkg dm; glycogen, mm01 glucosyl unitdkg dm; anaerobic ATP provision, m m t / k g dm. Blanks indicate no determination;

--, indicates no change from pretraining condition. *No significant difference from pretraining value.

resting substrate content and anaerobic ATP provision is unclear for anaerobic glycolysis, a higher PCr concentration content translates into a greater anaerobic ATP potential during intense activity. Boobis et d. (1983) sprint-trained seven males on a cycle ergometer and reported unchanged resting ATP concentration while PCr concentration decreased and glycogen increased (Table 4). The amount of anaerobic ATP provided per unit muscle mass from glycolysis, PCr degradation, and the ATP store during 30 s of maximal dynamic cycling was not significantly changed by training, although the glycolytic contribution and average power output increased by 8%. In a second study, four male and four female recreational runners sprinttrained for 8 weeks and no significant training-induced changes in muscle metahlites were reported. The average power output during a 30-s maximal run increased by 6% following training. Anaerobic ATP provision from glycolysis was significanay increased (20%) during the spring following training ,while the K r and ATP store contributions were unchanged. Consequently, the total anaerobic ATP provision during the 30-s sprint increased by 14% . There were no training-associatd changes in anaerobic ATP provision during a 2-min run at 110% of the pretraining Vo2max.

Medbo and Burgers (1990) examined the effects of sprint training on the anaerobic capacity of men and women during a 2- to 3-min treadmill run to exhaustion. Anaerobic capacity was estimated indirectly by measuring the accumulated O2 deficit. The subjects were recreationally active but none had recently participated in any form of anaerobic training. Following 6 weeks of sprint training (three sessions/week), anaerobic capacity increased 18%.There was no difference in the %naerobic capacity of a separate untrained group and a group of endurance trained subjects, while a group of track sprinters had a 30% higher anaerobic capacity.

Anaerobic energy from ph~sphwreatirae This section examines issues related to PCr degradation that are relevant to both the rate of ATP provision and the capacity of ATP derived from this source. Resting phosphocreatine content Several human investigations using 3BPnuclear magnetic resonance (NMR) have suggested that the resting muscle PCr concentration is considerably higher than when measured chemically in biopsy samples (Chance et al. 1982; Edwards et al. 1982; Taylor et d. 1986). This could result from dismp-

CAN. J. PHYSIQL. PHARMACOL. VOL. 70, 1992


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tion of muscle membranes during the biopsy procedure, with the release of Ca2+ activation of actomyosin ATPase and subsequent PCr degradation to maintain the ATP concentration prior to the freezing of the sample. The freezing procedure itself may also result in some PCr degradation. If the resting PCr coneewtration was greater than reported with traditional biochemical analyses, estimates of anaerobic ATP provision from PCr degradation would be underestimations. However, the validity of the NMR measurements has been questioned, as no quantitative calibration of chemical compunds in skeletal muscle with this technique presently exists. Calibration is attempted by assuming the area under the ATP peaks in the NMR spectra are equal to the chemically determined ATP content. If the NMW technique does not see all of the intracellular ATP, the estimated P e r concentration will be too high. Murphy et al. (1988) reported that the NMR technique was unable to observe all the existing ATP in ischemic liver tissue. The seriousness of this problem is evidenced by the fact that several investigators have reported resting PCr contents that are much higher (- 170 inmollkg dry muscle; Bun et al. 1976; Chance et d. 1982; Taylor et aB. 1986; Thon~senet al. 1989) than reported values for biochemically determined total Cr content ( 115- 140 mmol/kg dry muscle, Nevill et al. 1989; Ren et al. 1988; Spriet et d. 1989; Tesch et d. 1989). As a result of this calibration problem, several investigators report their NMR data only as a percentage of resting levels (Miller et al. 1988; Sinoway et al. 1989; Wilson et a%.1988). An equally serious problem with NMR spectra is the difficulty in determining the true baselines of the ATP and PCr peaks for subsequent integration of the areas under the peak. Multman and Sjoholm (1983a) have also argued that the degradation rate of P e r during the 3 -5 s it takes to obtain and freeze a biopsy sample would need to be as great or greater than the rates measured during maximal contractile activity ( 10 mmol - kg- d m s-I) for the NMR estimates of PCr concentration to be correct. This seems highly unlikely. Soder%undand Hultman (1986) examined the effects of delaying the freezing procedure itself on the resting PCr concentration in biopsy samples. If freezing was delayed by I min following the biopsy, PCr content increased from 72 to 85 mmollkg dm 16$0). Further delays for up to 6 rnin had no additional effect. The freezing procedure itself had no effect on resting PCr concentratioh. The authors concluded that the biopsy procedure resu%tsin only a small degradation of PCr, which was much less than suggested by NMR studies. Presumably this effect would be even smaller in exercised muscle where the PCr concentration was lower at the time of sampling.

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Stngk fiber phosgkscreatine and AT.. contents, rates of aatikizatiopa during intense activity, a d rates of recovery Recent investigations have separated single fibers and rneasured BCr and ATP contents either in pools of one fiber type or individual fibers sf ih h o w n fiber type. Tesch et d -(1989) reported P e r values of 82.7 _+ 11.2 and 73.1 $ 9.5 mrnollkg dm in pools of fast twitch (FT)and slow twitch (ST) fibers from the VL muscle of men. This difference was significant and consistent with an earlier report of a similar but nonsignificant trend reported by Essen (1978). Edstroran et al. (1982) also reported a lower resting PCr concentration in the psedominantly S% soleus muscle as compared with the VL in humans. The PCr concentration in FT and ST fibers decreased to

25.4 _+ 19.8 and 29.7 f 14.4 rnmol/kg dm, respectively, following 30 maximal isokinetic knee extensions (Tesch et al. 1989). The corresponding values after 1 min of recovery were 41.3 %2.6and 49.6 11.7 mmollkg dm. The data suggest that the PCr degradation rate is slightly higher in FT fibers during intense activity and that PCr may recover more quickly in ST fibers. Soderlund and Hultrnan ('1990) recently reported no difference in the resting ATP concentrations in single FT and ST fibers from VL muscles (25.9 3.6 and 25.2 _$ 4.8 mrnollkg dm). Electrical stimulation for 83 s (1-6 s on, 1.4 s off) at 20 Ha with an occluded circulation decreased the respective ATP contents to 15.9 6.2 and 16.1 f 3.8 mmollkg dm. Following 15 rnin sf recovery, ATP had fully recovered in the ST fibers (25.6 +_ 3.3 mmollkg dm) but was significantly below resting level in FT fibers (23.3 3.3 rnrnol/kg dm). It is interesting that the authors in these studies concentrated on the differences between the fiber types when the differences were nonexistent or very small, especially if compared with the differences reported in the fiber types of rat skeletal muscle (Spriet 1989, 1990). In fact, it is noteworthy that the human FT and ST fibers are remarkably similar in their high energy phosphate profiles. Very little work has appeared with respect to the rates of FT and ST recovery of PCr following different intensities of activity. It is important to h o w what effect the following factors have on the rate of PCr resynthesis: the type of contractile activity (isometric versus isotonic), the magnitude of the P e r decrease, the effect of a concomitant decrease in ATP concentration, and the effect of active versus passive recoveries. Complete and rapid recovery of PCr is essential in activities where repeated maximal contractions are required.

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Anaerobic energy from glycogenslysis- glycolysis The most significant source of anaerobic ATP during intense activities lasting longer than 10-28 s is from glycolysis. In 3 min, glycolysis provides exhausting exercise lasting 88% of the totd anaerobic ATP provided. While the energy yield of 3 mmol ATPIrnmsl g%ucosyIunit is low when compared with the aerobic energy yield, anaerobic glycolysis is quickly activated during intense activity? provides ATB at high rates, and has a much larger capacity than Per degradation. The predominant source of glucose moieties is from muscle glycogen as the rate that exogenous glucose can be taken up by muscle is severalfold lower than the flux through the pathway during maximal exercise (Katz et al. 1986). In addition, the accumulation of glucose 6-phosphate (G-6-P) is believed to inhibit glucose phosphorylatisn in the cell (Colowick 1943). Cilycol$is is also associated with the accumulation of lactate and hydrogen (H+) ions. Interestingly, the anaerobic glycolytic capacity during maximal contractile activity is reached well before the muscle glycogen store is depleted in well fed and previously rested subjects. Glycogenolytic-glycolytic regulation has received considerable attention in rn attempt to account for the several huwdrdfold increase in flux which occurs at the onset of maximal activity. The key regulatory or rate-limiting enzymes in this pathway are glycogen phosphorylase (PHOS) and phosphofmctokinase (PFK). Both are nonequilibrium enzymes and PHOS is also flux-generating as the Km of the enzyme for glycogen is 1-2 mM (Newsholme and Start 1973). It is not pssible in this paper to present a complete review

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of the regulation of these enzymes during maximd or nearmaximal muscuhr contractions. Instead, this section will briefly highlight areas of existing controversy and exciting new ideas that have appeared regarding the regulation of the flux-generating enzyme PHOS. The regulation of PFK in skeletal muscle will not be examined, as the literature in this area is extensive. Interested readers should consult the following references for discussions of PFK regulation during intense muscular contraction (Bobson et al. 1986; Spriet 1991; Uyeda 1979). Bhosphorybase reguhtion There are several potential mechanisms that may be responsible for the regulation of PHOS: substrate regulation, interconversion between less active and more active foms, allosteric regulation, bound and free f o m s of the enzyme, and glycogenglucose 1-phosphate (G-1-P) cycling. Substrate regulation For many years the two substrates of PHOS were not considered important in the regulation of the enzyme. In most situations glycogen is abundant in the muscle and it was believed that an adequate amount of inorganic phosphate (P,; dianion form (HP0,2-) is believed to be the active form) was present at all times in the cell. In vitro experiments have demonstrated that the K, of PHOS for glycogen is extremely low ( - 1-2 mM, Newsholme and Start 1973). The bulk of the literature on human data supports this finding, as glycogenolysis during shortterm, intense activity was unaltered following increases or decreases in the resting glycogen concentration (Boobis et al. 1983; Jacobs et al. 1981; Symons and Jacobs 1989). However, the human studies are confounded by extramuscular factors, although these should be minimal during intense exercise. Consequently, the relevance of the in vitro results for in vivo situations was questioned by Richter and Galbo (1986). They controlled for extramuscular factors by perfusing rat hindlimb muscles with media of identical composition and reported that an elevated glycogen content enhanced glycogenolysis during 1%min of intense electrical stimulation. However, the 15-min tetanic stimulation protocol used in their study included an early anaerobic phase when glycogenolysis is rapid and a longer aerobic phase when the rate of glycogen breakdown is low. Spriet et d.(1990) reported that increased and decreased resting glycogen concentration had no effect on glycogenolysis during the early anaerobic phase of tetmic stimulation. The results support the suggestion that in vivo PHOS activity is not regulated by the content of its substrate glycogen during shortterm tetanic stimulation. A series of studies from Dr. Eric Hultman's laboratory demonstrated the importance of Pi in the regulation of PHOS in human skeletal muscle, although it had been implied by Cori et al. (1956) many years earlier. Chasiotis et al. (1982) demonstrated that the fraction of PHOS in the more active a form was -20% at rest, although measured in vivo PHOS activity was low. In addition, infusion of epinephrine greatly increased the fraction of PHQS a , yet the rate of glycogenolysis remained low (Chasiotis et al. 1983a). These results were explained by the low concentration of Pi in resting muscle and demonstrated that transformation of PHOS from the less active b form to the a form is not synonymous with enhanced glycsgenolysis. During intense contractile activity, Pi rapidly accumulates in muscle at a rate which is approximately proportional to the rate of PCr degradation and permits rapid glycsgenolysis to occur.

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However, a more recent study from the same laboratory has questioned the high resting levels of PHOS a and has reported that 5 - 10% may be more realistic (Ren et al. 1988). A second study demonstrated that elevated Pi concentration is not the only factor required for glycogenolysis to proceed during intense activity. Ren and Multman (1989) stimulated human muscle for 10 s to increase the Pi concentration, then occluded the circulation for 1 min while the muscle was at rest to prevent the recovery of metabolites. Epinephrine was also infused and present in the blood trapped in the leg to ensure a high proportion of PHOS a. Despite high levels of Pi and a high PHOS a! fraction, glycogenolysis did not occur. In a third study epinephrine was infused during 10 s of stimulation at 15 Hz and 10 s at 50 Hz with the circulation occluded (Ren and Hultman 1990). The fraction of PHOS a was constant at 86 -92 % in both stimulation conditions, yet the glycogenolytic rate, force generation, and total anaerobic ATP provision were twofold higher during stimulation at 50 Hz. These experiments demonstrate that rapid glycogenolysis during intense muscular contractions is not only due to transformation of PHQS b to a! and increases in Pi concentration. Clearly, modulators associated with the rate of ATP utilization are also required to match the rate of glycogenolysis to the demand for ATP. Interconvertible forms It is generally believed that the transformation of PHOS b to cs during intense activity occurs through activation of PHOS b finase via Ca2+ liberated by contraction. It is known that epinephrine can also activate the transformation by increasing the concentration of cyclic AMP via an enzyme cascade. However, this mechanism does not appear important during intense contractions, as cyclic AMP concentration did not increase during an isometric contraction at 66% maximum voluntary contraction to fatigue (Chasiotis et al. 1983~).However, the role of epinephrine in the activation of glycogenolysis during intense muscular contractions has not been systematically studied in the human. There are suggestions that H+ may play a role in interfering with activation of PHOS in human skeletal muscle. Chasiotis et al. (19836) demonstrated that the transformation of PHOS b to a and the accumulation of cyclic AMP were depressed when muscles were acidotic. Spriet et al. (1989) maximally exercised subjects for 30 s on an isokinetic cycle ergometer three times with 4 min of rest between bouts. Estimations of muscle acidity and glycogenolysis during each bout demonstrated a relationship between increasing acidity and an inability to reactivate glycogenolysis with successive bouts. However, it is not possible to conclude whether it is the H+ or some other contraction-mediated event that interferes with the conversion of PHOS b to a. The initial conversion of PHOS from b to a during intense activity is reversed with sustained activity and PHOS is converted back to the b form (Chasiotis et al. 2982, 2983~).It is not known what causes the reconversion ~f PHOS back to the b form and whether the glycogenolysis that persists can be accounted for by the PHOS remaining in the a form or by an increased activity of the b form. Allosteric regulation Most of the work examining allosteric regulation sf this enzyme is associated with the b form. The a form appears to be active in the absence of allosteric regulators, although AMP decreases the K, of the enzyme for Pi. The b form requires dlosteric regulators to achieve significant activity; ATP,

CAW. J. PHYSIOL. BHARMACOL.

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G-6-P, and H+ are believed to be negative modulators and ADP, AMP, IMP, and Pi have been reported to positively modulate the enzyme (Aragon et al. 1980; Chasiotis ea al. 1982; Newsholme and Leech 1983). A considerable amount of work is needed to quantify the actual activiQ of PHOS b throughout a bout of intense activity and to determine the in vkvo significance of these putative modulators.

Other regda~orymechanisms There have been suggestions that PHOS is bound to a glycogen -protein -~rcoplasmicreticulum complex in viva (Entman et al. 1980). With sustained intense activity, PHOS may become unbound from this complex and reduce the ability of Ca2" to catalyze PHOS & to a transformation. A more detailed examination sf the potential significance of enzyme binding in glycolytic control is presented in another paper from this symposium. Newsholme and Leech (1983) have also proposed that a cycling between glycogen and G-1-P could increase the sensitivity of the system to the energy demands of the cell. Summary This paper has examined directly obtained infomation regarding the ability of human skeletal muscle to provide ATP anaerobically during short-term, intense contractions. The capacity of muscle to provide anaerobic ATP is -370 mm~l/kgdm during dynamic exercise lasting 3 min. Anaerobic glycolysis provided 80% and Pglr degradation contributed 16% of the total. When the blood Wow to the exercising muscle is reduced or totally occluded, the anaerobic ATP capacity decreases to -310 mmol/kg dm. This reduction is due to a lower glycolytic capacity associated with an inability to remove lactate from the muscles. The only study that compared direct and indirect estimations of the anaerobic capacity suggests that O2 deficit measured at the mouth is a good predictor of the anaerobic capacity in a single muscle group of man and that O2 debt is not a good predictor. Maximal rates s f directly measured anaerobic ATP provision B'rsm PCr degradation and glycolysis during intense muscular activity are each 9 - 10 mmol kg-' dm . s-l* Evidence suggests that both PCr and glycolysis are activated instantanesuslv with the onset of maximal activity. Surprisingly, sprint training does little to increase the resting PCr content and produces, in some instances, 18-28% increases in glycolytic ATP provision and 6 - 16% increases in total anaerobic ATP provision. Several issues related to the capacity of the PCr and glyccsgenolytic-glycolytic systems to provide ATP during intense activity are unresolved and require more investigation. Much effort is now being directed to understanding the in situ or in viva regulation of the regulatory and flux-generating enzyme, phosphorylase, in human skeIetd muscle.

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