Biochemistry of exercise

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1 Biochemistry of exercise Regulation in Metabolism Group Colloquium Organized by E. A. Newsholme (Oxford) and P. H. Sugden (London) and Edited by P. H. Sugden, 637th Meeting held at the University of Birmingham, 1820 December I990 Energy metabolism and fatigue during intense muscle contraction Eric Hultman, Paul L. Greenhaff,* JanMing Rent and Karin Soderlund Department of Clinical Chemistry II, Huddinge University Hospital, Karolinska Institutet, S I4 I 86 Huddinge, Sweden Introduction Intense exercise results in a marked acceleration of the ATP degrading processes, including increases in the activities of myosin, Ca2+ and Na+, K+ ATPases. A continuation of exercise at the required intensity necessitates a corresponding increase in the processes which rephosphorylate the ADP formed. Rapid anaerobic ADP rephosphorylation occurs at the beginning of exercise when the O2 availability in muscle is low; however, even when O2 transport to the muscle is maximal, the rate of oxidative ADP phosphorylation is limited. Evidence suggests that 8090% of the ATP resynthesized during near maximal contraction is derived from anaerobic processes. Indirect estimates of the anaerobic capacity of skeletal muscle can be obtained with the use of the 02deficit technique. The method has been used in several studies [ 141. During submaximal exercise a linear relationship exists between O2 uptake and exercise intensity. This relationship can be used to predict the energy demand of power outputs which utilize ATP at a rate above that achievable by the maximal O2 uptake of the exercising muscle. The O2 deficit during intense exercise can then be estimated as the difference between energy demand and the aerobic energy production. In a recent study by Bangsbo et al. [S], the contribution made by anaerobic and aerobic sources to energy production, calculated from the O2 deficit, was found to be 80%/20% in the initial 30 s, 45%/55% from 60 to 90 s and 30%/70% from 120 to 192 s. The work intensity in this study Abbreviation used: PCr, phosphocreatine. *School of Sport and Exercise Science, University of Birmingham, Birmingham B 15 2TT, U.K. TWashington University School of Medicine, Box 81 13, St Louis, MO , USA. corresponded to 130% of V,,,,, and was maintained for a duration of 192 s. Muscle tissue samples were also obtained during this study and, from the accumulation of muscle metabolites, the contribution made by anaerobic metabolism to energy production was calculated. There was good agreement between the two methods in the estimated anaerobic energy production. The exercise studies discussed below deal with muscle contractions of an even higher intensity (near maximal intensities with durations of s). The contribution of oxidative metabolism to ATP production in this situation will probably be much lower than the 20% observed in the study by Bangsbo et al. [ 51. Anaerobic energy provision In the 1960s, Margaria et al. [6, 71 reported that supramaximal exercise of 1015 s duration could be performed without significant elevation of blood lactate above resting conditions. The total energy provision during this short period of exercise was attributed to the splitting of the intramuscular stores of ATP and phosphocreatine (Per). It was postulated that only when the muscle store of PCr became depleted was glycogenolysis activated to provide continued ATP supply through glycolysis. However, it was shown in an earlier study by Pernow & Wahren [8], that 1 min after the termination of 5 s of intense contraction, the lactate content of both arterial and deep venous blood was increased. The musclebiopsy technique introduced by Jonas Bergstrom in 1962 [9] made it possible to investigate in some detail the metabolic changes occurring in skeletal muscle during exercise. With the use of this technique, it was shown that 6.6 s of isometric contraction decreased the PCr content by 20% and increased lactate content by 15 mmol kg ' 347

2 Biochemical Society Transactions 348 of dry muscle [lo]. The same technique was used later by a series of workers and confirmed the above finding (Table 1). Boobis et al. [ 131 reported a marked increase in muscle lactate concentration after 6 s of cycling at a high power output. The PCr concentration in the postexercise biopsy sample was 35% lower than the value measured at rest. Jacobs et al. [ 14, 191 systematically examined the question whether anaerobic glycolysis started before the depletion of the muscle PCr store. They showed a lactate accumulation of 51 mmol kg' of dry muscle and a PCr decrease of 60% after 30 s of maximal dynamic exercise. Furthermore, a high accumulation of lactate had already occurred after 10 s of exercise. Similar results were reported by Jones et al. [15] after 10 s of isokinetic exercise (Table 1). Another way to study the metabolism of contracting muscle is to stimulate the muscle electrically. The muscle blood flow to the quadriceps femoris muscle can be occluded, inhibiting transport to and from the muscle compartment. In tandem with the musclebiopsy technique, this provides a versatile method to investigate the utilization of substrates and accumulation of products during muscle contractions of varying durations and intensities. A nearmaximal contraction intensity can be Table I Rates of anaerobic ATP provision from PCr degradation and glycolysis during intense contraction Type of exercise ATP production (mmol kg' s') from: Duration (4 PCr glycolysis Ref. Intermittent electrical stimulation. 50 Hz occluded circulation 0 I [I Intermittent electrical stimulation. 50 Hz occluded circulation [I I 2. I 3.7 Isometric contraction Cycling Cycling (male) Cycling (female) I I lsokinetic cycling 60 revlmin I40 revjmin I I ~ Cycling [I61 lsokinetic cycling I ~ 7 1 Running I [I81 Cycling Running I.3 I ~ I POI Volume 19

3 Biochemistry of Exercise achieved using a stimulation frequency of 50 Hz. At this frequency, a stimulation for 1.28 s was enough to produce a PCr degradation of 11 mmol kg of dry muscle and a lactate accumulation of 2 mmol kg ' of dry muscle [ 113. When the contraction time was increased to 2.56 s, the PCr content fell further and the lactate accumulation amounted to 8.5 mmol kg of dry muscle. During 5 s of stimulation of the quadriceps muscle group at a frequency of 50 Hz with muscle blood flow occluded, the PCr utilization rate was 5.3 mmol sl kgl. With continued stimulation, the rate decreased progressively averaging 2.2 mmol sl kg' between the 10th and 20th second of contraction and 0.2 mmol s' kg' between the 20th and the 30th second of contraction. The anaerobic energy provision from glycolysis was 4.5 mmol of ATP sl kgl during the initial 20 s, but decreased thereafter to 2.1 between the 20th and 30th second of contraction. Force generation declined during the first 20 s of contraction to 80% of the initial value and decreased further to about 60% of the initial value after 30 s of contraction [121. A series of similar studies has been performed involving different types of maximal dynamic exercise (Table 1). In some of these studies, biopsy samples were obtained after 610 s of exercise and then again after 30 s [13, 151. The PCr degradation rate was very high initially, decreasing the PCr store in the muscle by more than 50% during the first 10 s of contraction. The rate of ATP provision from glycolysis showed a similar pattern of change with a high initial rate during the first 10 s of contraction, decreasing significantly as exercise was continued (Table 1). It is obvious that the rate of ATP formation from both PCr degradation and glycolysis decreases with the continuation of the intense contraction. Whether this decrease is caused directly by the observed reduction in muscle force generation, i.e. a decreased demand for ATP resynthesis or by an insufficient ATP resynthesis rate, resulting eventually in decreased capacity to generate force, cannot be determined from these studies. It is interesting to note, however, that the glycogen store in the whole muscle sample is still sufficiently abundant to fuel ATP formation, while the PCr store is practically depleted. Energy metabolism in type I and type II fibres The quadriceps femoris muscle of man is composed of two principal fibre types. These have been characterized as type I (slow contracting, highly oxidative and fatigue resistant) and type II (fast contracting, highly glycolytic and rapidly fatigued) fibres. The two fibre types have different maximal rates of ATP utilization and also different capacities to produce initial and sustained power output (for references see [21]). Most of the studies investigating the functional and biochemical differences between the two fibre types have been performed using rat soleus (composed predominantly of type I fibres) and gastrocnemius (composed predominantly of type 11 fibres) muscle. Such studies have demonstrated that the comparatively higher initial isometric force generating capacity of predominantly fastcontracting muscle is accompanied by a corresponding high rate of ATP utilization and glycolysis [ In a study by Faulkner et al. [26], involving the stimulation of bundles of human skeletal muscle fibres, it was estimated that the peak power output of type I1 fibres could be 34 times higher than that of type I fibres. The authors also presented evidence demonstrating that the initial power output of the fasttwitch motor units could not be sustained for longer than a few seconds after the initiation of contraction, but was well maintained in type I motor units. The calculated timerelated forcepower in Faulkner's study was very similar to the change in force measured during electrical stimulation of the knee extensors in man [ 12,271. In an attempt to relate the energy metabolism of individual type I or type I1 fibres to the decline in whole muscle force during stimulation, musclebiopsy samples were obtained before and after 10 and 20 s of intermittent electrical stimulation (1.6 s contraction, 1.6 s rest, at a frequency of 50 Hz) to the quadriceps muscle group with open circulation. After freezedrying, fragments of individual muscle fibres (n =about 50) were dissected free from each biopsy sample. After weighing and characterization, fibres of each type were analys'ed for singlefibre PCr and ATP concentrations. The PCr and ATP concentrations after 10 and 20 s of stimulation are shown in Fig. l(a). During the first 10 s of stimulation, the rate of PCr degradation in type I1 fibres averaged 5.3 mmol sl kg' of dry muscle. The corresponding rate in type I fibres was 3.3 mol s' kgi of dry muscle. During the period of 1020 s stimulation, the rate of PCr degradation in type I1 decreased to 2.1 mmol sl kg' of dry muscle and in type I fibres to 2.8 mmol sl kgi of dry muscle. At the end of the stimulation period the PCr store in type 11 fibres was nearly totally depleted. It is therefore plausible to suggest that continued contraction beyond 20 s is limited to the use of PCr at a rate 349

4 Biochemical Society Transactions Fig. I PCr and ATP concentrations after 10 s and 20 s of stimulation 350 (a) Whole muscle force (x) and singlefibre PCr (A. A) and ATP (0,.) concentrations at rest and after 10 and 20 s of intermittent electrical stimulation at 50 Hz. Open symbols denote type I fibres; closed symbols denote type II fibres. (b) Glycogenolytic rates in type I and type II fibres during the 20 s stimulation period. The open bar denotes type I fibres; the closed bar denotes type I1 fibres. I E M Y P) 0 I 0. Y 0 0 I0 20 Y Stimulation time (5) I T corresponding to that eventually formed by mitochondrial ADP phosphorylation. The glycogen content of single fibres obtained from the same musclebiopsy samples was also determined and the glycogenolytic rates of the two fibre types were estimated (Fig. lb). The very high glycogenolytic rate of 6.3 mmol sl kgi of dry muscle in type I1 fibres contrasted with the negligible rate of 0.6 mmol s' kgi of dry muscle in type I fibres. This finding is in agreement with animal studies demonstrating a very high capability for anaerobic energy supply via glycogenolysis in type IIa and IIb skeletal muscle fibres [28]. Recent histochemical studies of human skeletal muscle, obtained after repeated bouts of shortterm maximal exercise (about 200% Vo,,,,x,), suggest that the glycogen degradation rate is higher in type I1 fibres compared with type I fibres during this type of exercise [29]. The measurements made, however, gave only a semiquantitative estimation of the rate of glycogen degradation. In an attempt to study the glycogenolytic mechanism of the two fibre types further, we stimulated both legs of five subjects intermittently at a frequency of 50 Hz for a total stimulation time of 30 s, with blood flow intact. One leg was stimulated without simultaneous adrenaline infusion and the remaining leg was stimulated with continuous infusion of adrenalin (0.14 pg of adrenalin minl kg' body wt.). A further study using the same stimulation protocol was undertaken, but on this occasion Fig. 2 The glycogenolytic rate in type II fibres is unchanged by adrenaline infusion and only slightly increased by occlusion of blood flow during the contraction Glycogenolytic rates in type I (0) and type II (.) fibres during 30 s of intermittent electrical stimulation at 50 Hz. Two experiments were performed, one without and with adrenaline infusion in the same subjects and the other with circulation (circ.) occluded in a separate group of subjects. 10 T Open circ. Open circ. + Occluded circ. adrenaline blood flow to the leg was occluded. The occlusion was initiated 30 s before the start of the stimulation and continued during the stimulation period. The results are shown in Fig. 2. The glycogenolytic rate in type I1 fibres was unchanged by adrenaline infusion and was only marginally increased by occlusion of the blood flow during the contraction. This finding could be interpreted to mean that the rate of glycogenolysis is already close to maximum in type Volume 19

5 Biochemistry of Exercise II fibres during stimulation when circulation is intact, and thus it is not possible to increase the rate markedly by adrenaline infusion or ischaemia. This suggestion is supported by the finding that the rate of glycogenolysis recorded in these fibres was close to the apparent V,,, of phosphorylase measured in type I1 fibres [ 301. In type I fibres, however, bothadrenaline infusion and ischaemia increased the glycogenolytic rate above the extremely low rate recorded during stimulation with circulation intact and without adrenaline infusion (0.15 pg min' kgi). The glycogenolytic rate during contraction in type I fibres was increased 6fold by adrenaline infusion and 11fold by ischaemia. Even during ischaemic stimulation, however, the rate was still only half of that observed in type I1 fibres, but is in good agreement with the estimated maximal activity of the phosphorylase in type I human fibres [ 301. It is generally accepted that the primary determinant of the glycogenolytic rate during contraction is the degree of transformation of inactive phosphorylase b to the active a form. However, it has also been demonstrated that the activity of phosphorylase a, and thereby the rate of glycogenolysis, is also dependent upon the availability of inorganic phosphate (Pi) and AMP [ Additionally, a high accumulation of IMP will result in the activation of phosphorylase b [ 341. The most potent allosteric activator of phosphorylase a is AMP [31], the concentration of which is dependent on the rate of ADP formation in relation to its rate of rephosphorylation. It has been recently demonstrated [33], that the glycogenolytic rate of skeletal muscle is directly related to the intensity of the contraction and thus to the rate of ATP turnover. In this particular study, the muscle was stimulated electrically with frequencies from 15 Hz to 50 Hz and the mole fraction of phosphorylase a was kept at 8590% of total by continuous adrenaline infusion. The glycogenolytic rate varied from 0.5 to 3.5 mmol kg' min'. It was suggested that as the availability of free AMP probably increased with the increased ATP turnover rate, the free AMP concentration of the muscle was controlling the glycogenolytic activity of phosphorylase a. During the present series of experiments it is probable that the transformation of phosphorylase b to a was complete in type I1 fibres, as no further increase in glycogenolytic rate was observed in these fibres after adrenaline infusion. As fasttwitch muscles are known to have a high rate of ATP turnover [21, 221 and a high capacity to generate AMP [35], it is also probable that the rapid rate of glyco genolysis recorded in these fibres is attributable to a free AMPinduced activation of phosphorylase a. This is supported by the findings that the decline in ATP in type 11 fibres was close to 5 mmol kg' when circulation was open and was in excess of 10 mmol kgi when circulation was occluded. The decrease in ATP during intense contraction has been shown to result in a stoichiometric rise in IMP formed from the accumulation of free AMP. The lower ATP turnover rate of type I fibres, together with the oxidative resynthesis of formed ADP, explains the observed low rate of glycogenolysis in these fibres with open circulation. It is logical also that the increased rate of glycogenolysis observed in these fibres following adrenaline infusion was attributable to an increased degree of phosphorylase transformation. When the blood flow was occluded, the oxidative resynthesis of ADP was inhibited resulting in an increase in ADP availability as substrate for adenylate kinase. It is postulated that the resulting increased formation of free AMP was of a sufficient quantity to activate phosphorylase a. The marked decrease in ATP in type I fibres during occlusion (about 6 mmol kg') supports this suggestion. Furthermore, the recently reported [36] increase in calculated free AMP in ischaemic slowtwitch rat muscle was of a sufficient magnitude (about 5 pmol 1 of intracellular water) to support the above hypothesis. In accordance, it is also probable that the slightly higher rate of glycogenolysis observed in type I1 fibres during occlusion can be attributed to the activation of phosphorylase a in type IIa fibres via an ischaemiainduced increase in the availability of free AMP. A marked increase in free AMP available has been reported in contracting oxidative fasttwitch skeletal muscle after the onset of occlusion [35]. The rapid decrease in PCr in both fibre types suggests that the availability of Pi is not a limitation to glycogen phosphorylation. Possible relationship between energy metabolism and fatigue Force generation (measured as power output during cycling or as contraction force during electrical stimulation) showed a similar pattern of change during the 30 s work periods in the studies outlined in Table 1. In all studies, the force had decreased by about 40% after 30 s of work, but the shape of the force decay curve varied with the type of exercise performed. The force generation during the initial 20 s of electrical stimulation in the present series of studies is shown in Fig. 1. Parallel with the decreasing force generation is a decrease in 35 I

6 Biochemical Society Transactions 352 anaerobic ATP provision both from PCr degradation and from glycolysis (Table 1). The fastest drop in energy provision is from the PCr store, which during the first second of contraction apparently compensates for the short delay in glycolytic ATP production. This is especially the case in type 11 fibres, where after 10 s of contraction 70% of the PCr store is utilized and after 20 s the store is near depletion. At this point it would appear that glycogenolysisglycolysis has already reached maximal activity in type I1 fibres, and thus cannot increase further in spite of an abundant availability of glycogen. There is, therefore, apparently no mechanism by which the type I1 fibres can increase their glycolytic ATP resynthesis rate to compensate for the lack of available PCr. Taken together, this means that if the force had been kept constant an imbalance between ATP utilization and ATP resynthesis would have occurred. This imbalance could consequently be the reason for the decreased force production, i.e. a fatigue mechanism in this type of intense exercise. Conclusion The initial high force generation during nearmaximal contraction force seems to rely mainly on type I1 fibre contraction, utilizing both PCr degradation and glycolysis for ATP resynthesis. The rapid utilization of PCr limits the ATP provision from this store after 10 s of stimulation. The glycogenolysisglycolysis pathway is working at a maximum rate already within the first second and cannot be increased to compensate for the falling rate of ATP resynthesis from PCr. Type I fibres show a very low glycogenolytic rate, but also in these fibres the PCr store is rapidly utilized. The large difference in glycogenolysisglycolysis rate between the two fibre types depends both on differences in total phosphorylase activity and on differences in ATP turnover rates, which probably determine the glycogenolytic activity of phosphorylase. This regulation is suggested to be mediated via the sarcoplasmic concentration of free AMP. The free AMP concentration is itself determined by the rate of ADP formation and rephosphorylation and also by the activity of AMP deaminase, which is known to be high in type I1 fibres and activated by acidosis. The increasing acidosis during intense contraction could thus partly explain the observed fall in the glycogenolytic rate of these fibres resulting from a decreased AMP activation of phosphorylase a. 1. Hermansen, L. (1969) Med. Sci. Sports 1, Pate, R. R., Goodyear, L., Dover, V., Dorociak, J. & McDaniel, J. (1983) Med. Sci. Sports Exerc. 15, Astrand, P.O., Hultman, E., JuhlinDannfelt, A. & Reynolds, G. (1986) J. Appl. Physiol. 61, Medbo, J. I., Mohn, A.C., Tabata, I., Bahr, R., Vaage, 0. & Sejersted, 0. M. (1988) J. Appl. Physiol. 64, Bangsbo, J., Gollnick, P. D., Graham, T. E., Juel, C., Kiens, B., Minuzo, M. & Saltin, B. (1990) J. Physiol. 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7 Biochemistry of Exercise 26. Faulkner, J. A., Claflin, D. R. & McCully, K. K. (1986) in Human Power Output (Jones, N. L., McCartney, N. & McComas, A. J., eds.), pp. 8191, Human Kinetic Publ., Champaign, IL 27. Hultman, E. & Spriet, L. L. (1986) J. Physiol. (London) 374, Baldwin, K. M., Winder, W. W., Tejung, R. L. & Holloszy, J. 0. (1973) Am. J. Physiol. 225, Fridin, J., Seger, J. & Ekblom, B. (1989) Acta Physiol. Scand. 135, Harris, R. C., Essin, B. & Hultman, E. (1976) Scand. J. Clin. Lab. Invest. 36, Lowry, 0. H., Schultz, D. W. & Passonneau, J. V. (1964) J. Biol. Chem. 239, Chasiotis, D. (1983) Acta Physiol. Scand. Suppl. 518, Ren, J. M. & Hultman, E. (1990) J. Appl. Physiol. 69, Aragon, J. J., Tornheim, K. & Lowenstein, J. M. (1980) FEBS Lett. 117, K56K Tullson, P. C. & Terjung, R. L. (1990) Int. J. Sports Med. 11, S47S Tullson, P. C., Whitlock, D. M. & Terjung, R. L. (1990) Am. J. Physiol. 258, C258C265 Received 19 November Control of energetic processes in contracting human skeletal muscle Kent Sahlin Department of Clinical Physiology, Karolinska Institute, Huddinge University Hospital, S I41 86 Huddinge, Sweden Introduction Skeletal muscle is unique in its ability to switch rapidly from a low to a highenergy turnover, and this necessitates an intricate system for control of the flux through the energetic pathways. The ultimate process for ATP formation in aerobic cells is the oxidation of different substrates with O2 being the final electron acceptor. For a limited period of time ATP can also be produced through nonoxygen requiring processes [i.e. formation of lactate and breakdown of phosphocreatine (PCr)]. The maximal rate at which ATP can be formed by the anaerobic processes is at least two times higher than ATP formation by oxidative phosphorylation. However, the amount of energy that can be formed is restricted by the cellular content of PCr and by feedback inhibition of glycolysis by H+, which is formed stoichiometrically with lactate. Formation of ATP through the anaerobic processes is therefore of great importance during short bursts of highintensity exercise or during ischaemic conditions, but negligible in terms of energy equivalents during sustained exercise. The present paper will discuss some general aspects of how the energetic processes are controlled in skeletal muscle during exercise. Special emphasis will be placed on the role of adenine nucleotides and 0, availability. Abbreviations used: PCr, phosphocreatine; Cr, creatine; CK, creatine kinase; AK, adenylate kinase; PFK, phosphofructokinase; IMP, inosine monophosphate. A critical note on the calculation of ADP from the creatine kinase equilibrium The concentrations of the adenine nucleotides have a major influence on the energetic processes, the control being exerted both through allosteric and substrate/product effects. Evidence has been presented [ 1, 21 that changes in the PCr/creatine (Cr) ratio in skeletal muscle are not associated with similar changes in the ATP/ADP ratio and it was suggested that the adenine nucleotides are functionally compartmentalized in the cell [l, 21. From these studies and others, it was concluded that a large part (> 90%) of the cellular content of ADP and AMP is bound to proteins or otherwise sequestered in the cell [3, 41. The major binding sites for ADP are considered to be actin and myosin, but since brain tissue and liver exhibit a similar degree of functional compartmentation, it was suggested that part of the inactive ADP was located within the mitochondria [4]. Extraction of muscle tissue with ethanol/citrate and perchloric acid has shown that the proportion of ADP bound to proteins is about 50% of the total cellular ADP content [S]. Postmortem changes in the adenine nucleotides suggest a similar amount of ADP bound to proteins [6]. The fractions of ADP and AMP which are not bound or otherwise sequestered (i.e. ADP,, and AMP,,,) are the metabolically active forms and are conventionally calculated from the massaction ratios of the creatine kinase (CK) and the adenylate kinase (AK) reactions: