glycerol-i-phosphate, lactate and malate contents were lower after both work and

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1 J. Physiol. (1978), 281, pp With 5 text-ftigure8 Printed in Great Britain REGULATION OF GLYCOLYSIS IN INTERMITTENT EXERCISE IN MAN BY BIRGITTA ESSRN AND LENNART KAIJSER From the Department of Clinical Physiology, Karolinska Hospital, S Stockholm, Sweden (Received 26 January 1978) SUMMARY 1. Seven healthy male volunteers performed intermittent exercise (15 see work - 15 see rest) at a high work load for 60 min and six subjects performed continuous exercise at an equally high load to exhaustion, which occurred after 4-6 min. 2. Muscle biopsies were obtained from the lateral portion of the quadriceps muscle before intermittent exercise and after the end of a work period and the end of the subsequent rest period at 5, 15, 30 and 60 min of exercise, as well as before, immediately after and about 15, 30, 60 and 180 see after continuous exercise. 3. The reduction in glycogen content was smaller and glucose-6-phosphate, glycerol-i-phosphate, lactate and malate contents were lower after both work and rest periods in intermittent compared with continuous exercise, indicating a lower rate of glycolysis. 4. ATP and CP levels had decreased at the end of work periods in intermittent exercise but increased to slightly below basal in the subsequent rest periods. A still larger decrease in ATP and OP levels was found after continuous exercise to exhaustion and a progressive increase occurred over the 3 min of recovery. 5. In each rest period during intermittent exercise citrate levels increased to reach above basal. They increased also in the recovery phase after continuous exercise, although more slowly. 6. The findings support the assumption that ATP, COP and citrate act as regulatory factors of glycolysis in human muscle by retarding certain rate limiting steps. The increase in G-6-P/F-1-6-P2 ratio in rest periods of intermittent intense exercise and in the recovery phase of continuous intense exercise suggests that glycolysis is retarded at the phosphofructokinase reaction. 7. The factors mentioned may therefore contribute to the relative increase in lipid utilization during intense intermittent compared to continuous exercise. INTRODUCTION Intense physical exercise is accompanied by rapid glycogen depletion and lactate accumulation in the working muscle, indicating a high rate of glycolysis (Saltin & Karlsson, 1971; Karlsson, Diamant & Saltin, 1971). However, if work of high intensity is performed as intermittent exercise with short work periods (5-20 see) interrupted by short (5-20 see) rest periods, the decrease in glycogen is slower and the lactate accumulation smaller (Saltin & Ess6n, 1971; Edgerton, Essen, Saltin & Simpson, 1975). In addition the respiratory exchange ratio is relatively low, suggest-

2 500 B. ESSEN AND L. KAIJSER ing a substantial contribution of lipids to oxidative metabolism (Christensen, Hedman & Saltin, 1960). Further studies have shown that the relative contribution of lipids and carbohydrates to oxidative metabolism of the leg muscle is the same during 1 hr of intermittent (15 see work - 15 see rest) exercise at a high work load (300 W) as during 1 hr of continuous exercise at half that load (Essen, Hagenfeldt & Kaijser, 1977). Thus, if performed intermittently, work at a high load can be maintained with a lower rate of glycolysis than work at the same load performed continuously. This suggests that in the rest periods of intermittent exercise, regulatory factors are brought into play, retarding the rate of glycolysis and increasing the contribution of lipids to oxidative metabolism. Several feedback mechanisms have been proposed for the regulation of glycolysis and they might affect the relation between lipid and carbohydrate utilization. In the isolated perfused rat heart, increased oxidation of fatty acids and ketone bodies is associated with decreased carbohydrate utilization, produced by a retardation of glycolysis at the phosphofructokinase step, this being mediated by an increased citrate level (Newsholme, Randle & Manchester, 1962; Garland, Randle & Newsholme, 1963; Parmeggiani & Bowman, 1963). In contrast to heart muscle, most studies with skeletal muscle preparations have failed to show a decreased carbohydrate utilization and increased citrate levels when fatty acid availability is increased (Beatty & Bocek, 1971; Berger, Hagg, Goodman & Ruderman, 1976). However, in well oxygenated 'red' skeletal muscle the citrate level was found to increase and carbohydrate utilization to decrease upon augmented availability of fatty acids (Rennie & Holloszy, 1977), suggesting that citrate may have a similar effect in skeletal as in heart muscle. The aim of the present study was to examine possible regulatory factors of glycolysis in working human skeletal muscle which produce the altered relationship between carbohydrate and lipid utilization in intermittent exercise. This was done by studying fluctuations in levels of metabolites, which might be of importance in the regulation of glycolysis, between rest and work periods during intermittent exercise and, for comparison, in the rest period after continuous exercise to exhaustion at the same high work load. METHODS Thirteen healthy male volunteers participated in the study. Mean values and ranges were for: age 23 years (20-40), height 180 cm ( ), weight 74 kg (65-87) and maximal oxygen uptake /min ( ). All subjects took part in recreational sports but none at a competitive level. All subjects were informed about the experimental procedures and possible risks involved before giving their voluntary consent to participate. The maximal oxygen uptake (V02,OX) of each subject was determined during bicycle exercise in a preparatory test. Seven subjects were then studied during intermittent exercise (15 see work - 15 see rest) at an average work load of 284 W (range ) for 60 min, while the remaining six subjects performed continuous heavy exercise to exhaustion (4-6 min duration) at a work load of 280 W (range ). The loads in both intermittent and continuous exercise were selected so that the aerobic demand in performing it as continuous work would equal the maximal oxygen uptake of the subject. Exercise was performed on a Siemens-Elema bicycle ergometer at a pedalling rate of 60 rev/min. Heart rate was determined from the e.c.g. recorded by a Siemens-Elema Mingograf at 5 min intervals. During intermittent exercise oxygen uptake was determined at 15, 30 and 55 min. Muscle biopsies were taken from the vastus lateralis of m. quadriceps femoris by a needle-

3 REGULATION OF GLYCOLYSIS IN EXERCISE biopsy technique (Bergstrom, 1962). Two or three cutaneous incisions were made in each leg and one or two muscle samples were obtained from each incision. In the intermittent exercise experiments muscle biopsies were obtained before exercise (basal), within 5 see after the end of a work period and at end of the subsequent rest period at approximately 5, 15, 30 and 60 min of exercise. In the continuous exercise experiments muscle biopsies were obtained before exercise, at exhaustion and at about 15, 30, 60 and 180 see after the end of work. On some occasions it was not possible to obtain the biopsy at exactly the intended point of time or the sample was not large enough to permit all analyses which resulted in some missing values during both intermittent and continuous exercise. In recovery after continuous exercise it was not crucial that biopsies were obtained at the beforehand determined intervals. The individual values are entered into the figures at the actual times at which the biopsies were taken. The muscle samples were frozen immediately in liquid nitrogen and stored at -800C until analysed. Gas analyses Expired air was collected in Douglas bags and analysed by the Haldane technique. Analyses of muscle samples The muscle samples were weighed in a cryostat at -20 'C. In order to estimate water content the samples were freeze-dried overnight and weighed again at 21 0C in 35 % humidity. Under these conditions, water absorption by the muscle sample was assumed to be a few percent of its weight and was not corrected for. The samples were dissected free of blood and connective tissue as carefully as possible under a microscope. 1-3 mg of dry tissue was extracted with Id. 1-5 m-ice-cold perchloric acid. After 30 min in ice water, #1. 2 M-potassium bicarbonate was added to neutralize the samples and the precipitated potassium perchlorate was removed by centrifugation in the cold. Aliquots were taken from the supernatant for analyses of adenosine triphosphate (ATP), creatine phosphate (CP), glucose-6-phosphate (G-6-P), fructose-1-6- diphosphate (F-I1-6-P2), lactate, glycerol- 1-phosphate (Gl-1-P), malate and citrate. Glycogen was analysed as glucose on the precipitate following hydrolysis with 1 M-hydrochloric acid for 2 hr at 100 0C. All analyses were determined fluorimetrically by enzymatic methods, as described by Lowry & Passonneau (1973). Analyses of muscle samples from the intermittent and continuous experiments were performed on different occasions, which may explain slight differences in absolute values. Muscle contents of substrates and metabolites are given in m-mole/kg dry wt. Skshtisc Standard statistical methods and paired t test have been used in the analyses of data. Results in the text are given as mean values ± s.e. of mean unless otherwise stated. Individual data are given in the Figures. RESULTS Oxygen uptake, respiratory exchange ratio (R) and heart rate Oxygen uptake during intermittent exercise corresponded to 55-65% of Vo2 max and was after 15 min /min (range ), 30 min /min ( ) and 55 mmi /min ( ). The R value was 0-88 ( ) during the whole period of intermittent exercise, which suggests that 60% of oxidation was covered by carbohydrates and 40 % by lipids. Heart rate was 138 beats/min ( ) after 15 min and had increased to 159 beats/min ( ) at the end of exercise. Heart rate at the end of continuous heavy exercise was 180 beats/min ( ). Adenosine triphosphate (ATP) and creatine phosphate (CP) (Fig. 1) Intermittent exercise. Basal ATP content was m-mole/kg dry weight and basal CP content m-mole/kg dry wt. At the end of the work periods the 501

4 502 B. ESSJ9N AND L. KAIJSER ATP level was on average 10% (P < 0 05) and the CP level 45% (P < 0 01) below basal. At the end of a subsequent rest period both ATP and CP levels had been partially restituted, but ATP was still about 5% (P < 0.05) and CIP 25% (P < 0 001) below basal. The increase, which occurred with each rest period, was significant for ATP at 15 and 60 min and for CP on all occasions (P < 0.05). Adenosine triphosphate Adenosine triphosphate o~15-1 E 10 o=end of work period 10 E =end of rest period * * * * period t. + I ' Basal Basal 4-6 min Creatine phosphate 100 * 100 * Creatine phosphate :5Q ~ Ef 1 Work period Basal Basal 4-6 min Intermittent exercise (min) (15 sec work-1 5 sec rest) Continuous exercise Fig. 1. Adenosine triphosphate and creatine phosphate concentration before exercise (basal) and at the end of a work bout and the subsequent rest period after 5, 15, 30 and 60 min of intermittent exercise and before and at intervals in recovery after continuous exercise to exhaustion. In one subject biopsies were obtained only immediately and 3 min after continuous work and since the different variables did not recover linearly these points were not connected with a line. d.w., dry weight. Continuous exercise. Basal ATP content was m-mole/kg dry wt. and basal CP m-mole/kg dry wt. At the end of work the ATP level was 35% and the CP level '60% below basal (P < ). A gradual recovery occurred for both ATP and OP and sec after work the ATP level was 8% (P < 0-01) below basal, while the CIP level was 45% below (P < 0-05). After 3 min of recovery, ATP and CP levels were no longer significantly lower than basal. Glycerol-i-phosphate (Gl-1-P) and lactate (Fig. 2) Intermittent exercise. Basal lactate content was m-mole/kg dry wt. and basal Gl-1-P content m-mole/kg dry wt. At the end of the work period after 5 min an increase had occurred in lactate, to m-mole/kg dry wt

5 REGULATION OF CL YCOL YSIS IN EXERCISE 503 (P < 0.001), and in Gl-I-P to m-mole/kg dry wt. (P < 0-01). Thereafter both lactate and Gl-1-P contents decreased progressively with the duration of exercise, to become significantly lower (P < 0.05) at 60 min, when the lactate content at the end of the work period was m-mole/kg dry wt. and the Gl-1-P content m-mole/kg dry wt. A decrease in lactate occurred in the rest period at 5 min, by 3-4 m-mole/kg dry wt. (P < 0.05), and at 60 min, by 2-3 m-mole/kg dry wt. (P < 0.05). A corresponding decrease in Gl-1 -P by m-mole/kg dry wt. occurred at 15, 30 and 60 min (P < 0.05). Lactate o=end of work period *=end of rest period 100 ' 0~ Lactate E E * *~~~~~~~j Work1 period ' Basal Basal 4-6 min Glycerol-i -phosphate =8-8 Glycerol-i -phosphate Work * period ' I Basal Basal 4-6 min Intermittent exercise (min) Recoverv (sec) (1 5 sec work-1 5 sec rest) Continuous exercise Fig. 2. Lactate and glycerol-l-phosphate concentration before exercise (basal) and at the end of a work bout and the subsequent rest period after 5, 15, 30 and 60 min of intermittent exercise and before and at intervals in recovery after continuous exercise to exhaustion. Continuous exercise. Basal lactate content was m-mole/kg dry wt. and basal Gl-1-P content m-mole/kg dry wt. Immediately after exercise a large increase was observed in lactate, to m-mole/kg dry wt., and in Gl-1-P to m-mole/kg dry wt. (P < 0-001). Lactate increased further to m-mole/kg dry wt see (P < 0.05) after exercise and decreased from this value to m-mole/kg dry wt. (P < 0-05) after 3 min recovery. Gl-1-P content was m-mole/kg dry wt. after sec recovery and had decreased to m-mole/kg dry wt. after 3 min (P < 0-05).

6 504 B. ESSJN AND L. KAIJSER Citrate and palate (Fig. 3) Intermittent exercise. The basal citrate content was m-mole/kg dry wt. The citrate level after work periods at 15, 30 and 60 min was higher than basal (P < 0.05). On all occasions (5, 15, 30 and 60 min) the citrate content had increased, by a mean of 25 %, at the end of rest compared to the end of work periods (P < 0.05). Basal malate content was m-mole/kg dry wt. and increased to *17 m-mole/kg dry wt. (P < 0.01) at end of the work period at 5 min. Malate content was significantly lower at the end of the work period at 60 min than at 5 min (P < 0.05). Malate content had increased by 0-48 m-mole/kg dry wt. (P < 0.05) at the end of rest compared to the end of the work period at 5 min. 5* i,, 3 0 a 20 E E ) 0) 0 E. w I 1a6 r 1-2 F *4 I 0 S Malate )=end of work period * =end of rest. period I,-0_ Basal Citrate Baa + 1 Basal Intermittent exercise (min) (15 sec work-1 5 sec rest) r 4*0 I 3*0 L 2-0 1*0 F 1-6 r v-- r- Iti Work period -. Malate Basal 4-6 min O-A -- Citrate a 6 Work f~ ~~~~~ periodcd Basal 4-6 miin Continuous exercise Fig. 3. Malate and citrate concentration before exercise (basal) and at the end of a work bout and the subsequent rest period after 5, 15, 30 and 60 min of intermittent exercise and before and at intervals in recovery after continuous exercise to exhaustion. Continuous exercise. At the end of continuous exercise citrate content was similar to the basal value of m-mole/kg dry wt. During recovery after exercise a gradual increase occurred to m-mole/kg dry wt. after 1 min (P < 0 05) and 1* m-mole/kg dry wt. after 3 min (P < 0-001). Basal malate content was '06 m-mole/kg dry wt. and increased to m-mole/kg dry wt. immediately after work (P < 0-001). A further increase to m-mole/kg dry wt. had occurred after 1 min recovery (P < 0 05). It remained at that level 3 min after work.

7 REGULATION OF GLYCOLYSIS IN EXERCISE 505 Glucose-6-phosphate (G-6-P) and fructose-1-6-dipho8phate (F-1-6-P2) (Fig. 4) Intermittent exercise. Basal G-6-P content was m-mole/kg dry wt. G-6-P increased to its highest value in the early phase of exercise and was at end of the work period at 5 min m-mole/kg dry wt. (P < 0.01). At 60 min the G-6-P content was significantly lower than at 5 min (P < 0 05) and did not differ from the basal level. Higher values were found at the end of rest than the end of preceding work periods on two thirds of the occasions. Basal F-1-6-P2 content was m-mole/kg dry wt. and increased after work periods to a mean of m- mole/kg dry wt. (P < 0.05). It decreased significantly to m-mole/kg dry 20.0 Glucose-6-phosphate o =end of work period *=end of rest period cn 120 0) o 80 E E 4.0 Q a E04- Basal E asal Fructose-1-6-diphosphate Intermittent exercise (min) (1 5 sec work-1 5 sec rest) C F 16 r 1 2 F 08 F G lucose-6 -phosphate *; * L I WorkI period i_.. Basal 4-6 min ['ts Work Fructose-1-6-diphosphate Basal 4-6 min Continuous exercise Fig. 4. Glucose-6-phosphate and fructose-1-6-diphosphate concentration before exercise (basal) and at the end of a work bout and the subsequent rest period after 5, 15, 30 and 60 min of intermittent exercise and before and at intervals in recovery after continuous exercise to exhaustion. wt. after rest periods (P < 0-01; 5, 15, 30 and 60 min data combined). The G-6-P/ F-1-6-P2 ratio was higher at the end of rest than at the end of the preceding work period in fifteen out of twenty-one occasions (P < 0.05). Continuous exercise. Basal G-6-P content was m-mole/kg dry wt. The increase in G-6-P was more pronounced after continuous than intermittent exercise (P < 0.001) and was immediately after work m-mole/kg dry wt. and after sec recovery m-mole/kg dry wt. A decrease then occurred to rm-mole/kg dry wt. after 1 min (P < 0 05) and m-mole/kg dry wt. after

8 506 B. ESSflN AND L. KAIJSER 3 min (P < 0.05). The F-1-6-P2 content had increased to m-mole/kg dry wt. immediately after work compared to a basal level of m-mole/kg dry wt. (P < 0-01) but decreased again to m-mole/kg dry wt. after 3 min recovery (P < 0.05). The G-6-P/F-1-6-P2 ratio had increased after 1 min recovery compared to immediately after work in all subjects. Glucose of=end of work period 16 *=end of rest period Glucose 0 8 ~~~~~~~~~8 U F periods - Basal Basal4-6 min Glycogen ~ O200 Glycogen E *W*r. +,* + + -, I ~~~~period Basal Basal 4-6min Intermittent exercise (min) (15 sec work-1 5 sec rest) Continuous exercise Fig. 5. Glucose and glycogen concentration before exercise (basal) and at the end of a work bout and the subsequent rest period after 5, 15, 30 and 60 min of intermittent exercise and before and at intervals in recovery after continuous exercise to exhaustion. Glycogen and gluco8e (Fig. 5) Intermittent exercise. The basal glycogen content was m-mole/kg dry wt. The decrease in glycogen content after 60 min was m-mole/kg dry wt. (P < 0.001). The decrease in glycogen was most rapid in the early phase of work and the depletion was after 5 min m-mole/kg dry wt. (P < 0.05), after 15 min m-mole/kg dry wt. and after 30 min m-mole/kg dry wt. (P < 0-001). The muscle glucose content was increased fold above a mean basal value of m-mole/kg dry wt. (P < 0-05) during the whole period of intermittent exercise, with r o significant difference between work and rest periods. Continuous exercise. The basal glycogen content was m-mole/kg dry wt., and had decreased by m-mole/kg dry wt. at exhaustion (P < 0-001). Glycogen content did not change in the 3 min recovery phase. Basal muscle glucose content was m-mole/kg dry wt. and had increased to m-mole/kg

9 REGULATION OF GLYCOLYSIS IN EXERCISE 507 dry wt. immediately after exercise (P < 0 001). Throughout the 3 min post-exercise period the glucose content remained at about 5 times the basal level (P < 0.001) and was after 3 min recovery P9 m-mole/kg dry wt. Water content. The basal water content of the muscle sample was /. In intermittent exercise it had increased to % at 5 min (P < 0.05) and 76* % after 60 min (P < 0.05). No difference was found between work and, rest periods. Immediately after continuous exercise the water content had increased to % (P < 0.01) and remained at this level after 3 min recovery. DISCUSSION Methodological considerations A number of difficulties are involved in the interpretation of muscle biopsy data. Although the biopsies are obtained within a few seconds after the muscle has ceased working, changes in metabolite concentrations might already have commenced. Furthermore it is possible that on different occasions the samples are obtained from parts of the muscle which have not been involved in work to exactly the same extent, and in addition the muscle contains a mixture of fibre types with different metabolic characteristics and their proportion may vary between samples. Finally the analyzing technique available does not permit the determination of the subcellular localization of the metabolites. Several of these are present both intra- and extramitochondrially and a metabolite might have different effects in the mitochondrion than in the cytosol. But although the biopsy technique has limitations, it has been possible to determine significant changes in metabolites occurring over as short a period as 15 sec and the direction of these changes points to an important role in the regulation of specific metabolic steps. Glycolyszi in intermittent versus continuous exercise During continuous exercise at a load which demands maximal oxygen uptake, rapid glycogen depletion and a large accumulation of Gl-1-P as well as lactate and malate, metabolites which are involved in reoxidation of NADH formed in glycolysis were found to occur in the working muscle. In contrast, when work at the same load was performed intermittently, the rate of glycogen break-down and Gl-1-P, lactate and malate accumulation were far lower. This suggests a lower rate of glycolysis during intermittent than continuous exercise in spite of a similar, high energy demand during work bouts. This may be due to a smaller total substrate demand or increased utilization of other substrates than glycogen, notably lipids. The rest periods during intermittent exercise seem to permit a reloading of intramuscular oxygen stores, thereby allowing a greater aerobic energy release. As the yield of energy with aerobic combustion of glycogen is more than 10 times higher than with lactate formation, the total utilization of glycogen for a given work should be lower. The greater accumulation of lactate in and the greater release of lactate from exercising muscles during continuous compared with intermittent heavy exercise indicates that this mechanism is actually of importance (cf. Kaijser, 1970; Ess6n et al. 1977). Furthermore, oxidative metabolism may be covered to a greater extent by lipids in intense intermittent than continuous exercise. The contribution of lipids

10 508 B. ESSItN AND L. KAIJSER to oxidative metabolism during intermittent exercise at maximal work load is shown to be about 40 % and similar to continuous exercise at half that work load (Ess6n et al. 1977). From the respiratory exchange ratio it can be calculated that the relative contribution of lipids to oxidative metabolism decreases with increased work intensity, at least up to about two thirds of maximal oxygen uptake (Christensen & Hansen, 1939; Pruett, 1970). At higher work loads the respiratory exchange ratio may no longer reflect muscle metabolism, due to lactate release and hyperventilation. Furthermore, the limited duration of continuous intense work makes direct measurements of substrate exchange in working muscle difficult. However, using the present data on glycogen depletion and lactate accumulation together with data found in the literature on lactate release, glucose uptake and integrated leg oxygen uptake, a rough estimate suggests that oxidative metabolism during 5 min of continuous work at maximal intensity may be covered almost entirely by carbohydrates (Astrand & Saltin, 1961; Kaijser, 1970; Essen et al. 1977). Thus it might be concluded that a shift towards greater lipid utilization also contributes to the smaller glycogen breakdown in intermittent exercise. Such a shift is in all probability the result of regulatory factors retarding the rate of glycolysis. Since the load during actual work bouts was the same in intermittent as in continuous heavy exercise, metabolic changes responsible for such a shift should be found in the intervening rest periods. Regulatory factors Hexokinase, phosphorylase and phosphofructokinase (PFK) activities are all inhibited by ATP (Morgan & Parmeggiani, 1964; Passoneau & Lowry, 1963). CP has been shown to inhibit PFK and also to potentiate its inhibition by ATP (Storey & Hochachka, 1974). Citrate has been shown to be a potent inhibitor of PFK (Parmeggiani et al. 1963; Garland et al. 1963; Passoneau et al. 1963) as well as of pyruvate dehydrogenase (Taylor & Halperin, 1973). The inhibiting effect of citrate on PFK depends on a simultaneous inhibiting effect by ATP on PFK (Newsholme & Start, 1974). Thus the specific effect of citrate is to potentiate the ATP inhibition of PFK. The rate limiting steps of glycolysis are also known to be stimulated by increased levels of other metabolites, such as ADP, AMP, inorganic phosphate and fructose-1-6- diphosphate, and these metabolites also deinhibit the effect of citrate and ATP on PFK (Passoneau et al. 1963). With muscle contraction, ATP and CP are consumed and a rise occurs in ADP, AMP and inorganic phosphate (Newsholme et al. 1974). The decrease in ATP, CP and citrate which occurred with work periods in intermittent exercise and to a still greater extent with continuous exercise would thus stimulate glycolysis, whereas the increase in levels of these metabolites during rest periods would retard glycolysis. The repeated periods of rest in intermittent exercise would then contribute to maintain rather high average ATP, CP and citrate levels and consequently a comparatively high degree of inhibition of glycolysis, especially at the onset of each subsequent work bout. The tendency to an increased G-6-P/ F-1-6-P2 ratio at the end of rest compared to work periods found in intermittent exercise may further support the assumption that inhibition may occur at the PFK step. At the end of continuous heavy exercise a still higher G-6-P/F-1-6-P2 ratio was found than in intermittent exercise. However, this cannot be taken as evidence of

11 REGULATION OF GL YCOL YSIS IN EXERCISE 509 a high degree of PFK inhibition. It is probably merely a reflexion of a higher rate of glycolysis with greater activation of phosphorylase; since PFK is considered to be a rate limiting step of glycolysis, a high ratio will then be found also with maximal stimulation of PFK. The more pronounced decrease in ATP, CP and citrate levels at the end of continuous than intermittent exercise suggests a stronger stimulation of glycolysis during the preceding work, as is also reflected by the greater depletion of glycogen and accumulation of Gl-1-P as well as lactate. This is also supported by the increase in Gl-1-P and lactate levels over the first sec of recovery, which indicates that glycolysis continues at a substantial rate; moreover, the progressive return of ATP and CP and the increase in citrate that occurred over 180 see of recovery support the view that the rate of glycolysis is only gradually retarded in this situation. The progressive increase in G-6-P/F-1-6-P2 ratio that occurred over the first minute of recovery is furthermore an indication that the PFK step is affected. The increase in ATP with rest periods retards glycolysis not only directly but also through retardation of citric acid cycle activity by an inhibition of the isocitrate dehydrogenase step (Johnson & Hansford, 1975), thus contributing to the production of the increased citrate level. Continuous production of acetyl-coa from fatty acid oxidation may contribute to the increased citrate level with rest periods. Increased citrate levels require availability of oxalacetate. It has been suggested that this is generated by the transport of malate into the mitochondrion (Maizels, Ruderman, Goodman & Lau, 1977), which may be facilitated by the increased malate level produced by glycolysis. Oxalacetate may also be formed from aspartate by transamination (Bowman, 1966). It may be added that citrate and CP not only inhibit PFK activity but also have been shown to stimulate fructose-1-6-diphosphatase activity (Fu & Kemp, 1973). Consequently, the Gl-1-P which accumulated during each work period and disappeared during each rest period may have been utilized in glycogen resynthesis, thus adding to the glycogen saving effect in intermittent exercise. However, such a mechanism is probably less important, since CP did not return to basal in the rest periods. G-6-P has been shown to inhibit hexokinase and phosphorylase B (Crane & Sols, 1953; Morgan et al. 1964). The finding in intermittent exercise of the greatest increase in G-6-P level in the early phase, with a gradual return to basal level after 60 min of exercise, would thus indicate inhibition of hexokinase and phosphorylation of glucose early in exercise. This is in agreement with the observation, in a previous study of intermittent exercise, that glucose uptake was lowest in the early phase and increased gradually over 60 min of exercise (Ess6n et at. 1977). The high G-6-P level which was found after continuous intense exercise indicates inhibition of hexokinase. The simultaneous high intramuscular glucose level in this situation supports limited phosphorylation of glucose by inhibition of hexokinase. Conclsion The significant increase in citrate together with the increase in ATP and CP content which occurs in each rest period of intermittent exercise is in accordance with the assumption that the combined influence of these metabolites is important in retarding glycolysis at the start of each new work period and hence responsible for the shift to

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13 REGULATION OF GLYCOLYSIS IN EXERCISE 511 PRuETT, E. D. R. (1970). FFA mobilization during and after prolonged severe muscular work in men. J. apple. Phy8iol. 29, RENNIN, M. J. & HoiLoszy, J. 0. (1977). Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle. Biochem. J. 168, SATIN, B. & E5&AN, B. (1971). Muscle glycogen, lactate, ATP and CP in intermittent exercise. In Muscle Metaboliem during Exercise, ed. PERNOW, B. & SAI.Tim, B., pp New York: Plenum. SAI~N, B. & KAnLssoN, J. (1971). Muscle glycogen utilization during work of different intensities. In Muscle Metaboliem during Exercise, ed. PERNOW, B. & SALTIN, B., pp New York: Plenum. STOREY, K. B. & HOcHACUIXA, P. W. (1974). Activations of muscle glycolysis: A role for creatine phosphate in phosphofructokinase regulation. FEBS Lett. 46, TAYLOR, W. M. & HALPERIN, M. L. (1973). Regulation of pyruvatedehydrogenase in muscle. J. biol. Chem. 248,