Metabolic adaptations to training in muscle mitochondrial capacity

precede changes

H. J. GREEN, R. HELYAR, M. BALL-BURNETT, N. KOWALCHUK, S. SYMON, AND B. FARRANCE Department of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada GREEN, H.J.,R. HELYAR, M. BALL-BURNETT,N.KOWALCHUK, S. SYMON, AND B. FARRANCE.~M~~~~~Z~~adaptations to training precede changes in muscle mitochondrial capacity. J. Appl. Physiol. 72(2): 484-491, 1992.-To determine whether increases in muscle mitochondrial capacity are necessary for the characteristic lower exercise glycogen loss and lactate concentration observed during exercise in the trained state, we have employed a short-term training model involving 2 h of cycling per day at 67% maximal 0, uptake (vo2,) for 5-7 consecutive days. Before and after training, biopsies were extracted from the vastus lateralis of nine male subjects during a continuous exercise challenge consisting of 30 min of work at 67% vo2followed by 30 min at 76% VO, max. Analysis of samples at 0,15,20, and 60 min indicated a pronounced reduction (P < 0.05) in glycogen utilization after training. Reductions in glycogen utilization were accompanied by reductions (P < 0.05) in muscle lactate concentration (mmol/kg dry wt) at 15 min [37.4 t 9.3 (SE) vs. 20.2 * 5.31, 30 min (30.5 t 6.9 vs. 17.6 t 3.8), and 60 min (26.5 t 5.8 vs. 17.8 & 3.5) of exercise. Maximal aerobic power, 00, m8x(Vmin) was unaffected by the training (3.99 * 0.21 vs. 4.05 t 0.26). Measurements of maximal activities of enzymes representative of the citric acid cycle (succinic dehydrogenase and citrate synthase) were similar before and after the training. It is concluded that, in the voluntary exercising human, altered metabolic events are an early adaptive response to training and need not be accompanied by changes in muscle mitochondrial capacity. exercise; training; activities

muscle metabolism; adaptation;

enzymatic

ONE OF THE MOST widely held beliefs in exercise biochemistry is that endurance training by virtue of increasing the potential of the citric acid cycle, ,&oxidation and other mitochondrial enzyme systems, and components in skeletal muscle modifies the response to prolonged exercise. These adaptations are believed to decrease glycolysis and glycogen utilization and to shift substrate preference away from carbohydrates to a greater lipid utilization (9,20). However,‘must this be so? In previous work we showed, by the use of a short-term training program consisting of 3 days of prolonged exercise (13), that reductions in glycogen utilization occur during prolonged exercise that was unaccompanied by alterations in substrate preference, as indicated by the respiratory exchange ratio (R), or in glycolysis, as indicated by the muscle and blood concentration of lactate. When the training program was extended to lo-12 days, additional energy metabolic adaptations typical of the trained state 484

developed, namely, a lower muscle lactate concentration and a shift toward an increased lipid utilization as suggested by the lower submaximal exercise R (16). These changes were unaccompanied by’elevations (P > 0.05) in the maximal enzymatic activities of 3-hydroxyacyl CoA dehydrogenase (HAD), a representative enzyme of ,&oxG dation, and citrate synthase or succinic dehydrogenase, two enzymes of the citric acid cycle (15). Moreover, indications that the metabolic events may occur independently of alterations in mitochondrial capacity during submaximal exercise have been published previously (22), where reductions in blood lactate concentration have been reported within the 1st wk of training. Collectively, these studies suggest that the metabolic alterations observed during voluntary submaximal exercise in humans need not occur simultaneously and need not be intimately linked to changes in muscle mitochondrial capacity. In this study, we have employed a 5- to 7-day training model to determine whether the metabolic alterations are fully manifested within this time. We were particularly interested in more clearly delineating a dissociation between alterations in mitochondrial capacity and metabolism. Although the enzymes of P-oxidation and the citric acid cycle were not different (P > 0.05) after training in our previous study (15), elevations did occur, suggesting the possibility that lo-12 days of training in fact did induce an increase. With the more abbreviated training, the dissociation between the metabolic and enzymatic events was clearly evident, leading us to conclude that elevations in muscle mitochondrial capacity need not be linked to alterations in metabolism in the voluntary exercising human. METHODS

Experimental design. The basic experimental design was essentially the same as that employed in previous studies (13, 14). Approximately l-2 wk before training began, the subjects reported to the laboratory for measurement of maximal aerobic power (VOLT,). VO,, was reevaluated within 2 days of the final training session. To determine the effects of training on the metabolic response, the subjects exercised on a cycle ergometer at two different exercise intensities, first for 30 min at 67% vo 2 m8xfollowed by 30 min at 76% VO, max. The exercise was continuous except for brief interruptions to secure muscle samples. Immediately before and at 25 and

0161-7567192 $2.00 Copyright 0 1992 the American Physiological Society

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ENERGY

METABOLISM

AFTER

55 min of exercise, respiratory gas concentrations and ventilatory volumes were measured for determination of 0, uptake (VO,), CO2 output (VCO,), minute ventilation, and R (21). Muscle biopsy samples were obtained from three different regions of the vastus lateralis muscle by the use of the Bergstrom technique (1). These samples were used for determination of glycogen, glycolytic intermediates, and high-energy compounds as well as the measurement of the activities of enzymes representative of different metabolic pathways. Approximately 48 h after the last training session, the exercise challenge was repeated along with the schedule for sampling the respiratory gases, blood, and muscle. The training program consisted of 5-7 consecutive days of cycling for 2 h/day at ~67% VO, M8X. Water was provided ad libitum, and the subjects were allowed brief rest pauses if they were unable to perform’ the 2 h of exercise continuously. Both training and testing were performed at temperatures ranging between 24 and 26OC dry bulb and at 4959% relative humidity. Subjects. The volunteer subjects were nine active, but untrained, males ranging in age from 18 to 20 yr. During the experimental period, the subjects were requested to refrain from engaging in any heavy exercise outside of the study. Testing occurred at approximately the same time of day and 24 h after the ingestion of any food. No dietary manipulations were attempted during the study; the subjects were simply asked to follow their normal practice. After approval of the study by the Office of Human Research, all subjects were fully informed of the experimental procedures and the risks involved before obtaining written consent. Analytic techniques. Peak aerobic power (VO,,,) was determined as previously described (18) with the use of an electrically braked cycle ergometer (Quinton 870), with power output increased progressively in step increments of 100 kg. m-l min-l (16.3 W) each minute. The test continued until exhaustion. The Vozrnax value was recorded as the. highest value obtained. Ventilation and gas exchange (VO,, VCO,) were determined with the use of an open-circuit system (13, 21). Heart rate (HR) was recorded by standard electrocardiographic techniques. Details of the procedures have been published (13, l

14, 21).

During the submaximal exercise challenge before and after training, gas exchange ventilation and HR were monitored before the exercise and at selected time intervals (25 and 55 min) for a 3- to 5-min period. Measurements made before exercise were obtained after the subjects had been sitting stationary for a period of 215 min. Two muscle samples were obtained from each of the preselected and prepared regions of the vastus lateralis before the exercise and at 15, 30, and 60 min. The first sample, obtained as quickly as possible after the subject stopped cycling in the case of the exercise biopsies, was immediately plunged into liquid nitrogen, stored at -8O”C, and subsequently analyzed for glycogen, the labile metabolites, and high-energy phosphates, with the use of fluorometric techniques (26) as modified in our laboratory (19). In addition, determinations of ATP, ADP, AMP, and inosine 5’-monophosphate (IMP) were made from the same homogenate with the use of ion-pair

SHORT-TERM

TRAINING

485

reversed-phase high-performance liquid chromatography techniques (23), as detailed in previous publications (17). All values were corrected to total creatine (Cr) for each subject to adjust for contamination by blood and connective tissue. Because glucose, lactate, and pyruvate (PYR) exist in the extracellular space as well as in the muscle cell, it was necessary to correct for extracellular water content in the muscle sample and for extracellular concentrations of the three metabolites. Essentially, the procedure employed by Katz et al. (24) was followed. These investigators used values of 0.30 and 0.48 l/kg dry muscle for extracellular water content at rest and during submaximal exercise on the basis of figures published by Sjogaard et al. (32). The extracellular concentration of glucose, lactate, and PYR was considered to be approximated by the plasma concentration. The plasma concentrations of these metabolites were actual values measured during the study (H. Green, unpublished observations). The free concentrations of ADP (ADP,) and AMP (AMP,) were estimated on the basis of the near equilibrium nature of the creatine phosphokinase reaction and the adenylate kinase reaction, respectively. In the case of ADP,, the calculation was made by the use of the measured values of ATP, phosphocreatine (PCr), and Cr. The pH and H+ concentrations were estimated from muscle PYR and lactate concentrations applying the regression equation established by Sahlin et al. (29) for dynamic work. The Kobs value employed was 1.66 X 10' M. AMP, was calculated by the use of the determined ATP concentration, the calculated ADP, value, and a Kobs value of 1.05 (8). Free phosphate concentration (Pi,) at rest was assumed to be 2.5 mmol/kg wet wt (and 10.8 mmol/kg dry wt), whereas during exercise the Pi, was estimated as the difference between resting and exl ercise PCr concentrations plus the resting Pi,f concentrations (8). Calculations of the lactate-to-pyruvate and NAD+-toNADH ratios were also made. In the case of the lactateto-pyruvate ratios, actual determinations were employed. However, for the cytosolic redox potential, cytosolic NAD+/NADH values were calculated with the use of the lactate dehydrogenase equilibrium reaction and a constant value of 1.11 X lo-l1 M as the equilibrium

(30, 36).

The second biopsy, extracted from the same site immediately after the first biopsy, was used for biochemical determinations of enzyme activities. The enzymatic profile was established so that representation could be given to the main metabolic pathways involved in metabolism. These included total phosphorylase (PHOSPH), pyruvate kinase, phosphofructokinase (PFK), c\r-glycerophosphate dehydrogenase, lactate dehydrogenase, hexokinase (HK), HAD, succinate dehydrogenase (SDH), citrate synthase (CS), and creatine phosphokinase. Complete details of the enzymatic assays have been published previously (12). Changes in muscle mitochondrial capacity were estimated from changes in two enzymes of the citric acid cycle, SDH and CS. Although these enzymes may not be rate limiting in the muscle’s respiratory capabilities, they can provide an estimate of changes in mitochondrial capacity with training given that the

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486

ENERGY

METABOLISM

AFTER

1. Effects of short-term training on maximal aerobic power, heart rate, and ventilation l/min

Pre Post

3.99t0.21 4.05t0.26

% maxt

ml kg-’ l

l

min-l

55.0t0.04 54.1t0.03

HR,

beatdmin

19ltl.9 184t2.1

VE,

l/min

0 min

15 min

30 min

60 min

24.6t0.53 24.3t0.61

25.5t0.88 24.5tl.l

24.8t0.85 23.8t0.90

22.9tl.O 23.6t0.88

81.4tl.2 77.0t2.6

49.8t4.3 57.9t4.3

44.2t4.1 62.0t3.1

32.1t3.4 47.1t4.3

38.7tl.2 43.2t2.6

70.324.4 62.3t4.4

76.0t4.1 58.2t3.1

88.0t3.4 73.124.3

35.9tl.4 33.4tl.6

58.2k5.4 44.5t5.7

65.123.8 38.024.0

81.01t4.5 55.7t5.1

BTPS

12lt3.3 122t6.9

Values are means t SE; n = 9. VO, max,maximal-o, uptake; HR, heart rate; iTE, minute ventilation; Pre, pretraining; Post, posttraining.

stoichiometry of mitochondrial functions is preserved (20). Protein was measured by the Lowry technique with the use of the modification of Schacterle and Pollack (31). As with the metabolite samples, all samples for a given individual, both pre- and posttraining, were measured during the same session. Only in the case of the metabolites was a correction made to the concentration on the basis of total Cr content. Statistical procedures. A two-way analysis of variance for repeated measure was employed to determine the effects of exercise and training. The Newman-Keuls technique was selected to compare specific means where significance was indicated. In the case of the enzyme data, where measures were only available before and after training, Student’s t tests for correlated samples were used. The level of confidence was standardized at 95% for all comparisons. RESULTS . vo 2maxand related values. As indicated

in Table 1, the short-term training program had no effect in altering vo 2max or the HR (beats&in) and ventilation (Urnin, kTPS) observed at VO, m8x. VO, maxwas 3.99 l/min before training and 4.05 l/min after training. Gas exchange during submaximal exercise. TO, during the first 30 min of exercise amounted to 2.66 t 0.07 (SE) l/min or 67% TO 2 maxand 3.04 t 0.18 l/min or 76% i702 max during the final 30 min of exercise before training (Table 2). These values were unchanged with training. VCO, and R were also unaffected by the training. In contrast, HR was reduced (P < 0.05) after training, with the reduction amounting to between 11 and 12 beats/min.

ATP Pre Post PCr Pre Post Cr Pre Post PHOS Pre Post

Values are means t SE expressed in mmol/kg dry wt; n = 9. Time main effect (P < 0.05) was found for PCr, Cr, and PHOS. Training main effect (P < 0.05) was also found for PCr, Cr, and PHOS. No interaction effects were found. PCr, phosphocreatine; Cr, creatine; PHOS, phosphagen.

Muscle metabolites. Neither exercise nor training resulted in a change in ATP (Table 3). However, PCr concentration was reduced (P < 0.05) with exercise regardless of the training state. Before training, the reduction ranged between 31.5 and 37.2 mmol kg dry wt-l min-l during the first 30 min of cycling. When the intensity of exercise was increased during the final 30 min of exercise, a further reduction in PCr occurred. The reductions in PCr were accompanied by an approximately stoichiometric increase (P < 0.05) in both Cr and Pi (Table 3). Training resulted in alterations in all three of these variables: PCr, Cr, and Pi. With exercise after training, PCr concentration was higher, whereas both Cr and Pi were lower. There was no statistical evidence that the training effect was specific to a given time point. Both exercise and training failed to result in changes in the high-performance liquid chromatography determined concentration of ATP, ADP, or AMP (Table 4). In contrast, IMP was altered by both exercise and training. Before training, IMP increased during exercise but not until 60 min of exercise had been completed. After trainl

4. Alterations in adenine nucleotides and IMP during exercise after short- term training

2. Short-term training, submaximal exercise, and respiratory gas exchange 0 min

30 min

60 min

0.397t0.02 0.400t0.02

2.66t0.07 2.6320.08

3.04t0.14 3.05t0.13

0.39t0.02 0.37t0.02

2.4420.06 2.4120.06

2.80t0.10 2.75t0.11

0.98t0.07 0.92zko.04

0.92t0.01 0.92t0.02

0.92t0.02 0.92t0.01

71k3.4 73t4.5

17124.2 160t4.9

188t2.9 176t3.3

V02,

Values are means * SE; n = 9. For both VO, and hoz, main effect (P < 0.05) for time was found. OO,, 0, uptake; R, respiratory exchange ratio.

l

TABLE

TABLE

l/min Pre Post VC02, l/m& Pre Post R Pre Post HR, beatslmin Pre Post

TRAINING

TABLE 3. Effects of short-term training on high-energy phosphagen concentration during submaximal exercise

TABLE

fTQa Lnax,

SHORT-TERM

ATP Pre Post ADP Pre Post AMP Pre Post IMP Pre Post

0 min

15 min

30 min

60 min

26.5t0.86 25.9t0.70

26.6tl.O 25.920.84

26.4t0.97 25.0t0.79

24.0t0.87 24.621.0

4.31t0.28 4.75k0.46

4.74t0.32 4.61k0.23

4.64t0.32 4.4020.34

4.65t0.41 4.75k0.28

0.6lt0.08 0.90to. 13

0.92t0.28 1.020.2

0.99&0,3 0.95&O. 14

0.70tO.l 1.23rtO.2

0.327+0.03-f 0.354t0.03

0.612+0.08t 0.494t0.20

0.626k0.57 0.458~10.12

1.19t0.07 0.47lt0.29*

Values are means t SE expressed in mmol/kg dry wt; n = 9. For IMP, main effects of condition and time were found (P < 0.05). Also there was interaction effect (P < 0.05). Significant effects were not found for other variables. IMP, inosine 5’-monophosphate. * Significantly different from Pre (P < 0.5); t significantly different from 60 min (P < 0.05).

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ENERGY

METABOLISM

AFTER

5. Alterations in calculated values of ADP,, AMPf, and Pit, during exercise after short-term training TABLE

0 min

ADPf Pre Post AMPf Pre Post P i,f Pre Post ATP/ADPf Pre Post

15 min

30 min

SHORT-TERM

6. Effects of short-term training on changes in glucose and selected glycolytic intermediates during submaximal exercise TABLE

60 min 0

82.1t5.8 103t9.3 0.25t0.03 0.42t0.06

207t47 1751k26 2.00t0.93

1.28kO.36

487

TRAINING

248228 139t15

392t61 215k43

2.4620.56 0.79+0.19

GLUC Pre Post Post

43.1k4.7 30.4k5.6

48.2k4.6 2l.Ot4.6

61.9k5.1 35.0t7.5

G-6-P

321kl4 256223

164t29 175225

118t13 190t15

74.6t13 203286

F-6-P

Values are means t SE expressed in pmol/kg dry w-t for ADPf and AMP, and mmol/kg dry wt for Pif; n = 9. For ADPf, AMPf, Pif, and ATP/ADPf, main effect (P < 0.05) for exercise was found. For ADPf, AMP,, and Pi f, main effect (P < 0.05) for training was found. No interaction effects were observed. ADP,, AMPf, and Pi,f, free ADP, AMP, and phosphate, respectively.

15

Pre Post Pre Post F-1,6-P Pre Post PYR Pre Post

min

30

min

60

min

2.05t0.60

4.49t1.3 3.06tl.l

2.79i20.78 2.5220.53

0.08t0.01 O.llt0.03

0.43t0.16 0.24t0.11

0.25kO.06 0.14t0.04 0.28-1-0.07 0.22t0.05

l.OOt0.15 1.47t0.24

5.4321.7 2.87zkO.70

3.45kO.80 3.6920.94

1.64t0.35 1.98t_O.41

0.12t0.02 0.20t0.03

0.58t0.10 0.32t0.07

0.34-t-0.07 0.41kO.11

0.25t0.08 0.18t0.13

0.97t0.42

7.19tl.9 G-1-P 2.20t0.68 Pre

10.8 10.8

min

1.69tl.l 2.52tl.O

0.39t0.10

0.7720.29

0.43-t-0.10

0.43t0.10

0.30t0.07

0.37-t-0.06

0.25t0.04 0.28kO.05

0.15t0.04 0.14t0.04

0.39-+0.07 '0.353-0.05

0.36-t-0.09 0.37r40.08

0.25t0.10 0.40t0.08

Values are means t SE expressed in mmol/kg dry wt; n = 9. Main effect (P < 0.05) for time was found for all variables except glucose. No training effects were observed. GLUC, glucose; G-l-P, glucose l-phosphate; G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6P, fructose 1,6diphosphate; PYR, pyruvate.

- 320 ;. 1 'O *i 280 m t

T

-

r,----__a--____-_----__------_ i POST / ,I8,#’ ----w--

0

10

20

30

40

50

60

TIME (lain) FIG. 1. Changes in muscle glycogen concentration during exercise before (0) and after training (0). Values are means t SE (n = 9). Main effects (P < 0.05) for both time and training were found. No interaction effects were observed.

ing, IMP concentration was not different between rest and exercise. The calculated ADP,, AMP,, and Pi were affected by both exercise and training (Table 5). For both ADPf and Pi f, exercise-induced increases existed at 15 min, remained elevated over the ensuing 15 min of exercise, and then increased further after 60 min of exercise. In general, a similar pattern was observed for AMP,; however, in this case the metabolite increased after 60 min of exercise. The ATP-to-ADP, ratio, in contrast, declined at 60 min of exercise compared with rest and 15 and 30 min of exercise. Training significantly altered the response pattern for ADP,, AMP,, and Pi f. In all cases this was a generalized effect and not isolated to specific time points. With regard to glycogen and the glycolytic intermediates examined, exercise resulted in changes in all of them. Before training, pronounced reductions in glycogen were evident after 15 min of exercise, resulting in a concentration ~20% lower than rest (Fig. 1). Thereafter, glycogen continued to decline, ultimately reaching a level

I

I

0

10

I

I

I

20 30 40 TIME (min)

I

I

50

60

FIG. 2. Changes in muscle lactate concentration during exercise before (0) and after training (0). Values are means t SE (n = 9). Main effects (P < 0.05) for both time and training were found. No interaction effects were observed.

of 62.7 t 13 mmol. glucosyl units-l. kg-’ or 21% of the preexercise level. Similarly for the glycolytic intermediates, glucose l-phosphate, glucose 6-phosphate, fructose 6-phosphate (F-6-P), fructose 1,6-diphosphate (F1,6-P), PYR, and lactate, increases (P < 0.05) were found during the initial period of exercise (Table 6 and Fig. 2). In case of lactate and PYR, the concentration was unchanged (P > 0.05) during the remaining period of exercise but declined (P < 0.05) in the case of the other intermediates. Muscle glucose was not altered by exercise or training. Training had a pronounced effect on glycogen concentration, resulting in a reduced depletion (Fig. 1). After 60 min of exercise, glycogen concentration after training was -2.5-fold higher than before training. Of the glycolytic intermediates investigated, only lactate was modi-

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488

ENERGY

METABOLISM

AFTER

7. Effects of short-term training on N-AD+/ lactatelpyruvate, and pH during

TABLE

NADH,

submaximal exercise

Lactate/pyruvate Pre Post NAD+/NADH Pre Post PH Pre Post

0 min

15 min

30 min

60 min

53.4t20 28.3k6.0

107t34 36.7k7.3

64.1t9 46.426.8

96.2t16 45.1t6.3

256k66 428-+10

159t30 334t71

185t25 247t47

144t36 261t59

7.04t0.01 7.04t0.06

6.95kO.03 7.00t0.01

6.94t0.03 6.99t0.02

6.95kO.03 6.99t0.02

Values are means t SE expressed in mmol/kg dry wt; n = 8. Main effect (P < 0.05) for time was found for lactate/pyruvate, NAD+/ NADH, and pH. In addition, main effects (P < 0.05) for training were also found for these variables. No interaction effects were recorded.

fied with training (Fig. 2). Concentrations ranged from 32 to 46% lower during the exercise after training. Muscle lactate-to-PYR ratios were increased during exercise only in the pretrained state (Table 7). Before training, the increase in the lactate-to-PYR ratio observed at 15 min of exercise persisted throughout the exercise. Compared with the pretraining values, the posttraining lactate-to-PYR ratios were persistently lower. In comparison, the NAD+-to-NADH ratio calculated from lactate dehydrogenase reaction decreased with exercise. However, the decrease was not as pronounced after training. Reductions in pH occurred with exercise, with the magnitude of the change being unrelated to the time of exercise. After training, the pH levels were higher than before training. Figure 3 contains the crossover plots for the glycolytic intermediates examined at l&30, and 60 min of exercise before and after training. Each point represents the ratio of the exercise to rest values. The crossover plot in particular at 15 min of exercise before training suggests an inhibition at the level of PFK. Early in the exercise after training, an increased inhibition of phosphorylase activity is clearly evident and an inhibition of PFK is suggested. Maximal enzymatic activities. The maximal activities of SDH and CS were unchanged (P > 0.05) after training (Fig. 4). Similarly, the maximal activity of the enzyme selected to represent ,&oxidation, HAD, remained unaltered (P > 0.05). The enzyme selected to represent glycolysis, PFK, was depressed with the short-term training, with the reduction in maximal activity amounting to 122 pm01 mg protein-l min-’ or 38%. For the remaining two enzymes examined, PHOSPH, used to represent the glycogenolytic potential, and HK, used to represent the potential for glucose phosphorylation, no change (P > 0.05) was found.

SHORT-TERM

TRAINING

potential for P-oxidation. Thus in the voluntary exercising human, enhancement of mitochondrial capacity is not a prerequisite for the alterations in metabolism and endogenous glycogen depletion that exists after training (20). The findings of this study do not refute the well-established association between the metabolic changes and muscle respiratory potential during the imposed patterns of activity in isolated muscle, nor does it negate the possibility that with prolonged training programs in9

A

8

7 6

ok





Ot,’



































7 6

C

5i

l

l

DISCUSSION

The results of this study confirm our previous finding (15) that reductions in muscle glycogen loss and lactate concentration occur during submaximal exercise after short-term training in the absence of an increase in the capacitv for flux through the citric acid cvcle or in the

Ott”“““*“’ GLY yp

66-P

F1,6BP F6-P

CAP DHAP

3PGA 1,3DPG

PEP 2PGA

LACT PYR

FIG. 3. Crossover plots obtained from determining exercise-to-rest ratio for each metabolite at 15 (A), 30 (B), and 60 (C) min of prolonged exercise. Closed circles, ratios obtained before training. Closed triangles, ratios obtained after training. Glucose ratios are indicated as open circles before training and open triangles after training. Gly, glycogen; Glu, glucose; G-l-P, glucose l-phosphate; G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-biphosphate; Pyr, pyruvate; Lact, lactate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; l,SDPG, 1,3diphosphoglycerate; SPGA, 3-phosphoglycerate; 2PGA, .2-phosphoglycerate; PEP, phosphoenolnvruvate.

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ENERGY

METABOLISM

AFTER

SHORT-TERM

489

TRAINING

350

t 250 err E \ 5L 200 n ua

FIG.

4.

enzymes (H) and a ent from

,150

Maximal activities of representative ways before antly differ-

0

E q 100 50

0

SDH

cs

HAD

HEX

PHOSPH

creases in the potential for aerobic metabolism are linked to decreases in glycogenolysis and glycolysis and increases in lipid utilization (9,20). However, our findings suggest that other adaptive strategies may prevail early in training to promote some of the same metabolic effects as observed after the enhancement of the muscle mitochondrial protein concentration. There are several explanations for the lower net glycogen loss and muscle lactate concentration found in this study after training. One hypothesis is that both glycogenolysis and glycolysis were reduced. Alternatively, both glycogenolytic and glycolytic rates may not have changed with training. The lower net glycogen loss could have resulted from increased uptake and utilization of glucose by the working muscle either directly in the glyco,lytic pathway or indirectly via glycogenesis. Reductions in lactate concentration, on the other hand, could result from a greater clearance rate of lactate from the muscle or a decrease in lactate formation secondary to increased ) PYR disposal via mitochondrial oxidation. Unfortunately, in the absence of measures of turnover rates using isotopes, one could only speculate as to the mechanisms involved. Reductions in muscle lactate concentration and in net glycogen loss during exercise after training have been interpreted as a coordinated event occurring as a result of a depression in glycogenolysis and glycolysis (9, 20). The depression in glycogenolysis and glycolysis has been postulated to occur as a result of reductions in PHOSPH and PFK activities secondary to changes in one or more of the many modulators of these two enzymes (20). Support for this hypothesis is suggested by the relative changes in some of the glycolytic intermediates that occurred from rest. to exercise after training. Before train-

PFK

ing, exercise resulted in a greater increase in F-6-P compared to F-1,6-P, particularly during the early stages. This suggests an inability of PFK to phosphorylate F-6P at a rate equivalent to its production. A depression in the increase in F-6-P in conjunction with other metabolites, such as glucose l-phosphate and G-6-P, early in exercise after training is consistent with training-induced depression in glycogenolysis. A comparison of the crossover plots for F-6-P and F-1,6-P after training, particularly early in exercise, also suggests a reduction in the PFK activation. The hypothesis of a reduction in glycogenolysis secondary to changes in phosphorylase activity is supported by the changes in several variables known to affect phosphorylase a and phosphorylase b activities, namely Pi, AMP,, pH, and the ATP-to-ADP, ratio (28). All were depressed after training. The reduction in the adrenergic response with exercise after short-term training as indicated by the pronounced reductions in blood concentrations of both epinephrine and norepinephrine (H. Green, unpublished observations) may also contribute to a reduced glycogenolysis. Increases in these hormones have a potentiating effect on the activation of glycogenolysis (10). In addition, it has been found that during progressive exercise the inflection point for both blood catecholamines and, in particular, epinephrine concentration was closely correlated with the blood lactate threshold (27). It is possible that after training the depression in adrenergic drive with exercise may have blunted glycogenolysis and reduced the flux through glycolysis. Evidence for a greater inhibition of PFK after training is supported by the changes occurring in several modulators. The concentrations of Pi f 9AMP, and ADP,, and the ATP-to-ADP, ratio, all regarded as stimulatorv to PFK

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activity (6, 28), were lower after training. Lower PFK activities could have resulted from training-induced alterations in the enzyme itself. When measured in vitro, we found that training resulted in a 38% decrease in maximal activity. Although the evidence presented favors the hypothesis that glycolysis is reduced with training, other possibilities must be acknowledged. As an example, reductions in lactate could have been mediated by an increased uptake of PYR by mitochondria and used as a substrate for mitochondrial respiration. However, this explanation does not seem plausible because R was unaffected by training, suggesting that the proportion of fats and carbohydrates utilized were unchanged. In addition, because VO, and, therefore, mitochondrial respiration, were unaffected by training, it is improbable that PYR oxidation would have increased. Enhanced clearance of lactate from the working muscle remains as a potentially viable alternative mechanism to explain the lower posttraining lactate concentration (7). A decrease in glycogenolysis could explain the increased sparing of glycogen concentration that occurs with exercise after training. The glycogen-sparing effect may be coupled to an increased uptake and utilization of glucose. Transport of glucose across the muscle membrane is facilitated during exercise very early in training (34). This, in combination with the pronounced trend toward a lower G-6-P concentration after training, may result in increased HK activity and increased phosphorylation of glucose (28). The increase in G-6-P could be used as a substrate directly in glycolysis or resynthesized to glycogen via glycogenesis. A number of recent studies using isotopes, as summarized by Bonen et al. (2), have concluded that during prolonged exercise, glycogen synthesis may occur concomitantly with glycogenolysis in working muscle. Interestingly, a reduction in glycolysis does not seem to be a prerequisite for the glycogen-sparing effect noted with short-term training. We have previously shown (13) with the use of a 3-day training model that muscle glycogen depletion was attenuated in the absence of changes in muscle lactate concentration. It seems improbable from the present study that the increase in ATP production occurring via glycolysis during exercise before training was necessary to maintain ATP concentration, because ATP concentration during exercise was similar after training when glycolysis appeared to be reduced, and no compensatory increases were evident in oxidative phosphorylation. If glycolysis is not important in supporting ATP homeostasis at submaximal work intensities, a view that has been supported by others (3, 4, 30), the question remains as to why it is accelerated during aerobic-based exercise. A number of theories have been proposed (3,5,30,35). One is based on the view that there is a lack of control over glycogenolysis and glycolysis, resulting in a mass action effect with production of PYR and NADH by the Embden-Meyerhoff pathway in excess of what can be removed by the mitochondria (9, 35). Alternatively, increases in glycolysis have been proposed as being necessary in supplying reducing equivalents to support the increase in mitochon-

SHORT-TERM

TRAINING

drial respiration that occurs with exercise (5, 30). However, differences in opinion exist as to whether or not cellular hypoxia is present when glycolysis is accelerated 6,30). The changes in the lactate-to-PYR ratio and in the calculated cytosolic NAD+-to-NADH ratio could imply cellular hypoxia. With exercise, the increase in the lactate-to-PYR ratio that occurred and that has been observed previously (25) has been interpreted to reflect an accumulation of cytosolic NADH as a consequence of a limitation of the mitochondria to accept reducing equivalents from the cytosol. The reductions in the calculated cytosolic redox potential observed during exercise are also consistent with this notion. A training-induced reduction in anaerobic glycolysis occurring as a consequence of cellular hypoxia would be expected to be reflected in lower lactate-to-PYR ratios and higher calculated cytosolic NAD+-to-NADH ratios. This is essentially what was found. The lactate-to-PYR ratio was reduced, and the calculated cytosolic NAD+-to-NADH ratio was significantly increased. However, the issue of whether cellular hypoxia exists has been challenged from several perspectives. In two studies using two different techniques to estimate mitochondrial redox potential during increased contractile activity (11,33), no evidence of mitochondria hypoxia could be documented. Further, Connett (4) demonstrated that at least in stimulated dog red muscle, glycolysis occurs in the absence of any evidence of cellular oxygen availability. Connett et al. (5) emphasized that increases in glycolysis and the cytosolic redox potential may be necessary for the efficient recruitment of mitochondrial oxidative phosphorylation (4,5). If this hypothesis is correct and in view of the fact that glycolysis and the redox potential was apparently lowered with training in the face of a constant oxidative phosphorylation, then additional adaptations would have to occur to maintain the redox gradient across the mitochondrial membrane (4,5). The nature of these adaptations remains uncertain. In summary, the major finding of this study is that muscle glycogen loss and lactate concentration during exercise are depressed with training and that this response is an early adaptive event occurring well in advance of increases in muscle mitochondrial capacity. Although yet to be proven experimentally, the reduction in muscle lactate concentration is strongly suggestive of a blunting of lactate production via glycolysis and not enhanced removal of PYR either via muscle mitochondria or by extramuscular sites. The reduction in several putative modulators of both phosphorylase and PFK is consistent with a depression of both glycogenolysis and glycolysis. The reduction in many of these modulators, such as ADP,, AMP,, and Pi, after training occurs as a consequence ,of a more conserved PCr concentration. As evidenced by the muscle metabolite profile, these adaptations appear to be mainly manifest soon after the onset of exercise. This study was supported by the Natural Sciences and Engineering Research Council of Canada.

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METABOLISM

AFTER

SHORT-TERM

Address reprint requests to H. J. Green.

145-149,1984. 19.

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Metabolic adaptations to training precede changes in muscle mitochondrial capacity.

To determine whether increases in muscle mitochondrial capacity are necessary for the characteristic lower exercise glycogen loss and lactate concentr...
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