Early muscular and metabolic adaptations to prolonged exercise training in humans H. J. GREEN, Department
S. JONES,
M. E. BALL-BURNETT,
D. SMITH,
J. LIVESEY,
AND B. W. FARRANCE
of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
prolonged training are well documented (21, 22, 36). On the basis of association between the training-induced alteration in energy metabolic behavior and the enzymatic activity (21, 22), it has been postulated that a primary mechanism accounting for the depression in anaerobic glycolytic flux rate observed during exercise is a reduced activation of phosphofructokinase (PFK). According to this schema, the activation of PFK is attenuated after training as a consequence of smaller exercise-induced changes in the metabolic effecters of PFK, namely the high-energy phosphate compounds such as ADP and AMP and phosphate (22). The reduced dependence on carbohydrate during exercise is believed to result not only from a blunting of the glycolytic response but, in addition, from an accelerated uptake and utilization of free fatty acids by the mitochondria in the trained muscle cells (22, 36). Although the fundamental mechanisms for the alteration in energy metabolic behavior and substrate utilization in the working muscle appear to be well established (31), particularly in “in vitro” animal preparations (7, 11), and are closely associated with changes in enzymatic potential (7,10, ll), such is not the case during voluntary exercise. During voluntary exercise, numerous other adaptations external to the working muscle cell, such as alterations in hormonal balance, substrate mobilization, and neuromuscular recruitment patterns, could conceivably account for the changed bioenergetic behavior. In previous work (14), we have been able to demonstrate substantial reductions in the blood catecholamines, epinephrine and norepinephrine, during prolonged exercise after only 3 days of training. In addition, a pronounced short term; energy metabolism; peripheral adaptations; en- reduction in muscle glycogen utilization was evident (14) zymes;fiber types without any apparent change in maximal enzymatic activity of any of the major energy metabolic pathways and segments (2). Collectively, these studies suggest that other adaptive strategies may prevail particularly during AMONG THE MOST PROMINENT adaptations reported to result from sustained increases in activity level are re- the early part of training and that the reduction in anaerobic glycolysis and carbohydrate utilization noted with ductions in anaerobic metabolism and carbohydrate utitraining need not be irrevocably linked to increases in the lization (22,36). These changes are readily apparent durmitochondrial enzymatic potential. ing prolonged exercise of moderate intensity and appear In this paper, we have concentrated on describing the to be manifested in the working muscle both by a reduced alteration that occurs in energy metabolism during volunendogenous glycogen utilization and a reduced muscle tary exercise and the degree to which these changes are lactate concentration (22). Collectively, these adaptations have been attributed to increases in the potential of related to adaptations in the muscle cell. To investigate these relationships we have employed a lo- to la-day the muscle cell for both aerobic metabolism and ,&oxidation (22, 36). Increases in the maximal activity of repretraining period, involving 2 h of prolonged exercise sentative enzymes of these metabolic pathways after per day. GREEN,H.J.,S. JONES,M.E. BALL-BURNETT,D. SMITH, J. LIVESEY,AND B. W. FARRANCE.Early muscular and metabolic adaptations to prolonged exercise training in humans. J. Appl. Physiol. 70(5): 2032-2038, 1991.-A short-term training program involving 2 h of daily exercise at 59% of peak 0, uptake (vozmax)repeated for lo-12 consecutive days was employed to determine the significance of adaptations in energy metabolic potential on alterations in energy metabolism and substrate utilization in working muscle. The initial vozrnax determined before training on the eight male subjectswas 53.0 t 2.0 (SE) ml kg-l. min-‘. Analysis of samplesobtained by needlebiopsy from the vastus lateralis musclebefore exercise (0 min) and at 15, 60, and 99 min of exercise indicated that on the average training resulted (P < 0.05) in a 6.5% higher concentration of creatinephosphate, a 9.9% lower concentration of creatine, and a 39%lower concentration of lactate. Training had no effect on ATP concentration. These adaptations were also accompanied by a reduction in the utilization in glycogen such that by the endof exerciseglycogen concentration was47.1% higher in the trained muscle.Analysis of the maximal activities of representative enzymes of different metabolic pathways and segments indicated no changein potential in the citric acid cycle (succinate dehydrogenase, citrate synthase), P-oxidation (3-hydroxyacyl CoA dehydrogenase),glucosephosphorylation (hexokinase), or potential for glycogenolysis (phosphorylase) and glycolysis (pyruvate kinase, phosphofructokinase, ar-glycerophosphate dehydrogenase, lactate dehydrogenase). With the exception of increasesin the capillary-to-fiber area ratio in type IIa fibers, no changewas found in any fiber type (types I, IIa, and IIb) for area, number of capillaries, capillary-to-fiber arearatio, or oxidative potential with training. It is concluded that the altered metabolism, probably indicative of a depressionin anaerobicglycolysis, neednot be accompaniedby peripheral adaptations in the musclemetabolic potential. l
2032
0161-7567/91 $1.50 Copyright 0 1991 the American Physiological Society
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METHODS
Experimental design. The training model utilized in this study consisted of lo-12 days of daily cycling at 59% of peak aerobic power (VO 2 m,) (determined before training) for 2 h/day. Before and after the training, gas exchange, ventilatory, and heart rate responses were determined during progressive cycle exercise to exhaustion. In addition, the effects of training on changes in gas exchange, ventilation, cardiac output, and core temperature were investigated during prolonged exercise performed at the same absolute power output before and after training. The procedures used to measure these parameters have been published previously (15). The data from both the progressive and prolonged exercise tasks are published elsewhere (12). The prolonged exercise task was also used to investigate the adaptations that occurred at both the blood and muscle levels. The blood data, including the response to a variety of metabolites, hormones, protein, and osmolality, constitute a second paper in this series (16). In this paper, we have concentrated on the energy metabolic aspects and selected adaptations to the muscle cell. As in the previous papers, the data are based on eight healthy males between the ages of 19 and 30 yr. 00, max in these active but untrained volunteer subjects averaged 3.94 t 0.11 (SE) l/min or 53.0 t 2.0 ml. kg-l min-‘. Extraction of the muscle samples from the vastus lateralis muscle was performed both before and during the exercise by the technique of Bergstrom (3). Approximately 90 min before the beginning of exercise, the subjects reported to the laboratory for insertion of a venous catheter in a dorsal hand vein and for preparation for the muscle biopsies. During this time four small incisions were made, two on each leg, for extraction of the muscle samples. One incision was used for the initial resting biopsy, whereas the remaining three were used to obtain samples at 15,60, and at pretraining fatigue or 120 min of exercise, whichever came first. Pretraining and posttraining biopsies were obtained at identical time points for each subject. The final biopsy was obtained at 99 t 5.6 min, the average exercise time for the subjects. Muscle biopsies representative of the exercise state were obtained by interrupting the ride briefly after all other relevant measurements at a given time point had been completed. This involved, in the order done, measurement of gas exchange, cardiac function, and core temperature and blood sampling. At a preset time the individual stopped cycling and fell back into the arms of an assistant while a second assistant simultaneously elevated the leg designated for the biopsy to a horizontal position. A third individual rapidly extracted the muscle sample with the use of suction and immediately plunged the needle into liquid N,. The tissue obtained from this biopsy was used for analysis of glycogen, selected glycolytic intermediates, and high-energy phosphate compounds. Immediately after the first biopsy, a second biopsy was obtained and used for histochemical measurements and biochemical determinations of enzyme activities. The tissue from both biopsies was stored at -80°C before analysis. Analytic procedures. Measurements of glycogen, glycol
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lytic intermediates, ATP, and creatine phosphate (CrP) were performed on freeze-dried tissue extracted according to the procedures of Harris et al. (20), with the use of fluorometric techniques (29) identical to those used previously in our laboratory (14,19). The concentrations of all variables with the exception of glucose, lactate, and pyruvate were corrected to the total creatine concentration, determined on each individual by averaging the values obtained on all biopsies. In the absence of changes in total creatine with exercise or training, neither of which was found to occur in this study, this procedure helps correct for discrepancies in nonmuscle components, such as fat and connective tissue, not visible in the freezedried tissue. Glucose, pyruvate, and lactate were not corrected because these compounds exist both in the muscle and in the interstitial space (26). To investigate the adaptations in the muscle in response to short-term, prolonged training, several different properties were examined. These properties included measurements of the maximal activities of several enzymes representative of different energy metabolic pathways and segments in mixed homogenates. In addition, we also measured, in fiber-specific types, the oxidative potential, size, and capillarization. Muscle samples for biochemical analysis were homogenized (1: 100 dilution) at 0-4OC in 0.17 M phosphate buffer at pH 7.4 that contained 0.2% bovine serum albumin (BSA) and 5 mM 2mercaptoethanol. The maximal activities of all the selected enzymes were measured fluorometrically and at room temperature (22-23°C) with the use of the linear portion of the reaction time curve. Complete details of all the enzyme assays have been published previously (13, 18). The values are expressed in prnol. g protein-l min-l. Protein was measured by the Lowry technique with the use of the modification of Polachek and Cabib (34). For a given individual, all enzyme measurements, both pre- and posttraining, were performed on a single day without freezing the homogenate. Histochemical properties were determined on cross sections of tissue cut in a cryostat maintained at -20°C. Muscle fiber separation and typing was based on the procedure of Brooke and Kaiser (5) as modified by Doriguzzi et al. (9). The oxidative potential of individual fiber types was determined with the use of microphotometric procedures (32) according to modifications published previously (17). With this procedure, only end-point determinations are employed with measurements of optical density (OD) recorded from the core of the fiber. We have been able to obtain very high correlations between measurements of OD determined for periods of up to 45 min after the initiation of the reaction and the initial reaction rate velocity determined by multiple measurements during the first 2 min of the reaction (17). Oxidative potential was assessed with the use of histochemical stains for both succinic dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). For all enzymes, the OD measured for the blank, representing the mounting medium, glass slide, and cover slip just outside the section being measured, was subtracted from the OD measured within the fiber core. In addition, for SDH we have also subtracted the change in OD due to “nothing dehydrogenase” activity l
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2034
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(i.e., the nonspecific reduction of the reaction indicator, nitro blue tetrazolium) (4). The value for nothing dehydrogenase was determined on separate pieces of tissue that had been stained under conditions as nearly identical as possible. This adjustment was necessary because it has been recently demonstrated that the nothing-dehydrogenase reaction may represent a substantial component of the OD of the tissue and not a minor fraction as previously assumed (32). Because no difference between fiber types was found for the nothing-dehydrogenase reaction, a constant value has been subtracted from each fiber. Tissue sections stained for NADH-TR were not corrected for nothing dehydrogenase because we have found the change in OD to be negligible. Muscle capillarization was demonstrated with the use of the amylase and periodic acid-Schiff (PAS) procedure (1) on sections cut at 16.pm thickness. Fiber sections were determined from the NADH-TR sections with the use of planimetry. Capillary density was determined by dividing the fiber area (FA) into the number of capillaries in contact with each fiber (CC). On the average, 75 type I, 80 type IIa, and 40 type IIb fibers were randomly selected for the measurements of histochemical properties. As a result of insufficient tissue, poor cross sections, or freeze artifact, equal subject numbers were not available for all histochemical stains. The subject number employed for each stain was that from which we had both a pre- and posttraining sample. Statistical procedures. To determine the effects of exercise time and training, a two-way analysis of variance (ANOVA) for repeated measures was employed. Where significance was indicated, post hoc analysis with the use of the Newman-Keuls technique was performed to compare specific means. Where only two measures were available, as with the enzymatic and histochemical data, Student’s t tests for correlated samples were used. A 95% level of confidence was accepted for all comparisons.
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A
30 I
I 15’
1
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I
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,,, \,\\ i ‘,, 4x I ,,I ,, -----0-0..--w_ -0---0--0--_----w---a---r P P
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40-
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$
60
RESULTS
Mu&e metabolites. As expected, the concentrations of ATP, CrP, and creatine were all significantly altered (P < 0.05) with exercise (Fig. 1). In the case of CrP, an ~38% reduction was observed by the time of the first exercise biopsy at 15 min. Thereafter, no further change in CrP was observed over the remaining period of exercise. The change in creatine demonstrated a similar time course and was approximately stoichiometric. With ATP, the change was more delayed, with reductions over rest values not being observed until the end of exercise, which averaged 99 t 5.6 min. Training was found to alter only the CrP and creatine concentrations (P < 0.05). With training, higher CrP and lower creatine concentrations were observed. Changes in several of the glycolytic intermediates were also found with exercise (Table 1). Glucose l-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6phosphate, pyruvate, and lactate were all increased with exercise (P < 0.05). However, differences were observed in the time at which the changes occurred. For glucose l-phosphate, glucose 6=phosphate, and fructose 6-phosphate, the increase was only evident at 15 min of exer-
0
15
30
45
TIME
60
75
90
105
(min)
FIG. 1. Changes in muscle ATP, creatine phosphate (CrP), and creatine concentrations during exercise before (Pre) and after training (Post). Values are means t SE of 8 subjs. Main effect for time (P < 0.05): CrP, 0 > 15 = 60 = 99 min; creatine, 0 < 15 = 60 = 99 min; ATP, 0 = 15 = 30 < 99 min. In addition, a main effect (P < 0.05) for training was found for CrP and creatine (P < 0.05). Interaction effects were not significant.
cise. For fructose 1,6-phosphate, exercise resulted in higher concentrations at 60 and 99 min of exercise over rest but not at 15 min. The increase in pyruvate noted at 15 min of exercise was sustained throughout the two remaining periods, whereas lactate increased at 15 min and then declined at 60 and 99 min. Despite the decline in lactate after 15 min of exercise, the concentrations at 60 and 99 min remained higher than the rest values. Of the glycolytic intermediates studied, training was found to modify only the lactate responses, with lower concentrations observed after the short-term training program (P < 0.05).
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AND METABOLIC
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TO TRAINING
TABLE 1. Effects of exercise and training on selected glycolytic intermediates in muscle Time, min 0
G-6-P Pre Post G-1-P Pre Post F-6-P Pre Post F-1,6-P Pre Post Pyruvate Pre Post Lactate Pre Post
1.38kO.31 0.79t0.17
15
60
99
2.4620.47
2.32kO.39
1.9lt0.40
2.87tl.l
1.43kO.32
1.58t0.25
0.050t0.01 0.049t0.01
0.116t,O.O2
0.117t0.02
0.125t0.05
0.073t0.01
0.118t0.03 0.070t0.02
0.199*0.05 0.1151kO.02
0.327t0.05 0.35lkO.12
0.276t0.05 0.194t0.04
0.232t0.05 0.208kO.04
0.299t0.07 0.137t0.03
0.330t0.05 0.326t0.08
0.605-t-0.14 0.385tO.11
0.439t0.08 0.383kO.10
0.190t0.04
0.439t0.09
0.388t0.09
0.48lt0.14
0.203t0.07
0.317t0.05
0.317t0.07
0.364t0.09
7.76tl.5 6.0821.43
29.0t7.0 15.0t5.9
16.5k3.1 10.4t2.0
60. I
8
17.lk5.0 11.222.2
Values are means t SE in mmol/kg dry wt; n = 8 subjs. Pre, pretraining; Post, posttraining. A main effect of time was found for glucose 6-phosphate (G-6-P), glucose l-phosphate (G-l-P), fructose 6-phosphate (F-6-P), fructose 1,6-phosphate (F-1,6-P), pyruvate, and lactate (P < 0.05). For G-6-P, G-l-P, and F-6-P, 15 > 0 = 60 and 99 min; for F-1,6-P, 60 = 99 > 0 = 15 min; for pyruvate, 15 = 60 = 99 > 0; for lactate, 15 > 60 and 99 > 0 min. For lactate, a main effect of training was also found (P < 0.05).
For two additional cellular compounds studied, glu10L . ’ 1 1 I I I 1 I cose and glycogen, only glycogen was modified by the 0 15 30 45 60 75 90 105 exercise and training (Fig. 2). With exercise, glycogen TIME (min) was reduced, with progressively lower (P < 0.05) values FIG. 2. Changes in muscle glucose and glycogen concentrations durobserved at each of the exercise time points. On average, ing exercise before and after training. Values are means t SE of 8 subjs. the concentration was reduced 20% at 15 min, 30% at 60 Main effects (P < 0.05) for time and training were found for glycogen min, and 43% by 99 min of exercise. Training resulted in only (P < 0.05). For glycogen, 0 > 15 > 60 > 99 min. Interaction effects were not significant. a persistently higher concentration of glycogen. Muscle enzymes. The enzymes selected for measureTABLE 2. Effect of short-term endurance training ment were designed to represent the metabolic pathways on maximal enzymatic activities and segments involved in glycogenolysis (total phosphorylase), glycolysis [pyruvate kinase (PK), PFK, aPre Post glycerophosphate dehydrogenase (a-GPDH), and lactate dehydrogenase (LDH)], glucose phosphorylation [ hexo67.5t8.8 SDH 76.9k7.5 kinase (HK)], P-oxidation [3=hydroxyacyl CoA dehydro48.924.4 cs 60.026.2 HAD 40.8t3.6 43.6t4.1 genase (HAD)], the citric acid cycle [SDH and citrate HEK 12.5tl.l 13.7kO.58 synthase (CS)], and high-energy phosphate transfer [crePHOS 106tll 96.325.4 atine phosphokinase (CPK)]. Analysis of biopsies from PK 1,507&115 1,558+150 pre- and posttraining indicated that the maximal activiPFK 293+24 259k16 ties of none of these enzymes were affected by training c~-GPDH 23.3tl.8 23.9H.7 LDH 1,776+194 1,666_+148 (P > 0.05). The most conspicuous increases were obCPK 9,076_+1,091 8,889+536 served for SDH and CS. However, in neither case was the difference sufficient to meet the level of confidence esValues are means t SE in pmol g protein-’ min-‘; n = 8 subjs. SDH, succinate dehydrogenase; CS, citrate synthase; HAD, 3-hytablished for the study (Table 2). droxyacyl CoA dehydrogenase; HK, hexokinase; PHOS, phosphoryMuscle histochemistry. The effects of training on fiber lase; PK, pyruvate kinase; PFK, phosphofructokinase; wGPDH, CYtype distribution, fiber type area, and capillarization are glycerophosphate dehydrogenase; LDH, lactate dehydrogenase; CPK, presented in Table 3. Training failed to alter the districreatine phosphokinase. bution of type I, type IIa, and type IIb fibers in the vastus lateralis muscle (P > 0.05). Similarly, no changes were capillaries per unit area, a training effect (P < 0.05) was observed, but only for the type IIa fibers. detectable in the area or in the number of capillaries surrounding these fiber specific types (P > 0.05). When Microphotometric determinations of NADH-TR and the area of each fiber type was divided into the number of SDH in fiber specific types were performed to evaluate capillaries to produce a ratio representing the number of whether training differentially affected the oxidative pol
l
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3. Effect of training on muscle fiber type, distribution, size, and capillarization TABLE
Fiber n
Fiber type, % Pre Post Area, pm2 Pre Post CAP Pre Post Cap/area, pm2/10w3 Pre Post Values are means cantly different from
I
Type IIa
7
45.4k3.0 44.9k3.6
39.3t2.8 41.1t3.5
6
4,539+476 4,462+478
5,346_+481 4,894+420
IIb
15.3t3.3 13.6-t-3.6 4,682-t406 4,365?276
5
4.3720.32 4.67kO.44
4.45t0.28 4.55kO.38
3.58t0.34 3.77kO.32
5
1.05t_o.o7 1.llt0.11
0.87t0.07 0.98t0.04*
O.SZt_O.OS 0.95t0.06
t SE; n, no. of fibers. Pre (P < 0.05).
Cap, capillaries.
* Signifi-
tential in the different fiber types (Table 4). Training was not observed to alter either the NADH-TR or SDH of any type I, type IIa, or type IIb fibers identified. DISCUSSION
This study has demonstrated that lo-12 days of prolonged cycle training induced an alteration in muscle energetics and endogenous glycogen such that the changes in both CrP and lactate concentration were attenuated and glycogen depletion was less emphasized during the moderate exercise challenge. In this respect, the training adaptations are consistent with what has previously been observed in exercising humans after training (21,25,37) and what has been demonstrated with the use of the technique of electrical stimulation to increase acutely muscle activity in animal preparations (7, 11). However, unlike previous studies, these metabolic changes were not accompanied by significant alterations in any of the energy metabolic enzymes that were measured and most particularly in the markers selected to represent oxidative potential, SDH and CS. Because only an increase in the capillary-to-fiber area ratio was found in the type IIa fibers after training without any change in the other fiber types on this parameter and without changes in remaining parameters of size, capillary number, and oxidative potential of the individual fiber types, it would appear that, in general, these measures alone cannot explain the change in substrate and metabolic behavior. However, some qualification of these conclusions appear appropriate. Within the statistical confines imposed, namely the selection of 295% confidence level for all comparisons, the conclusions stated are technically correct. As noted by the trends in some of the data, the direction of change is consistent with what has been found in previous training studies, particularly with regard to the increases in oxidative potential and capillaryto-fiber area ratios. For the enzymes used to represent oxidative potential, SDH and CS, the specific probabilities (P) were 0.096 and 0.111, respectively. Similarly for the measure of the number of capillaries per unit area, the calculated P values were 0.180 for type I fibers, 0.028 for type IIa fibers, and 0.095 for type IIb fibers. Whether
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these changes have physiological significance cannot be determined by this study. The only conclusion that can be made is that, with regard to the adaptive changes in muscle enzymology and histochemistry, the magnitude of the increase was not sufficient to provide the confidence needed that such changes did not happen by chance. Of particular significance to the conclusions of this study is the interpretation of the changes that occurred in energy metabolism and substrate utilization after training. Statistically, only main effects of training and exercise were demonstrated for the CrP, glycogen, and lactate data. A significant interaction effect between time and training was not found. Strictly interpreted, this means that the training effect was not expressed during exercise alone but during rest and exercise. However, analysis of the resting mean values before and after training for all three variables indicated that none of the differences was significant (P > 0.05) when compared with paired t tests. When ANOVA techniques were applied to the exercise samples only, a significant difference (P < 0.05) was observed between the pre- and posttraining samples for each of the variables. Accordingly, our interpretation of the data is consistent with this statistical finding, namely that training modified the exercise response only. A sparing of muscle glycogen utilization after training in conjunction with a lower muscle lactate concentration has most often been interpreted to mean that there has been an increase in free fatty acid utilization and a reduction in anaerobic glycolysis (7,21,23), a position that has been repeatedly emphasized in authoritative reviews (22, 36). These experimental training studies, particularly in regard to the human, have also been able to demonstrate that at the same absolute submaximal work rate before and after training steady-state 0, uptake (VO,) was not significantly elevated (21,25,37), a finding that supports the notion that the attenuated metabolic responses are not due to increased aerobic metabolism. Our study is also consistent with this finding (12). The lack of a change in aerobic metabolism during submaximal exercise after training occurred despite a small but significant increase of 4.3% in Vogmax induced by the training (12). The observation that exercise Vo2 did not change with training would also suggest that even with the increase in capillary density found for the type IIa fibers in this study, mitochondrial 0, utilization was not enhanced. In addition, a reduction in CO, production TABLE 4. Training-induced changes in fiber type oxidative and glycolytic potential Fiber I
NADH Pre Post SDH Pre Post Values
Type IIa
IIb
0.733t0.02 0.747t0.03
0.400t0.02 0.449t0.03
0.442t0.06 0.458kO.04
0.138t0.02 0.138t0.01
0.084t0.01 0.087t0.01
0.078t_O.O1 0.083t0.01
are means
t SE of 6 subjs.
Optical
density
measured
at 546
nm.
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(hog) and R have been reported (21,23) and the results of this study support these cha nges (12) The decrease in R has been used to suggest an increase 1.n fat utilization during exercise (21, 23, 30). However, other mechanisms, in addition to a reduced anaerobic glycolysis and increased free fatty acid utilization after training, may be equally plausible to account for the reduced muscle lactate and glycogen sparing effect. One possibility is that fuel utilization (carbohydrate vs. fat) was not altered after training, the lower muscle lactate concentration being due to an elevated removal by extramuscular tissues or oxidization within the muscle itself (8, 27) and the glycogen sparing occurring as a result of enhanced blood glucose uptake and utilization either for glycolysis or for glycogenesis. Although the design of this study was not appropriate for examining these possibilities, several observations seem appropriate. Glucose can represent an extremely important substrate in work of this intensity and duration and is capable of compensating for the reduction in muscle glycogen utilization (38). Furthermore, it has been reported that, during exercise, glycogenesis may occur in the working muscle with glucose acting as a substrate (24). Despite the fact that no training-induced changes were found in blood insulin concentration in this study (16), recent evidence would suggest that, with exercise and training, little if any insulin is needed to facilitate glucose transport across the cell membrane (33). Furthermore, it is possible that the lower R was not due to increased utilization of fat but was due to a reduction in excess CO, mediated by a reduced acidosis (28). In this study, this probability appears plausible because we have found a lower ventilation (VE) accompanied by a lower ventilatory equivalent for 0, (vE/vO,) and a lower arterialized blood lactate concentration (12, 16). Reduction in glycogenolysis and glycolysis as the major mechanism mediating the lower muscle lactate response with exercise after training remains the most popular interpretation (22,27,36), and there is some experimental evidence to support this contention at least in animal preparations subjected to involuntary increases in muscle activity (7,11). The reduced glycolytic flux rate has been proposed to result from an inhibition of PFK occurring as a result of a depressed increase in several metabolic effecters of the enzyme, such as ADP, AMP, and Pi and ammonia (10,22). Although the precise mechanism remains to be demonstrated, the changes in these metabolic byproducts are such as to support this hypothesis, at least in anesthetized rats after a strenuous running program (7). In this study, we have found a less pronounced reduction in CrP with exercise after training. However, ATP concentration was not altered. Whether the less pronounced reduction in CrP (and expected reduction in Pi) altered metabolic control is questionable. At least, on the basis of glycolytic intermediates that, with the exception of lactate, were unchanged with training, flux rate did not appear to be altered. It is possible, however, that at this work load our assays were not sensitive enough to detect the subtle decreases that might be expected in prolonged exercise after training. The significance of Pi in effecting changes in phosphorylase activity and glycogenolysis is well established (6),
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and it would be productive to determine whether the reduction in Pi after training has any significance in this regard. It has been suggested that the proposed reduction in anaerobic glycolysis after training is intimately coupled to increases in oxidative potential mediated by increases in size and number of mitochondria (22,36). This association, at least between lower muscle lactate concentration and increases in oxidative potential with training, is indisputable, particularly in nonhuman studies employing rats (7,11) and in the limited number of training studies on humans (21, 37). However, these associations have been established after relatively long-term training programs. These associations do not preclude the existence of other adaptive strategies, particularly early in training. That other adaptive strategies are possible is emphasized by the results of this study. As suggested, the results could simply be explained by a shift toward a greater dependence on blood glucose acting as a substrate for glycolysis and/or as a substrate for glycogenesis, a comparable glycolytic flux rate, and an enhanced lactate clearance after training. Alternatively, glycolytic rate could have been reduced as a consequence of a decrease in selected metabolic effecters as proposed but by an essentially different mechanism. Holloszy et al. (22) have speculated that, with the increase in mitochondria size and number, the increase in ADP and Pi need not be as great to stimulate increases in mitochondrial respiration necessary to attain the pretraining level of VO,. Essentially, the same mechanism could be operative after training without increases in oxidative potential if the neuromuscular recruitment strategy were altered. This could occur by distributing the work load to more muscles and muscle fibers, thereby lessening the work done by a single muscle fiber. The fact that considerable plasticity has been documented in the neural system as an early response to resistance training (35) makes such a hypothesis inviting. The authors thank Ian Fraser for the excellent technical assistance provided during the study. This study was supported by a grant from the National Science and Engineering Research Council (NSERC). Address reprint requests to H. J. Green. Received
26 June
1989; accepted
in final
form
17 December
1990.
REFERENCES 1.
2.
3. 4.
5. 6.
7.
ANDERSON, P., AND J. HENRIKSSON. Capillary
supply of the quadriceps femoris muscle of man. Adaptive response to exercise. J. whysiol. Lond. 270: 677-690, 1977. BALL, M. E., H. J. GREEN, AND M. E. HOUSTON. Alterations in human muscle fibre type distribution induced by acute exercise. Histochemistry 79: 53-57, 1983. BERGSTROM, J. Muscle electrolytes in man. Scand. J. Clin. Lab. Inuest. 68, Suppl.: l-110, 1962. BLANCO, C. E., G. C. SIECK, AND V. R. EDGERTON. Quantitative histochemical determination of succinic dehydrogenase activity on skeletal muscles. Histochem. J. 20: 230-243, 1988. BROOKE, M. H., AND K. K. KAISER. Muscle fibre types. How many and what kind? Arch. Neurol. 23: 369-379, 1970. CHASIOTIS, D.,K. SAHLIN,AND E. HULTMAN. Regulationofglycogenolysis in human skeletal muscle at rest and during exercise. J. Appl. Physiol. 53: 708-715, 1982. CONSTABLE, S. H., R. J. FAVIER, J. A. MCLANE, R. D. FELL, M. CHEN, AND J. 0. HOLLOSZY. Energy metabolism in contracting rat
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2038
8.
9.
10.
11.
12.
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14.
15.
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22.
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AND
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skeletal muscle: adaptation to exercise training. Am. J. Physiol. 253 (Cell Physiol. 22): C316-C322, 1987. DONOVAN, C. M., AND G. A. BROOKS. Endurance training affects lactate clearance not lactate production. Am. J. Physiol. 244 (EndocrinoZ. Metab. 7): E83-E92, 1983. DORIGUZZI, C., T. MONGINI, L. PALMUCCI, AND D. SCHIFFER. A new method for myofibrillar Ca2+-ATPase reaction based on the use of metachromatic dyes: its advantages in muscle fibre typing. Histochemistry 79: 289-294, 1983. DUDLEY, G. A., P. C. TULLSON, AND R. L. TERJUNG. Influence of mitochondrial content on the sensitivity of respiratory control. J. BioZ. Chem. 262: 9109-9114,1987. FAVIER, R. J., S. H. CONSTABLE, M. CHEN, AND J. 0. HOLLOSZY. Endurance training reduces lactate production. J. AppZ. Physiol. 61: 885-889,1986. GREEN, H. J., G. COATES, J. R. SUTTON, AND S. JONES. Early adaptations in gas exchange, cardiac function, and hematology to prolonged exercise training in man. Eur. J. AppZ. Physiol. Occup. Physiol. In press. GREEN, H. J., I. G. FRASER, AND D. A. RANNEY. Male and female differences in enzyme activities of energy metabolism in vastus lateralis muscle. J. Neural. Sci. 65: 323-331, 1984. GREEN, H. J., L. L. JONES, M. E. HOUSTON, M. E. BALL-BURNETT, AND B. W. FARRANCE. Muscle energetics during prolonged cycling after exercise hypervolemia. J. AppZ. Physiol. 66: 622-631, 1989. GREEN, H. J., L. L. JONES, AND D. C. PAINTER. Effects of shortterm training on cardiac function during prolonged exercise. Med. Sci. Sports Exercise 22: 488-493, 1990. GREEN, H. J., S. JONES, M. E. BALL-BURNETT, D. SMITH, J. LIVESEY, AND B. W. FARRANCE. Early adaptations in blood substrates, metabolites, and hormones to prolonged exercise training in man. Can. J. Physiol. Pharmacol. In press. GREEN, H. J., M. MORRISSEY, D. SMITH, AND I. FRASER. Relationship between microphotometric determinations of succinic dehydrogenase activity in single fibers using kinetics and end point criteria. Med. Sci. Sports Exercise 17: 192, 1985. GREEN, H. J., J. R. SUTTON, A. CYMERMAN, P. M. YOUNG, AND C. S. HOUSTON. Operation Everest II: adaptations in human skeletal muscle. J. AppZ. Physiol. 66: 142-150, 1989. GREEN, H. J., J. A. THOMSON, AND M. E. HOUSTON. Supramaximal exercise after training-induced hypervolemia. II. Blood/muscle substrates and metabolites. J. AppZ. Physiol. 62: 1954-1961, 1987. HARRIS, R. C., E. HULTMAN, AND L.-O. NORDES~O. Glycolytic intermediates and high energy phosphates determined in biopsy samples of musculus femnis of man at rest. &and. J. CZin. Lab. Invest. 33: 102-120,1974. HENRIKSSON, J. Training induced adaptation of skeletal muscle and metabolism during submaximal exercise. J. Physiol. Lond. 270: 661-675,1977. HOLLOSZY, J. O., AND E. F. COYLE. Adaptations of skeletal muscle
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24. 25.
26.
27. 28.
29. 30.
31. 32.
33.
34.
35.
36.
37.
38.
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to endurance exercise and their metabolic consequences. J. AppZ. Physiol. 56: 831-838, 1984. HURLEY, B. F., P. M. NEMETH, W. H. MARTIN III, J. M. HAGBERG, AND G. P. DALSKY. Muscle triglyceride utilization during exercise: effect of training. J. AppZ. Physiol. 60: 562-567, 1986. HUTBER, C. A., AND A. BONEN. Glycogenesis in muscle and liver during exercise. Acta Physiol. &and. 134: 159-160, 1988. KARLSSON, J., L.-O. NORDESJO, L. JORFELDT, AND B. SALTIN. Muscle lactate, ATP and CP levels during exercise after physical training in man. J. AppZ. Physiol. 33: 199-203, 1972. KATZ, A., S. BROBERG, K. SAHLIN, AND J. WAHREN. Leg glucose uptake during maximal dynamic exercise in humans. Am. J. Physiot. 251 (Endocrinol. Metab. 14): E65-E70, 1986. KATZ, A., AND K. SAHLIN. Regulation of lactic acid production during exercise. J. AppZ. Physiol. 65: 509-518, 1988. KOYAL, S. N., B. J. WHIPP, D. HUNTSMAN, G. A. BRAY, AND K. WASSERMAN. Ventilatory responses to the metabolic acidosis of treadmill and cycle ergometry. J. AppZ. Physiol. 40: 864-867, 1976. LOWRY, 0. H., AND J. V, PASSONNEAU. A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 146-218. MOORE, R. L., E. M. THACKER, G. A. KELLEY, T. I. MUSCH, L. I. SINOWAY, V. L. FOSTER, AND A. L. DICKINSON. Effect of training/ detraining on submaximal exercise responses in humans. J. AppZ. Physiol. 63: 1719-1724, 1987. NEWSHOLME, E. A., AND A. R. LEECH. Biochemistry for the Medical Sciences. New York: Wiley, 1983, p. 357-379. PETTE, D. Microphotometric measurement of initial reaction rates in quantitative enzyme histochemistry in situ. Histochem. J. 13: 319-327,1981. PLOUG, T., H. GALBO, AND E. A. RICHTER. Increased muscle glucose uptake during contractions. No need for insulin. Am. J. Physiol. 247 (Endocrinol. Metab. 10): E726-E731, 1983. POLACHEK, K. I., AND E. CABIB. A simple procedure for protein determination by the Lowry method in dilute solution and in the presence of interfering substances. AnaZ. Biochem. 117: 31 l-314, 1981. SALE, D. G. Neural adaptation in strength and power training. In: Human Muscle Power, edited by N. L. Jones, N. McCartney, and A. J. McComas. Champaign, IL: Human Kinetics, 1986, p. 281-305. SALTIN, B., AND P. D. GOLLNIGK. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. Skeletal MuscZe. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 10, p. 551-631. SALTIN, B., K. NAZAR, D. L. COSTILL, E. STEIN, E. JANSSON, B. ESSEN, AND P. D. GOLLNICK. The nature of the training response: peripheral and central adaptations to one-legged exercise. Acta Physiol. Stand. 96: 289-305, 1976. WAHREN, J., G. AHLBORG, P. FELIG, AND L. JORFELDT. Glucose metabolism during exercise in man. In: Muscle Metabolism During Exercise. New York: Plenum, 1970, p. 189-203.
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S. JONES,
M. E. BALL-BURNETT,
D. SMITH,
J. LIVESEY,
AND B. W. FARRANCE
of Kinesiology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
prolonged training are well documented (21, 22, 36). On the basis of association between the training-induced alteration in energy metabolic behavior and the enzymatic activity (21, 22), it has been postulated that a primary mechanism accounting for the depression in anaerobic glycolytic flux rate observed during exercise is a reduced activation of phosphofructokinase (PFK). According to this schema, the activation of PFK is attenuated after training as a consequence of smaller exercise-induced changes in the metabolic effecters of PFK, namely the high-energy phosphate compounds such as ADP and AMP and phosphate (22). The reduced dependence on carbohydrate during exercise is believed to result not only from a blunting of the glycolytic response but, in addition, from an accelerated uptake and utilization of free fatty acids by the mitochondria in the trained muscle cells (22, 36). Although the fundamental mechanisms for the alteration in energy metabolic behavior and substrate utilization in the working muscle appear to be well established (31), particularly in “in vitro” animal preparations (7, 11), and are closely associated with changes in enzymatic potential (7,10, ll), such is not the case during voluntary exercise. During voluntary exercise, numerous other adaptations external to the working muscle cell, such as alterations in hormonal balance, substrate mobilization, and neuromuscular recruitment patterns, could conceivably account for the changed bioenergetic behavior. In previous work (14), we have been able to demonstrate substantial reductions in the blood catecholamines, epinephrine and norepinephrine, during prolonged exercise after only 3 days of training. In addition, a pronounced short term; energy metabolism; peripheral adaptations; en- reduction in muscle glycogen utilization was evident (14) zymes;fiber types without any apparent change in maximal enzymatic activity of any of the major energy metabolic pathways and segments (2). Collectively, these studies suggest that other adaptive strategies may prevail particularly during AMONG THE MOST PROMINENT adaptations reported to result from sustained increases in activity level are re- the early part of training and that the reduction in anaerobic glycolysis and carbohydrate utilization noted with ductions in anaerobic metabolism and carbohydrate utitraining need not be irrevocably linked to increases in the lization (22,36). These changes are readily apparent durmitochondrial enzymatic potential. ing prolonged exercise of moderate intensity and appear In this paper, we have concentrated on describing the to be manifested in the working muscle both by a reduced alteration that occurs in energy metabolism during volunendogenous glycogen utilization and a reduced muscle tary exercise and the degree to which these changes are lactate concentration (22). Collectively, these adaptations have been attributed to increases in the potential of related to adaptations in the muscle cell. To investigate these relationships we have employed a lo- to la-day the muscle cell for both aerobic metabolism and ,&oxidation (22, 36). Increases in the maximal activity of repretraining period, involving 2 h of prolonged exercise sentative enzymes of these metabolic pathways after per day. GREEN,H.J.,S. JONES,M.E. BALL-BURNETT,D. SMITH, J. LIVESEY,AND B. W. FARRANCE.Early muscular and metabolic adaptations to prolonged exercise training in humans. J. Appl. Physiol. 70(5): 2032-2038, 1991.-A short-term training program involving 2 h of daily exercise at 59% of peak 0, uptake (vozmax)repeated for lo-12 consecutive days was employed to determine the significance of adaptations in energy metabolic potential on alterations in energy metabolism and substrate utilization in working muscle. The initial vozrnax determined before training on the eight male subjectswas 53.0 t 2.0 (SE) ml kg-l. min-‘. Analysis of samplesobtained by needlebiopsy from the vastus lateralis musclebefore exercise (0 min) and at 15, 60, and 99 min of exercise indicated that on the average training resulted (P < 0.05) in a 6.5% higher concentration of creatinephosphate, a 9.9% lower concentration of creatine, and a 39%lower concentration of lactate. Training had no effect on ATP concentration. These adaptations were also accompanied by a reduction in the utilization in glycogen such that by the endof exerciseglycogen concentration was47.1% higher in the trained muscle.Analysis of the maximal activities of representative enzymes of different metabolic pathways and segments indicated no changein potential in the citric acid cycle (succinate dehydrogenase, citrate synthase), P-oxidation (3-hydroxyacyl CoA dehydrogenase),glucosephosphorylation (hexokinase), or potential for glycogenolysis (phosphorylase) and glycolysis (pyruvate kinase, phosphofructokinase, ar-glycerophosphate dehydrogenase, lactate dehydrogenase). With the exception of increasesin the capillary-to-fiber area ratio in type IIa fibers, no changewas found in any fiber type (types I, IIa, and IIb) for area, number of capillaries, capillary-to-fiber arearatio, or oxidative potential with training. It is concluded that the altered metabolism, probably indicative of a depressionin anaerobicglycolysis, neednot be accompaniedby peripheral adaptations in the musclemetabolic potential. l
2032
0161-7567/91 $1.50 Copyright 0 1991 the American Physiological Society
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MUSCULAR
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METHODS
Experimental design. The training model utilized in this study consisted of lo-12 days of daily cycling at 59% of peak aerobic power (VO 2 m,) (determined before training) for 2 h/day. Before and after the training, gas exchange, ventilatory, and heart rate responses were determined during progressive cycle exercise to exhaustion. In addition, the effects of training on changes in gas exchange, ventilation, cardiac output, and core temperature were investigated during prolonged exercise performed at the same absolute power output before and after training. The procedures used to measure these parameters have been published previously (15). The data from both the progressive and prolonged exercise tasks are published elsewhere (12). The prolonged exercise task was also used to investigate the adaptations that occurred at both the blood and muscle levels. The blood data, including the response to a variety of metabolites, hormones, protein, and osmolality, constitute a second paper in this series (16). In this paper, we have concentrated on the energy metabolic aspects and selected adaptations to the muscle cell. As in the previous papers, the data are based on eight healthy males between the ages of 19 and 30 yr. 00, max in these active but untrained volunteer subjects averaged 3.94 t 0.11 (SE) l/min or 53.0 t 2.0 ml. kg-l min-‘. Extraction of the muscle samples from the vastus lateralis muscle was performed both before and during the exercise by the technique of Bergstrom (3). Approximately 90 min before the beginning of exercise, the subjects reported to the laboratory for insertion of a venous catheter in a dorsal hand vein and for preparation for the muscle biopsies. During this time four small incisions were made, two on each leg, for extraction of the muscle samples. One incision was used for the initial resting biopsy, whereas the remaining three were used to obtain samples at 15,60, and at pretraining fatigue or 120 min of exercise, whichever came first. Pretraining and posttraining biopsies were obtained at identical time points for each subject. The final biopsy was obtained at 99 t 5.6 min, the average exercise time for the subjects. Muscle biopsies representative of the exercise state were obtained by interrupting the ride briefly after all other relevant measurements at a given time point had been completed. This involved, in the order done, measurement of gas exchange, cardiac function, and core temperature and blood sampling. At a preset time the individual stopped cycling and fell back into the arms of an assistant while a second assistant simultaneously elevated the leg designated for the biopsy to a horizontal position. A third individual rapidly extracted the muscle sample with the use of suction and immediately plunged the needle into liquid N,. The tissue obtained from this biopsy was used for analysis of glycogen, selected glycolytic intermediates, and high-energy phosphate compounds. Immediately after the first biopsy, a second biopsy was obtained and used for histochemical measurements and biochemical determinations of enzyme activities. The tissue from both biopsies was stored at -80°C before analysis. Analytic procedures. Measurements of glycogen, glycol
ADAPTATIONS
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TRAINING
2033
lytic intermediates, ATP, and creatine phosphate (CrP) were performed on freeze-dried tissue extracted according to the procedures of Harris et al. (20), with the use of fluorometric techniques (29) identical to those used previously in our laboratory (14,19). The concentrations of all variables with the exception of glucose, lactate, and pyruvate were corrected to the total creatine concentration, determined on each individual by averaging the values obtained on all biopsies. In the absence of changes in total creatine with exercise or training, neither of which was found to occur in this study, this procedure helps correct for discrepancies in nonmuscle components, such as fat and connective tissue, not visible in the freezedried tissue. Glucose, pyruvate, and lactate were not corrected because these compounds exist both in the muscle and in the interstitial space (26). To investigate the adaptations in the muscle in response to short-term, prolonged training, several different properties were examined. These properties included measurements of the maximal activities of several enzymes representative of different energy metabolic pathways and segments in mixed homogenates. In addition, we also measured, in fiber-specific types, the oxidative potential, size, and capillarization. Muscle samples for biochemical analysis were homogenized (1: 100 dilution) at 0-4OC in 0.17 M phosphate buffer at pH 7.4 that contained 0.2% bovine serum albumin (BSA) and 5 mM 2mercaptoethanol. The maximal activities of all the selected enzymes were measured fluorometrically and at room temperature (22-23°C) with the use of the linear portion of the reaction time curve. Complete details of all the enzyme assays have been published previously (13, 18). The values are expressed in prnol. g protein-l min-l. Protein was measured by the Lowry technique with the use of the modification of Polachek and Cabib (34). For a given individual, all enzyme measurements, both pre- and posttraining, were performed on a single day without freezing the homogenate. Histochemical properties were determined on cross sections of tissue cut in a cryostat maintained at -20°C. Muscle fiber separation and typing was based on the procedure of Brooke and Kaiser (5) as modified by Doriguzzi et al. (9). The oxidative potential of individual fiber types was determined with the use of microphotometric procedures (32) according to modifications published previously (17). With this procedure, only end-point determinations are employed with measurements of optical density (OD) recorded from the core of the fiber. We have been able to obtain very high correlations between measurements of OD determined for periods of up to 45 min after the initiation of the reaction and the initial reaction rate velocity determined by multiple measurements during the first 2 min of the reaction (17). Oxidative potential was assessed with the use of histochemical stains for both succinic dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). For all enzymes, the OD measured for the blank, representing the mounting medium, glass slide, and cover slip just outside the section being measured, was subtracted from the OD measured within the fiber core. In addition, for SDH we have also subtracted the change in OD due to “nothing dehydrogenase” activity l
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2034
MUSCULAR
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(i.e., the nonspecific reduction of the reaction indicator, nitro blue tetrazolium) (4). The value for nothing dehydrogenase was determined on separate pieces of tissue that had been stained under conditions as nearly identical as possible. This adjustment was necessary because it has been recently demonstrated that the nothing-dehydrogenase reaction may represent a substantial component of the OD of the tissue and not a minor fraction as previously assumed (32). Because no difference between fiber types was found for the nothing-dehydrogenase reaction, a constant value has been subtracted from each fiber. Tissue sections stained for NADH-TR were not corrected for nothing dehydrogenase because we have found the change in OD to be negligible. Muscle capillarization was demonstrated with the use of the amylase and periodic acid-Schiff (PAS) procedure (1) on sections cut at 16.pm thickness. Fiber sections were determined from the NADH-TR sections with the use of planimetry. Capillary density was determined by dividing the fiber area (FA) into the number of capillaries in contact with each fiber (CC). On the average, 75 type I, 80 type IIa, and 40 type IIb fibers were randomly selected for the measurements of histochemical properties. As a result of insufficient tissue, poor cross sections, or freeze artifact, equal subject numbers were not available for all histochemical stains. The subject number employed for each stain was that from which we had both a pre- and posttraining sample. Statistical procedures. To determine the effects of exercise time and training, a two-way analysis of variance (ANOVA) for repeated measures was employed. Where significance was indicated, post hoc analysis with the use of the Newman-Keuls technique was performed to compare specific means. Where only two measures were available, as with the enzymatic and histochemical data, Student’s t tests for correlated samples were used. A 95% level of confidence was accepted for all comparisons.
ADAPTATIONS
TO
TRAINING
A
30 I
I 15’
1
I
I
I
I
6
90 r
,,, \,\\ i ‘,, 4x I ,,I ,, -----0-0..--w_ -0---0--0--_----w---a---r P P
O360. < H E” 50%E a &
1
’
40-
100F
C
90 80 I
$
60
RESULTS
Mu&e metabolites. As expected, the concentrations of ATP, CrP, and creatine were all significantly altered (P < 0.05) with exercise (Fig. 1). In the case of CrP, an ~38% reduction was observed by the time of the first exercise biopsy at 15 min. Thereafter, no further change in CrP was observed over the remaining period of exercise. The change in creatine demonstrated a similar time course and was approximately stoichiometric. With ATP, the change was more delayed, with reductions over rest values not being observed until the end of exercise, which averaged 99 t 5.6 min. Training was found to alter only the CrP and creatine concentrations (P < 0.05). With training, higher CrP and lower creatine concentrations were observed. Changes in several of the glycolytic intermediates were also found with exercise (Table 1). Glucose l-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6phosphate, pyruvate, and lactate were all increased with exercise (P < 0.05). However, differences were observed in the time at which the changes occurred. For glucose l-phosphate, glucose 6=phosphate, and fructose 6-phosphate, the increase was only evident at 15 min of exer-
0
15
30
45
TIME
60
75
90
105
(min)
FIG. 1. Changes in muscle ATP, creatine phosphate (CrP), and creatine concentrations during exercise before (Pre) and after training (Post). Values are means t SE of 8 subjs. Main effect for time (P < 0.05): CrP, 0 > 15 = 60 = 99 min; creatine, 0 < 15 = 60 = 99 min; ATP, 0 = 15 = 30 < 99 min. In addition, a main effect (P < 0.05) for training was found for CrP and creatine (P < 0.05). Interaction effects were not significant.
cise. For fructose 1,6-phosphate, exercise resulted in higher concentrations at 60 and 99 min of exercise over rest but not at 15 min. The increase in pyruvate noted at 15 min of exercise was sustained throughout the two remaining periods, whereas lactate increased at 15 min and then declined at 60 and 99 min. Despite the decline in lactate after 15 min of exercise, the concentrations at 60 and 99 min remained higher than the rest values. Of the glycolytic intermediates studied, training was found to modify only the lactate responses, with lower concentrations observed after the short-term training program (P < 0.05).
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MUSCULAR
AND METABOLIC
ADAPTATIONS
2035
TO TRAINING
TABLE 1. Effects of exercise and training on selected glycolytic intermediates in muscle Time, min 0
G-6-P Pre Post G-1-P Pre Post F-6-P Pre Post F-1,6-P Pre Post Pyruvate Pre Post Lactate Pre Post
1.38kO.31 0.79t0.17
15
60
99
2.4620.47
2.32kO.39
1.9lt0.40
2.87tl.l
1.43kO.32
1.58t0.25
0.050t0.01 0.049t0.01
0.116t,O.O2
0.117t0.02
0.125t0.05
0.073t0.01
0.118t0.03 0.070t0.02
0.199*0.05 0.1151kO.02
0.327t0.05 0.35lkO.12
0.276t0.05 0.194t0.04
0.232t0.05 0.208kO.04
0.299t0.07 0.137t0.03
0.330t0.05 0.326t0.08
0.605-t-0.14 0.385tO.11
0.439t0.08 0.383kO.10
0.190t0.04
0.439t0.09
0.388t0.09
0.48lt0.14
0.203t0.07
0.317t0.05
0.317t0.07
0.364t0.09
7.76tl.5 6.0821.43
29.0t7.0 15.0t5.9
16.5k3.1 10.4t2.0
60. I
8
17.lk5.0 11.222.2
Values are means t SE in mmol/kg dry wt; n = 8 subjs. Pre, pretraining; Post, posttraining. A main effect of time was found for glucose 6-phosphate (G-6-P), glucose l-phosphate (G-l-P), fructose 6-phosphate (F-6-P), fructose 1,6-phosphate (F-1,6-P), pyruvate, and lactate (P < 0.05). For G-6-P, G-l-P, and F-6-P, 15 > 0 = 60 and 99 min; for F-1,6-P, 60 = 99 > 0 = 15 min; for pyruvate, 15 = 60 = 99 > 0; for lactate, 15 > 60 and 99 > 0 min. For lactate, a main effect of training was also found (P < 0.05).
For two additional cellular compounds studied, glu10L . ’ 1 1 I I I 1 I cose and glycogen, only glycogen was modified by the 0 15 30 45 60 75 90 105 exercise and training (Fig. 2). With exercise, glycogen TIME (min) was reduced, with progressively lower (P < 0.05) values FIG. 2. Changes in muscle glucose and glycogen concentrations durobserved at each of the exercise time points. On average, ing exercise before and after training. Values are means t SE of 8 subjs. the concentration was reduced 20% at 15 min, 30% at 60 Main effects (P < 0.05) for time and training were found for glycogen min, and 43% by 99 min of exercise. Training resulted in only (P < 0.05). For glycogen, 0 > 15 > 60 > 99 min. Interaction effects were not significant. a persistently higher concentration of glycogen. Muscle enzymes. The enzymes selected for measureTABLE 2. Effect of short-term endurance training ment were designed to represent the metabolic pathways on maximal enzymatic activities and segments involved in glycogenolysis (total phosphorylase), glycolysis [pyruvate kinase (PK), PFK, aPre Post glycerophosphate dehydrogenase (a-GPDH), and lactate dehydrogenase (LDH)], glucose phosphorylation [ hexo67.5t8.8 SDH 76.9k7.5 kinase (HK)], P-oxidation [3=hydroxyacyl CoA dehydro48.924.4 cs 60.026.2 HAD 40.8t3.6 43.6t4.1 genase (HAD)], the citric acid cycle [SDH and citrate HEK 12.5tl.l 13.7kO.58 synthase (CS)], and high-energy phosphate transfer [crePHOS 106tll 96.325.4 atine phosphokinase (CPK)]. Analysis of biopsies from PK 1,507&115 1,558+150 pre- and posttraining indicated that the maximal activiPFK 293+24 259k16 ties of none of these enzymes were affected by training c~-GPDH 23.3tl.8 23.9H.7 LDH 1,776+194 1,666_+148 (P > 0.05). The most conspicuous increases were obCPK 9,076_+1,091 8,889+536 served for SDH and CS. However, in neither case was the difference sufficient to meet the level of confidence esValues are means t SE in pmol g protein-’ min-‘; n = 8 subjs. SDH, succinate dehydrogenase; CS, citrate synthase; HAD, 3-hytablished for the study (Table 2). droxyacyl CoA dehydrogenase; HK, hexokinase; PHOS, phosphoryMuscle histochemistry. The effects of training on fiber lase; PK, pyruvate kinase; PFK, phosphofructokinase; wGPDH, CYtype distribution, fiber type area, and capillarization are glycerophosphate dehydrogenase; LDH, lactate dehydrogenase; CPK, presented in Table 3. Training failed to alter the districreatine phosphokinase. bution of type I, type IIa, and type IIb fibers in the vastus lateralis muscle (P > 0.05). Similarly, no changes were capillaries per unit area, a training effect (P < 0.05) was observed, but only for the type IIa fibers. detectable in the area or in the number of capillaries surrounding these fiber specific types (P > 0.05). When Microphotometric determinations of NADH-TR and the area of each fiber type was divided into the number of SDH in fiber specific types were performed to evaluate capillaries to produce a ratio representing the number of whether training differentially affected the oxidative pol
l
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2036
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3. Effect of training on muscle fiber type, distribution, size, and capillarization TABLE
Fiber n
Fiber type, % Pre Post Area, pm2 Pre Post CAP Pre Post Cap/area, pm2/10w3 Pre Post Values are means cantly different from
I
Type IIa
7
45.4k3.0 44.9k3.6
39.3t2.8 41.1t3.5
6
4,539+476 4,462+478
5,346_+481 4,894+420
IIb
15.3t3.3 13.6-t-3.6 4,682-t406 4,365?276
5
4.3720.32 4.67kO.44
4.45t0.28 4.55kO.38
3.58t0.34 3.77kO.32
5
1.05t_o.o7 1.llt0.11
0.87t0.07 0.98t0.04*
O.SZt_O.OS 0.95t0.06
t SE; n, no. of fibers. Pre (P < 0.05).
Cap, capillaries.
* Signifi-
tential in the different fiber types (Table 4). Training was not observed to alter either the NADH-TR or SDH of any type I, type IIa, or type IIb fibers identified. DISCUSSION
This study has demonstrated that lo-12 days of prolonged cycle training induced an alteration in muscle energetics and endogenous glycogen such that the changes in both CrP and lactate concentration were attenuated and glycogen depletion was less emphasized during the moderate exercise challenge. In this respect, the training adaptations are consistent with what has previously been observed in exercising humans after training (21,25,37) and what has been demonstrated with the use of the technique of electrical stimulation to increase acutely muscle activity in animal preparations (7, 11). However, unlike previous studies, these metabolic changes were not accompanied by significant alterations in any of the energy metabolic enzymes that were measured and most particularly in the markers selected to represent oxidative potential, SDH and CS. Because only an increase in the capillary-to-fiber area ratio was found in the type IIa fibers after training without any change in the other fiber types on this parameter and without changes in remaining parameters of size, capillary number, and oxidative potential of the individual fiber types, it would appear that, in general, these measures alone cannot explain the change in substrate and metabolic behavior. However, some qualification of these conclusions appear appropriate. Within the statistical confines imposed, namely the selection of 295% confidence level for all comparisons, the conclusions stated are technically correct. As noted by the trends in some of the data, the direction of change is consistent with what has been found in previous training studies, particularly with regard to the increases in oxidative potential and capillaryto-fiber area ratios. For the enzymes used to represent oxidative potential, SDH and CS, the specific probabilities (P) were 0.096 and 0.111, respectively. Similarly for the measure of the number of capillaries per unit area, the calculated P values were 0.180 for type I fibers, 0.028 for type IIa fibers, and 0.095 for type IIb fibers. Whether
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these changes have physiological significance cannot be determined by this study. The only conclusion that can be made is that, with regard to the adaptive changes in muscle enzymology and histochemistry, the magnitude of the increase was not sufficient to provide the confidence needed that such changes did not happen by chance. Of particular significance to the conclusions of this study is the interpretation of the changes that occurred in energy metabolism and substrate utilization after training. Statistically, only main effects of training and exercise were demonstrated for the CrP, glycogen, and lactate data. A significant interaction effect between time and training was not found. Strictly interpreted, this means that the training effect was not expressed during exercise alone but during rest and exercise. However, analysis of the resting mean values before and after training for all three variables indicated that none of the differences was significant (P > 0.05) when compared with paired t tests. When ANOVA techniques were applied to the exercise samples only, a significant difference (P < 0.05) was observed between the pre- and posttraining samples for each of the variables. Accordingly, our interpretation of the data is consistent with this statistical finding, namely that training modified the exercise response only. A sparing of muscle glycogen utilization after training in conjunction with a lower muscle lactate concentration has most often been interpreted to mean that there has been an increase in free fatty acid utilization and a reduction in anaerobic glycolysis (7,21,23), a position that has been repeatedly emphasized in authoritative reviews (22, 36). These experimental training studies, particularly in regard to the human, have also been able to demonstrate that at the same absolute submaximal work rate before and after training steady-state 0, uptake (VO,) was not significantly elevated (21,25,37), a finding that supports the notion that the attenuated metabolic responses are not due to increased aerobic metabolism. Our study is also consistent with this finding (12). The lack of a change in aerobic metabolism during submaximal exercise after training occurred despite a small but significant increase of 4.3% in Vogmax induced by the training (12). The observation that exercise Vo2 did not change with training would also suggest that even with the increase in capillary density found for the type IIa fibers in this study, mitochondrial 0, utilization was not enhanced. In addition, a reduction in CO, production TABLE 4. Training-induced changes in fiber type oxidative and glycolytic potential Fiber I
NADH Pre Post SDH Pre Post Values
Type IIa
IIb
0.733t0.02 0.747t0.03
0.400t0.02 0.449t0.03
0.442t0.06 0.458kO.04
0.138t0.02 0.138t0.01
0.084t0.01 0.087t0.01
0.078t_O.O1 0.083t0.01
are means
t SE of 6 subjs.
Optical
density
measured
at 546
nm.
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MUSCULAR
AND
METABOLIC
(hog) and R have been reported (21,23) and the results of this study support these cha nges (12) The decrease in R has been used to suggest an increase 1.n fat utilization during exercise (21, 23, 30). However, other mechanisms, in addition to a reduced anaerobic glycolysis and increased free fatty acid utilization after training, may be equally plausible to account for the reduced muscle lactate and glycogen sparing effect. One possibility is that fuel utilization (carbohydrate vs. fat) was not altered after training, the lower muscle lactate concentration being due to an elevated removal by extramuscular tissues or oxidization within the muscle itself (8, 27) and the glycogen sparing occurring as a result of enhanced blood glucose uptake and utilization either for glycolysis or for glycogenesis. Although the design of this study was not appropriate for examining these possibilities, several observations seem appropriate. Glucose can represent an extremely important substrate in work of this intensity and duration and is capable of compensating for the reduction in muscle glycogen utilization (38). Furthermore, it has been reported that, during exercise, glycogenesis may occur in the working muscle with glucose acting as a substrate (24). Despite the fact that no training-induced changes were found in blood insulin concentration in this study (16), recent evidence would suggest that, with exercise and training, little if any insulin is needed to facilitate glucose transport across the cell membrane (33). Furthermore, it is possible that the lower R was not due to increased utilization of fat but was due to a reduction in excess CO, mediated by a reduced acidosis (28). In this study, this probability appears plausible because we have found a lower ventilation (VE) accompanied by a lower ventilatory equivalent for 0, (vE/vO,) and a lower arterialized blood lactate concentration (12, 16). Reduction in glycogenolysis and glycolysis as the major mechanism mediating the lower muscle lactate response with exercise after training remains the most popular interpretation (22,27,36), and there is some experimental evidence to support this contention at least in animal preparations subjected to involuntary increases in muscle activity (7,11). The reduced glycolytic flux rate has been proposed to result from an inhibition of PFK occurring as a result of a depressed increase in several metabolic effecters of the enzyme, such as ADP, AMP, and Pi and ammonia (10,22). Although the precise mechanism remains to be demonstrated, the changes in these metabolic byproducts are such as to support this hypothesis, at least in anesthetized rats after a strenuous running program (7). In this study, we have found a less pronounced reduction in CrP with exercise after training. However, ATP concentration was not altered. Whether the less pronounced reduction in CrP (and expected reduction in Pi) altered metabolic control is questionable. At least, on the basis of glycolytic intermediates that, with the exception of lactate, were unchanged with training, flux rate did not appear to be altered. It is possible, however, that at this work load our assays were not sensitive enough to detect the subtle decreases that might be expected in prolonged exercise after training. The significance of Pi in effecting changes in phosphorylase activity and glycogenolysis is well established (6),
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2037
TRAINING
and it would be productive to determine whether the reduction in Pi after training has any significance in this regard. It has been suggested that the proposed reduction in anaerobic glycolysis after training is intimately coupled to increases in oxidative potential mediated by increases in size and number of mitochondria (22,36). This association, at least between lower muscle lactate concentration and increases in oxidative potential with training, is indisputable, particularly in nonhuman studies employing rats (7,11) and in the limited number of training studies on humans (21, 37). However, these associations have been established after relatively long-term training programs. These associations do not preclude the existence of other adaptive strategies, particularly early in training. That other adaptive strategies are possible is emphasized by the results of this study. As suggested, the results could simply be explained by a shift toward a greater dependence on blood glucose acting as a substrate for glycolysis and/or as a substrate for glycogenesis, a comparable glycolytic flux rate, and an enhanced lactate clearance after training. Alternatively, glycolytic rate could have been reduced as a consequence of a decrease in selected metabolic effecters as proposed but by an essentially different mechanism. Holloszy et al. (22) have speculated that, with the increase in mitochondria size and number, the increase in ADP and Pi need not be as great to stimulate increases in mitochondrial respiration necessary to attain the pretraining level of VO,. Essentially, the same mechanism could be operative after training without increases in oxidative potential if the neuromuscular recruitment strategy were altered. This could occur by distributing the work load to more muscles and muscle fibers, thereby lessening the work done by a single muscle fiber. The fact that considerable plasticity has been documented in the neural system as an early response to resistance training (35) makes such a hypothesis inviting. The authors thank Ian Fraser for the excellent technical assistance provided during the study. This study was supported by a grant from the National Science and Engineering Research Council (NSERC). Address reprint requests to H. J. Green. Received
26 June
1989; accepted
in final
form
17 December
1990.
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