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Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada
Submitted 26 October 2007 ; accepted in final form 11 January 2008
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ABSTRACT |
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O2 peak) = 44.3 ± 2.3 ml·kg–1·min–1] cycled at 
91% O2 peak for 6 min once per hour for 16 h. Maximal enzyme activities (Vmax, mol·kg–1·protein·h–1) were measured in homogenates from tissue extracted from the vastus lateralis before and after exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). For the mitochondrial enzymes, exercise resulted in reductions (P < 0.05) in cytochrome-c oxidase (COX, 14.6%), near significant reductions in malate dehydrogenase (4.06%; P = 0.06) and succinic dehydrogenase (4.82%; P = 0.09), near significant increases in β-hydroxyacyl-CoA dehydrogenase (4.94%; P = 0.08), and no change in citrate synthase (CS, 2.88%; P = 0.37). For the cytosolic enzymes, exercise reduced (P < 0.05) Vmax in hexokinase (Hex, 4.4%), creatine phosphokinase (9.0%), total phosphorylase (13.5%), phosphofructokinase (16.6%), pyruvate kinase (PK, 14.1%) and lactate dehydrogenase (10.7%). Repetition-dependent reductions (P < 0.05) in Vmax were observed for CS (R1, R2 > R16), COX (R1, R2 > R16), Hex (1R, 2R > R16), and PK (R9 > R16). It is concluded that heavy exercise results in transient reductions in a wide range of enzymes involved in different metabolic functions and that in the case of selected enzymes, multiple repetitions of the exercise reduce average Vmax. maximal activity; metabolic pathways; cycling
There is evidence particularly in humans that alterations in intrinsic regulation, occurring during exercise, result in an increase in the maximal in vitro activity of citrate synthase (CS) (6, 26, 40), an enzyme of the citric acid cycle (CAC). These effects appear to be specific to either the metabolic pathway and/or the enzyme because no changes have been reported in 3-hydroxyacyl-CoA dehydrogenase (3-HAD) and phosphofructokinase (PFK) (40), enzymes of β-oxidation and glycolysis, respectively. It is possible that the specific metabolic pathways and/or enzymes affected could be dictated by the characteristics of the exercise task. Interestingly, in the human, the exercise tasks employed have emphasized prolonged cycling of moderate intensity where ATP synthesis would be expected to be dominated by oxidative phosphorylation (6, 40) or prolonged single-leg knee extensor exercise (26).
If the contractile activity is repeated on a regular basis, changes in the protein content of enzyme occur that alter the maximal activity of the enzyme. The specific changes that occur to the enzymes are to a large degree dependent on the strain imposed on the metabolic pathway as dictated by the type of metabolism, the metabolic rate, and the substrate used (32). The most documented adaptation has been in response to regular, submaximal exercise in which oxidative phosphorylation represents the dominant form of ATP resynthesis. This form of training results in an increase of mitochondrial content and increases in the maximal activity of a wide range of CAC and electron transport system (ETS) enzymes (18). As might be expected, training involving more intense activity, resulting in large increases in glycolysis, also results in an upregulation in the maximal enzyme activities of enzymes in this pathway, most probably as a result of increases in protein content (15).
A controversial and little-studied issue is the exercise dosage required to shift from a metabolic regulation that is based predominately on intrinsic control to one that depends on increases in enzyme protein content-mediated changes in maximal enzyme activity. There is evidence, particularly in the human that some enzymes respond with increases in maximal activity soon after the onset of regular exercise. Hexokinase (Hex) appears to be one such enzyme. It has been reported that in human muscle a single session of prolonged exercise increased both the mRNA (23, 24) and the protein (23) level of this enzyme within 3 h following the exercise. These findings have also been extended to rat muscle where it has been reported that if the exercise is sufficiently prolonged, the increase in Hex mRNA and protein may actually occur during the exercise (30, 31). There is also evidence to indicate that other enzymes may not be as responsive since our laboratory (9, 33) as well as others (2, 34) have not reported any change in either mitochondrial or cytosolic enzymes within the first 5 days of daily prolonged cycle exercise. With this type of training, increases in mitochondrial enzymes appear to occur soon after this point (9, 39). It is possible that the enzymatic adaptations could occur earlier with optimization of the training stimulus. Indeed, a recent study has reported increases in CS with just six sessions of sprint interval training performed over 2 wk (1). It is possible that the transient changes induced by individual bouts of exercise could culminate in a more rapid increase in enzyme activity if the exercise were intermittent and performed in a much more abbreviated time frame.
The purpose of this study was to investigate the changes that occur in the maximal activity in a wide variety of enzymes representative of the various metabolic pathways and segments in working muscle to an intermittent session of repetitive heavy exercise. Our working hypothesis was that the performance of a session of heavy, intermittent exercise, designed to result in repeated large and rapid flux in both oxidative phosphorylation and glycolysis, would result in transient increases in the maximal activities in all representative enzymes. Moreover, with multiple repetitions of the task, the increase in select enzymes such as CS and Hex would persist during the recovery period between repetitions.
It should be noted that this study was part of a much larger study in which our laboratory has tried to characterize the changes that occur in a wide range of properties. Previous publications have addressed mechanical function and fatigue (10), Na+-K+-ATPase (11), sarcoplasmic reticulum Ca2+ cycling (19), metabolism (12), and glucose and lactate transporters (8).
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METHODS |
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O2 peak) of the participants (10 men and 3 women) was 3.02 ± 0.18 l/min. Physical characteristics of the group included age of 22.0 ± 0.92 yr, height of 173 ± 2.5 cm, and weight of 69 ± 3.7 kg.
Experimental design.
The cycling protocol used to investigate the regulation of enzyme activity involved 6 min of exercise, performed at 91%O2 peak, followed by 54 min of rest. This schedule was repeated over 16 h, resulting in 16 repetitions of the exercise. Tissue was obtained by needle biopsy from the vastus lateralis from preprepared sites before and after exercise at the first (R1), second (R2), ninth (R9), and sixteenth (R16) repetition. Because our protocol required the performance of eight biopsies, extracted from different sites, a number in excess of what could reasonably be expected, it was necessary to perform the experiment on two separate occasions separated by 30 days. During one session, the volunteers performed only the first two repetitions (R1 and R2) of the exercise. During the other visit, the full 16-h protocol was performed, but tissue was only extracted at R9 and R16. The order of conditions was randomly assigned. A potential limitation of this design is the change that may occur in muscle properties between conditions. Our laboratory used this type of design previously and found that provided exercise and dietary habits remain relatively constant, a range of muscle characteristics remain stable (4, 35). As a further verification that our experimental design had no effect, we examined the enzyme activity before exercise at R1 and R16. As expected, no differences were observed for any of the enzymes studied.
For each testing session, exercise began at 9:00 AM. At least 2 h before the start of testing on each day, participants were required to ingest a can of Ensure (250 kcal) meal replacement consisting of 9.4 g protein, 6.7 g of fat, and 38 g of carbohydrate (Ross Products Division, Saint-Laurent, QC, Canada). The Ensure was intended to replace breakfast and to standardize nutrient intake before each test session. All volunteers were instructed to abstain from vigorous exercise, alcohol, and caffeine for at least 24 h before each test session. The specific behavior requested from the volunteers was detailed in personal interview with the study investigators. During the interval between exercise bouts, water was allowed ad libitum following the first 2 h. During this time, participants were also allowed to consume selected vegetables, fruits, and Gatorade bars. Dietary composition and energy intake for each individual were recorded and are reported in a separate publication (10). Additional details regarding testing, tissue sampling, and environmental conditions appear in earlier publications (11, 12, 19).
Enzyme activities. The enzymes selected for measurement included three from the CAC [CS, succinic dehydrogenase (SDH), and malate dehydrogenase (MDH)], one from β-oxidation (3-HAD), and one from the ETS [cytochrome-c oxidase (COX)]. In addition, the glycogenolytic enzyme [total phosphorylase (TPhos)], the enzyme involved in glucose phosphorylation (Hex), an enzyme involved in high-energy phosphate transfer [creatine phosphokinase (CPK)], and three enzymes of glycolysis [PFK, pyruvate kinase (PK), and lactate dehydrogenase (LDH)] were selected.
With the exception of SDH and PFK, enzyme activities were measured in homogenates that were prepared from tissue (5–10 mg) that had been immediately frozen in liquid nitrogen after extraction from muscle and stored at –80°C. After preparation, the homogenate was frozen and stored at –80°C pending measurement. Because freezing the homogenate can result in reductions in the activity of SDH and PFK (16), these enzymes were measured on fresh homogenates immediately after preparation. The activity of each enzyme was assessed on tissue samples hand homogenized (0–4°C) in a phosphate buffer (pH 7.40) containing 5 mM mercaptoethanol, 0.5 mM EDTA, and 0.2% BSA. To further disrupt the mitochondrial membranes, the homogenate was sonicated for 20 s using a schedule of repeated 2 s bursts separated by 5 s. During sonication, the tube containing the homogenate was submersed in a beaker containing a mixture of water and ice. Homogenates (1:50) were diluted in 20 mM imidazole buffer with 0.2% BSA. Before freezing, each tissue sample was cleaned of visible blood and connective tissue.
All enzyme assays, with the exception of COX and PK, were performed at 22°C, according to previously published procedures (3, 16) as modified in our laboratory (9, 14), using an end-point assay. It has been shown (3) and confirmed by our laboratory (13) that near-perfect correlations between the end-point assay and assays based on first-order kinetics. The activity of PK was measured fluorometrically in a first-order kinetic reaction at 22°C (16). With the exception of COX, all enzymes were measured fluorometrically.
The assay for COX, the ETS complex selected, was performed at 37°C in a reaction mixture consisting of 10 mM potassium phosphate buffer and 1 mM solution of reduced cytochrome-c (Sigma C-2506). The diluted homogenate was added to the medium, and the decrease in absorbance was measured at 550 nm for 4 min in a Shimadzu UV 160U spectrophotometer (Shimadzu, Kyoto, Japan). Using the slope of the reaction and millimolar extinction coefficient (29.5) of reduced cytochrome-c at 550 nm, the units (µmol/min) of activity were calculated and converted to units per gram protein. These procedures are essentially as published previously by our group (5).
Protein was determined by the Lowry technique as modified by Schacterle and Pollock (36).
All samples for a given enzyme and for a given individual were analyzed in triplicate during the same analytic session. The intra-assay coefficients of variation (CVs) for the mitochondrial enzymes were the following: CS, 5.38%; MDH, 6.68%; SDH, 4.74%; 3-HAD, 4.70%; and COX, 4.05%. For the cytosolic enzymes, the CVs were the following: CPK, 6.54%; Hex, 3.80%; TPhos, 5.20%; PFK, 5.86%; PK, 2.96%; and LDH, 2.30%.
Data analysis. A two-way ANOVA for repeated measurements was used to determine the effects of time (number of repetitions) and exercise (before vs after). Where significance was found, the Newman-Keuls technique was applied to determine which means were significantly different. Statistical significance was accepted at P < 0.05. Where differences between means are indicated in the text, significance is implied. Where near significance was found for specific comparisons, the probability level has been indicated. Data are presented as means ± SE.
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RESULTS |
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Protein. The protein concentrations of the homogenates before and after exercise were 194 ± 6.2 and 199 ± 6.0, 188 ± 8.0 and 198 ± 5.2, 193 ± 4.0 and 203 ± 3.6, and 201 ± 5.6 and 189 ± 4.1 mg/g for R1, R2, R9, and R16, respectively. No changes were observed for either exercise or repetition.
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DISCUSSION |
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The cytosolic enzymes, regardless of metabolic pathway, all showed the same general effect of exercise, namely a depression in activity. With the exception of CPK, the activity of all enzymes recovered between repetitions until late in the protocol when a general depression was found. No such depression was found for CPK. As with the mitochondrial enzymes, these observations are also inconsistent with our hypothesis. It should be emphasized that our findings cannot be explained on the basis of changes in tissue water content because protein was measured and because we found no change in protein either with exercise or with repetition.
Our failure to find an increase in CS activity is at odds with previous studies in humans in which increases have been reported immediately after prolonged heavy cycling exercise (6, 40) and prolonged single-leg knee extension exercise (26). The increases appear to persist for a considerable time after exercise based on reports documenting elevations in CS activity at 1 h (40) and 3 h (6) of recovery. As we have observed in the present study, this behavior did not appear to be the case with the other CAC enzymes or with COX, part of the ETS, where near-significant or significant reductions were observed. Our failure to observe an increase in CS activity with our exercise protocol may be due to one or more factors. As indicated previously, increases in the activity of the enzyme could be due to changes in protein abundance and/or to changes in intrinsic regulation. Given the acute nature of the exercise protocol, increases in the amount of the enzyme protein appear unlikely in explaining the increases in CS (40). However, changes in the apparent mitochondrial concentration of the homogenate, as a result of changes in other cellular constituents, remain a possibility. Where increases in CS activity have been reported, measurements have either been expressed per unit wet weight (6, 40) or per unit dry weight (40) of tissue. When changes are expressed per unit protein content (40), the exercise-induced increase in CS appears to be substantially lower (6, 26, 40). Although several factors could contribute to the apparent decrease in nonmitochondrial cellular constituents, resulting in an apparent increase in CS activity when expressed per unit wet or dry weight tissue, the depletion of endogenous glycogen that occurs with exercise, is the most obvious (40). In previous studies reporting increases in CS activity, glycogen depletion in working muscle would be expected to be substantial given the nature of the exercise protocols employed (6, 26, 40). In the present study, we have reported extensive glycogen depletion with our 16 sessions of heavy exercise (12). Interestingly, we report no increase in CS activity with exercise regardless of the number of repetitions when our measurements are expressed per unit protein.
The CS activity could also be affected by the amount of enzyme available for measurement. Because CS is a mitochondrial enzyme, measurement procedures must ensure that the enzyme is liberated from the mitochondria into the homogenate for measurement. To accomplish this, the homogenate is sonicated (26), or the mitochondrial membrane is permeabilized with the use of Triton X-100 (40). We have used sonication. All other analytical aspects are essentially as used by others reporting increases in CS activity with exercise (26, 40). Whereas others have used a kinetic assay to assess enzyme activity (26), we have used an end-point assay, based on the close correlation that exists between the linear portion of absorbance vs time curve (13). Conceivably, this difference could explain the contradictory results between laboratories. However, this possibility appears remote because we have not found exercise-induced increases in CS activity following prolonged cycle exercise of moderate intensity when the same analytic protocols were employed (data not presented) as used by others (26, 40) who have reported increases in CS with this type of exercise.
Although studies have reported isolated increases in CS activity with acute exercise in the human muscle (6, 26, 40), this is not what has generally been reported on animals where a reduction in several mitochondrial enzymes have been found, including CS (20). These authors have attributed the depression to damage induced by increases in reactive oxygen species (ROS). It is possible that these results have application to our study, given the heavy exercise employed and the expected generation of large amounts of ROS (37, 38). Interestingly, the ETC is believed to be a major producer of ROS during exercise (37). It should be noted that not all mitochondrial enzymes respond the same way to acute exercise. The enzyme selected to represent β-oxidation, 3-HAD, appeared to be increased.
The acute effects of the exercise was clearly evident for the cytosolic enzymes measured. Approximately parallel decreases were observed in the activity of the enzyme to measure the glycogenolytic potential, TPhos, and the enzymes involved in glycolysis, PFK, PK, and LDH. The similarity in magnitude and the transient nature of the effect observed for these enzymes, particularly early in the protocol, would suggest that a common mechanism is involved that is reversed during the 54 min of recovery between exercise bouts. It is not clear whether the common mechanism involves enzyme modification as a result of increases in ROS. We have also found that both CPK and Hex, the enzymes involved in high-energy phosphate transfer and glucose phosphorylation, respectively, displayed a similar depression in Vmax with the exercise.
We had anticipated that for some enzymes such as Hex, our repetitive exercise protocol would result in an increase in Vmax. Previous studies have shown that a single session of exercise can result in an increase in both the mRNA (23, 24) and the Vmax of the enzyme (23). By employing repeated sessions of heavy exercise, known to result in large increases in glucose uptake (22), interspersed with a rest period in which carbohydrate supplements were supplied, which is also known to increase muscle glucose uptake (41), we had expected that Hex activity would increase to meet the increased demands for glucose phosphorylation. Our results indicate that at R16, the Vmax of Hex was reduced, an effect that appears to be due, in part, to insufficient recovery from the previous exercise sessions. The depression would suggest damage and/or degradation of the enzyme, possibly involving ROS.
An interesting issue is whether the reductions that we have observed in enzyme activity have any functional significance. Given the enzymes affected, a decrease in the potential for oxidative phosphorylation, glucose phosphorylation, glycogenolysis, and glycolysis is suggested to occur during the exercise. It could be possible that given the strain imposed on both the CAC and the ETS, as a result of our task (91% O2 peak), oxygen uptake (O2) might be compromised (42). However, this is not what we have found. With increasing repetitions of the exercise, O2 was increased (12). Alternatively, and assuming Michaelis-Menten kinetics, the reduction in oxidative potential might be manifested in a greater need to increase the free energy necessary to achieve a given level of mitochondrial respiration (27). Future well-controlled studies are desirable to assess the impact of the reduction in the maximal enzyme activities in the regulation of flux through the various metabolic pathways and segments. These studies should also incorporate other key enzymes, such as pyruvate dehydrogenase and creatine palmitoyltransferase I, viewed as key regulatory enzymes in carbohydrate oxidation and fat metabolism, respectively. Because oxoglutarate dehydrogenase has been proposed as limiting in maximum flux in the CAC (28), this enzyme should be included for study as well.
It is important to emphasize that the depression in the metabolic potential that we have observed also occurred in conjunction with a reduction in the maximal catalytic activity of the two cation pumps involved in excitation-contraction coupling, namely the Ca2+-ATPase (19) and the Na+-K+-ATPase (11), both of which utilize ATP for their transport functions.
In summary, in this study, we found that heavy exercise performed repetitively, does not result in an acute increase in CS activity as previously reported. In contrast, evidence is presented demonstrating that oxidative potential is transiently depressed as indicated by the lower COX activity. The exercise-induced inhibition of COX is also accompanied by transient reductions in the cytosolic enzyme activities involved in glucose phosphorylation (Hex), high-energy transfer (CPK), glycogenolysis (TPhos), and glycolysis (PFK). As shown by the preexercise activities, multiple repetitions of the exercise failed to induce an upregulation in maximal activity regardless of the enzyme studied.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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