It is well established from in vitro studies that the energy cost of contraction differs between muscle fiber types, with fast-twitch (FT) fibers having a larger V̇O2 and heat production per work unit than slow-twitch (ST) fibers at low contraction speeds (3,7,12). However, it is still debated whether recruitment of FT muscle fibers elevates energy requirement during dynamic exercise in humans. Several studies have demonstrated an inverse correlation between the fraction of FT fibers and muscular efficiency at cadences below 90 rpm (6,22), whereas others find no such relationship (18). Additionally, pulmonary V̇O2 per work unit has been reported to be higher during intense exercise than at moderate submaximal intensities (30), which may be related to an additional FT fiber recruitment with increasing power outputs (11,26). A more mechanistic approach to differentiate between the energy cost of different fiber types is to manipulate fiber type recruitment during dynamic exercise at a given intensity. Some studies have used intravenous infusion of curare to cause partial neuromuscular blockade, preferentially in ST fibers (2,10). Galbo et al. (10) demonstrated a twofold higher pulmonary V̇O2 during maximal semisupine cycling after curarization compared with a control bout at the same intensity. However, as curarization caused disturbances in the movement pattern and resulted in a higher metabolic stress, other factors than fiber type recruitment may have elevated pulmonary V̇O2.
Another way to manipulate fiber recruitment may be to perform moderate-intensity exercise with and without a previous exercise regime that specifically depletes glycogen stores of ST fibers in the active muscles (11,14). By determination of glycogen and CP content in individual muscle fibers from biopsies taken before and immediately after exercise, it is possible to evaluate the involvement of FT fibers (8,11,26,29). Thus, it can be investigated whether glycogen depletion of ST fibers results in a greater recruitment of FT fibers during subsequent moderate-intensity exercise and whether such a procedure elevates the energy requirement during dynamic exercise in humans at an intensity that causes little metabolic stress.
Recruitment of FT fibers has also been hypothesized to alter V̇O2 kinetics as a positive correlation has been observed between the fraction of FT fibers and the amplitude of the slow component V̇O2 (4). Furthermore, the magnitude of the slow component V̇O2 has been shown to be a function of the relative exercise intensity (22,27). However, it is not fully understood whether this latter relationship is predominantly caused by a greater activation of less efficient FT fibers, muscular acidosis, increased muscle temperature, additional cardiac and ventilatory work, or other factors, although it has been revealed that a majority of the slow component can be attributed to the active muscles (21). Several studies have shown that the amplitude of the V̇O2 slow component correlates to blood lactate (27,28), but this relationship may be coincidental rather than causal (9,20). Meanwhile, to our knowledge, no investigations have reported a slow component of V̇O2 below the lactate thresh-old. On this basis it would be of particular interest to investigate whether a V̇O2 slow component occurs when FT fibers are manipulated to be active during low-intensity exercise with no concomitant changes in blood lactate or muscle pH.
Thus, the aims of this study were to examine whether previous ST fiber glycogen depletion enhances FT fiber recruitment, elevates energy requirement and causes a slow component of V̇O2 during moderate-intensity exercise with no changes in blood lactate or muscular acidosis.
Twelve, healthy, male subjects ranging in age from 21 to 28 yr, with an average height of 181 (range: 174–189) cm, and an average body mass of 78.0 (67.5–99.1) kg participated in the study. V̇O2max was 3.85 ± 0.17 (±SEM) L·min−1 or 49.4 ± 2.7 (39.0–61.4) mL·min−1·kg−1. The fiber type distribution of the quadriceps femoris muscle was 56.5 ± 3.6% (29.8–71.4%) ST fibers, 29.1 ± 3.9% (11.4–66.2%) FTa fibers, and 14.3 ± 2.0% (3.9–23.7%) FTx fibers. All subjects were habitually physically active, but none trained for competition. The subjects were fully informed of any risks and discomforts associated with the experiments before giving their written consent to participate. The Ethics Committee of Copenhagen and Frederiksberg Municipalities approved the study.
All subjects visited the laboratory on several separate occasions for pretesting procedures. First, the subjects completed an incremental cycle ergometer test to exhaustion (6–10 min) to determine V̇O2max. Second, an individual power output-V̇O2 relation-ship was established by continuous cycle exercise at four different intensities to estimate the workloads for the exercise protocols on the preexperimental and main experimental days. Lastly, each subject carried out the same preex-perimental procedures and exercise protocols as in the main experiments (see below). Gas exchange data were collected during these trials.
Preexperimental glycogen manipulation.
The subjects went through three different preexperimental preparation regimes. On one occasion, they carried out the main experimental exercise protocol after having fasted for ~15 h from the afternoon on the previous day (CON, N = 12). On a second occasion, the exercise protocol was preceded by a 3-h cycle exercise at an external power output of 114 3 7(85–140) W or 340% of V̇O2max, followed by an overnight fast for the same duration as in CON (CHO-DEP, N = 12). This procedure was used in order to glycogen deplete a vast majority of the ST fibers in the muscles that were to be active in the main experiment. On a third occasion, the 3-h cycle exercise was followed by the intake of a carbohydrate-rich diet to reload the glycogen stores of ST fibers (CHO-RE, N = 6). The subjects were provided with a food package that they consumed within 6 h after exercise (8 MJ; 400 g of CHO, 25 g of protein, and 20 g of fat). The latter trial was carried out to separate the effects of depleted glycogen stores and previous long-term low-intensity exercise. The trials were performed within 3 wk in a randomized order. Subjects refrained from strenuous exercise and intake of alcohol for 72 h and from tobacco and caffeine for 24 h before the main experiments.
On the main experimental days subjects reported to the laboratory 1 h before the start of exercise. After 30 min of rest in the supine position, a catheter was inserted into an antecubital arm vein. Then two incisions were made in the medial part of m. vastus lateralis under local anesthesia (20 mg·mL−1 lidocaine without adrenaline) for later obtainment of muscle biopsies. The exercise protocol consisted of 20 min of cycling at a constant power output of 139 ± 4 (105–160) W, corresponding to ~50% of V̇O2max. The cycling cadence was 70 rpm throughout exercise. Breath-by-breath pulmonary V̇O2 was measured continuously in a 3-min period before and throughout exercise by a MedGraphics CPX/D breath-by-breath gas analyzing system (Saint Paul, Minneapolis, MN). Heart rate was recorded in 5-s intervals before and throughout exercise by a Polar Vantage NV heart rate monitor (Polar Electro Oy, Kempele, Finland). Blood samples (5 mL) were drawn from the antecubital arm vein at rest and after 1, 2, 3, 6, 9, 12, 15, 18, and 20 min of exercise and immediately placed in ice-cold water until analyzed. Needle muscle biopsies were obtained from the medial part of m. vastus lateralis at a depth of ~3 cm before and immediately after exercise in CON (N = 9), CHO-DEP (N = 9), and CHO-RE (N = 6) conditions. The postexercise biopsies were taken within 6 s of the cessation of exercise with the subjects still seated on the bike. The samples were frozen in liquid nitrogen within another 6 s. Muscle temperature was measured in m. vastus lateralis of the other leg before and after exercise in CON and CHO-DEP at a depth of ~3 cm by a needle thermistor with an accuracy of 0.1°C (MKA08050-A, Ellab A/S, Rødovre, Denmark).
At two separate occasions within 14 d of the last main experiment, seven subjects who had biopsies taken in the main experiment went through the same preexperimental procedures as in CON and CHO-DEP. Subsequently, they performed a 3-min exercise bout at the same intensity and frequency as in the main experiment. Muscle temperature was measured and muscle biopsies were obtained at rest as well as after exercise. Together with the data from the main experiments, these measurements made it possible to evaluate changes in muscle temperature and muscle metabolites from 0 to 3 and 3 to 20 min of exercise in CON and CHO-DEP.
Single muscle fiber analyses.
After freeze drying the muscle biopsy samples, fragments of 70–80 single muscle fibers from each biopsy were manually dissected under a low-power microscope. Five biopsies from each of seven subjects were included in analyses of single fiber CP and pooled fiber glycogen content, i.e., pre- and postexercise biopsies in CON and CHO-DEP as well as 3-min biopsies in CHO-DEP. For that purpose, more than 2500 fibers were dissected. The 3-min biopsy in CON was not included in the single fiber analyses due to lack of biopsy material. Three pieces of each fiber fragment were cut off and attached to separate glass plates in drops of distilled water for later triple identification of fiber type (ST or FT). This was done via staining for myofibrillar ATPase activity by preincubation at pH 10.30 and incubation at pH 9.40 (5). The remainder of each fiber fragment was weighed on a quartz fiber fish-pole balance (16). The quartz fiber balance was calibrated before and after the weighing procedure by a spectrophotometrical determination of weighed p-nitrophenol crystals. A minimum of 10 fibers fragments of each type weighing 1–4 μg were used for luminometric determination of ATP and CP in single fibers according to the luciferase method described by Wibom et al. (29). Briefly, each fiber fragment was extracted in 200 μL of trichloroacetic acid (2.5%) followed by neutralization in 20 μL of 2.2 M KHCO3. Then 50 μL of the extract was added to a sucrose buffer containing Dluciferin. The assay was carried out using a luminometer (1251, Bio Orbit Oy, Turku, Finland). The SD of the difference in duplicate determinations of ATP and CP content in the same fiber extract (N = 64) was 2.6 and 3.5 mmol·kg−1 d.w. with a coefficient of variance (CV) of 9.1 and 5.7%, respectively. The CV of ATP and CP determinations in two separate fragments from the same muscle fiber (N = 12) was 10.5 and 7.0%, respectively. Moreover, 7–15 ST and FT fibers from each biopsy were pooled (20–30 μg) for fluorometric analyses of glycogen using a modified version of the single fiber hexokinase method described by Essén and Henriksson (8). Briefly, the fibers were placed in a 1-mL Eppendorf tube to which 80 μL of 1 M HCl was added. After sealing and mixing the tube, the fibers were hydrolyzed at 100°C for 2 h. Subsequently, 20 μL of the solution was added to 850 μL of reagent solution and triplicate determination of glycogen content was performed using fluorometrical analyses. CV for triplicate glycogen analyses from the same fiber pool was 17.3% with a SD value of 31 mmol·kg−1 d.w.
One portion of the resting biopsy samples (N = 9) was mounted in an embedding medium (OCT Compound, Miels Tissue-Tek, Zoeterwoude, The Netherlands) and frozen in isopentane that was precooled with liquid nitrogen. These muscle pieces were used for histochemical determination of fiber type distribution and fiber type specific glycogen content. After storage at −80°C, five serial 10-μm-thick sections were cut at −20°C and stained for myofibrillar ATPase (5). Subsequently, about 150 fibers from each biopsy were classified under light microscopy as ST, FTa, and FTx fibers. Furthermore one 16-μm-thick transverse section was cut at −20°C and stained for relative glycogen content by the periodic acid-Schiff (PAS) reaction. Under light microscopy, the fibers were rated as full, partly full, almost empty, and empty based on the staining intensity. A naïve data collector made these analyses.
Muscle Biopsy Analysis
The frozen muscle samples were freeze dried and dissected free from blood and connective tissue. Approximately 2 mg d.w. of muscle was extracted in a solution of 0.6 M perchloric acid (PCA) and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3 and analyzed for creatine phosphate (CP) and lactate by fluorometric assays (16). Muscle pH was measured with a small glass electrode (Radiometer GK2801, Copenhagen, Denmark) after homogenizing ~2-mg d.w. muscle in a nonbuffering solution containing 145 mM KCl, 10 mM NaCl, and 5 mM iodoacetic acid. Another 1–2 mg of d.w. muscle was extracted in 1M HCl and hydrolyzed at 100°C for 3 h, and the glycogen content was determined by the hexokinase method (16).
Within 10 s of sampling, 100 μL of blood was hemolyzed with a 100-μL buffer solution containing 20 g·L−1 Triton X-100 (Yellow Springs Instruments, Yellow Springs, OH) to determine lactate and glucose concentrations (YSI model 23). The rest of the blood sample was rapidly centrifuged for 30 s, and the plasma was collected and stored at −80°C until analyzed. Plasma catecholamines were analyzed fluorometrically by the use of an enzymatic kit (WAKO Chemical, Neuss, Germany). Plasma ammonia (NH3) was determined spectrophotometrically as previously described (10).
Breath-by-breath gas exchange data from two repetitions per subjects were averaged in 15-s intervals. The CV for total exercise V̇O2 in repeated trials was 4% and CV values for V̇O2 after 1.5, 3, 6, and 20 min was 10, 6, 4, and 3%, respectively. Oxygen cost per unit of work was calculated for each subject as the exercise-induced V̇O2 (exercise minus preexercise V̇O2) divided by the external power output. Nonlinear regression (MathCad, Engberg, Hillerød, Den-mark) was used to fit the mean response to either a one- or two-component exponential function (10). An iterative process was used to minimize the sum of squared error between the fitted function and observed values. The one-component exponential model contains a baseline (BL), which was defined as the average V̇O2 during 3 min of seated rest, and fitting parameters for a primary component; i.e., one time delay (TD1), one amplitude (A1), and one time constant (τ1).The two-component model also contains fitting parameters for a slow component V̇O2, that is, a second independent time delay (TD2), a second amplitude (A2), and a second time constant (τ);
where V̇O2 (t) is the V̇O2 at any given time point. The least sum of squared error was used as criterion for using a one-or two-component exponential model. V̇O2 data were aver-aged in 30-s intervals starting from 15 s of exercise for determination of ΔV̇O2 (20–3min).
Two-way ANOVA with repeated measures was used for evaluation of changes during exercise and differences between CON and CHO-DEP as well as CON and CHO-RE. When a significant F value was observed, a Newman-Keuls post hoc test was used to locate the differences. A Wilcoxon signed rank test was used to evaluate differences in relative glycogen content of the individual fiber types before exercise in CON, CHO-DEP, and CHO-RE. A Student paired t-test was used to compare exponential fitting variables between CON and CHO-DEP. Oxygen uptake data points from 3 to 20 min of each trial were tested for a significant positive slope by Pearson’s linear regression test. CV was determined as SD of differences in test-retest results divided by the mean value of the measures and multiplied by 100%. Values are means ± SEM (N = 12) unless otherwise stated. A significance level of 0.05 was chosen.
Preexperimental Glycogen Manipulation
Fiber type specific glycogen content.
Before CON and CON-RE, less than 1% of the fibers were glycogen depleted, whereas 74 ± 13 and 71 ± 12% of the ST fibers and 80 ± 10 and 83 ± 6% of the FT fibers were rated as full with glycogen, respectively. Before CHO-DEP, 51 ± 14 and 44 ± 13% of the ST fibers were empty and almost empty of glycogen, respectively, but less than 2% of the FT fibers were empty of glycogen (Fig. 1). In accordance with the histochemical data, glycogen content was more than threefold higher (P < 0.05) in pooled ST fibers before CON compared with CHO-DEP (293 ± 24 vs 92 ± 22 mmol·kg−1 d.w.;Fig. 2a). Before CHO-DEP, glycogen content was 2.4-fold higher (P < 0.05) in FT than ST fibers, whereas glycogen content in FT and ST fibers was not different before CON (Fig. 2a).
Pooled fiber glycogen utilization.
Glycogen content of ST fibers decreased (P < 0.05) by 89 ± 29 mmol·kg−1 d.w. during CON, whereas no change was observed in FT fibers (Fig. 2a). In contrast, glycogen content was unaltered in ST fibers during CHO-DEP (92 ± 22 and 84 ± 13 mmol·kg−1 d.w.) and tended to decrease (P = 0.07) in FT fibers (220 ± 37 and 149 ± 18 mmol·kg−1 d.w.) (Fig. 2a).
Single fiber CP and ATP content.
Average CP content was unchanged in ST or FT fibers during CON, whereas average CP levels decreased (P < 0.05) by 28 ± 5% in ST fibers and 29 ± 7% in FT fibers during CHO-DEP (Fig. 2b). Average CP concentration after CHO-DEP was lower (P < 0.05) than after CON both for FT (50.1 ± 6.3 vs 72.3 ± 3.4 mmol−kg−1 d.w.) and ST fibers (42.5 ± 3.3 vs 56.4 ± 3.4 mmol−kg−1 d.w.) (Fig. 2b). Before and after CON, 9 and 25% of the ST fibers had CP levels below 49.5 mmol−kg−1 d.w. (i.e., resting mean 1 SD), with corresponding values for FT fibers (below 53.0 mmol·kg−1 d.w.) of 9 and 5% (Fig. 3). At rest and after 3 and 20 min of CHO-DEP, a CP value below resting mean 1 SD was observed in 12, 35, and 50%, respectively, of the ST fibers and 12, 30, and 56%, respectively, of the FT fibers (Fig. 3). Average single fiber ATP content was unaltered in both fiber types during CON and CHO-DEP with no differences between trials (Table 1).
Oxygen uptake was similar before CON and CHO-DEP but became higher (P < 0.05) in CHO-DEP than in CON from 2 to 20 min of exercise (20 min: 0.15 ± 0.03 L·min−1 or 8 ± 2%) (Fig. 4a). From 3 to 20 min of exercise, V̇O2 was unaltered in CON but increased (P < 0.05) by 0.11 ± 0.02 L·min−1 in CHO-DEP (Fig. 4a). Accordingly, exercise-induced V̇O2 per work unit was unaltered from 3 to 20 min of CON (10.8 ± 0.2 and 11.1 ± 0.2 mL·min−1·W−1) but increased (P < 0.05) from 11.3 ± 0.2 to 12.1 ± 0.4 mL·min−1·W−1 in CHO-DEP. The increase in V̇O2 from 3 to 20 min of CHO-DEP correlated with the fraction of FT fibers (r2 = 0.47, P < 0.05) but not with V̇O2max (r2 = 0.13, P > 0.05). No differences were observed in V̇O2 between CON and CHO-RE (Fig. 4b).
FIGURE 4Pulmonary O2...Image Tools
Kinetics of V̇O2 response.
Exponential fittings to the V̇O2 curves revealed a slow component only during CHO-DEP. The amplitude (A2), time delay (TD2), and time con-stant (τ2) of the V̇O2 slow component were 0.12 ± 0.04L·min−1, 299 ± 39 s, and 648 ± 220 s, respectively. The cardio-pulmonary time delay (TD1; 21 ± 2 and 24 ± 3 s) and the time constant of the rapid component (τ1; 34 ± 4 and 29 ± 2 s) were not different between CON and CHO-DEP, whereas the amplitude of the rapid component (A1) was greater (P < 0.05) in CHO-DEP than in CON (1.59 ± 0.06 vs 1.51 ± 0.05 L·min−1). Linear regression analysis revealed a positive V̇O2–slope of 6 mL·min2 from 3 to 20 min of CHO-DEP.
Ventilatory parameters and heart rate.
Ventilation increased (P < 0.05) gradually during CON and CHO-DEP with no differences between trials (Table 1). In CON, respiratory exchange ratio (RER) was higher (P < 0.05) from 1.5 to 20 min than in CHO-DEP with mean values of 0.86 ± 0.01 and 0.78 ± 0.01, respectively (Table 1). Heart rate was lower (P < 0.05) from 4.5 to 20 min in CON than in CHO-DEP with mean values of 121 ± 5 and 132 ± 5 bpm, respectively (Table 1). From 3 to 20 min, ventilation and heart rate increased (P < 0.05) to a similar extent in CON and CHO-DEP, whereas RER increased (P < 0.05) only in CHO-DEP (Table 1).
When comparing CON and CHO-RE, ventilation or RER were not different, whereas average heart rate was 8 ± 3 bpm higher (P < 0.05) in CHO-RE than in CON (Table 2).
Muscle homogenate metabolites and pH.
Before CON, muscle glycogen content was 2.3-fold higher (P < 0.05) than before CHO-DEP. Muscle glycogen breakdown was not significantly different between CON and CHO-DEP (56 ± 10 and 32 ± 17 mmol·kg−1 d.w.) (Table 1). Muscle CP levels were similar before CON and CHO-DEP with CP content being 17 and 20% lower (P < 0.05) after exercise, respectively (Table 1). Muscle lactate and pH were not different before CON and CHO-DEP and remained unaltered during exercise in both conditions (Table 1).
No differences were observed in muscle metabolite changes and concentrations or muscle pH between CON and CHO-RE, except that initial muscle glycogen was 26 ± 7% lower (P < 0.05) in CHO-RE than in CON (Table 2).
Blood lactate increased (P < 0.05) by 0.5 ± 0.1 mmol·L−1 during CON but remained at resting levels during CHO-DEP. Blood glucose was unchanged during CON but decreased (P < 0.05) by 0.7 ± 0.1 mmol·L−1 during CHO-DEP. Blood lactate and blood glucose were lower (P < 0.05) in CHO-DEP than in CON from 3 to 20 min of exercise (Table 1). Hematocrit was not different between trials (Table 1). Plasma catecholamines were not different between trials (Table 1), whereas plasma NH3 was higher (P < 0.05) after 3 and 20 min of CON than in CHO-DEP.
During CHO-RE, blood lactate and glucose remained unaltered throughout exercise with absolute values being lower (P < 0.05) than during CON (Table 2). No differences were observed in plasma catecholamines between CHO-RE and CON (Table 2).
Quadriceps muscle temperature was 37.0°C after 3 min of exercise in CON and CHO-DEP and increased (P < 0.05) by 1.1 ± 0.2 and 1.2 ± 0.2°C from 3 to 20 min of exercise, respectively (Table 1).
The major findings of the present study are that exercise-induced glycogen depletion of ST fibers leads to a greater FT fiber recruitment and an elevated O2 utilization during subsequent dynamic exercise. Moreover, it was observed that a slow component of V̇O2 occurs when FT fibers are active during moderate-intensity exercise despite no changes in muscular acidosis or blood lactate. Conversely, increases in muscle temperature, heart rate, ventilation, and catecholamines in the control trial did not result in a V̇O2 slow component. Together, these findings suggest that recruitment of FT fibers increase in vivo O2 cost of dynamic exercise and support an important role of FT fibers in producing the V̇O2 slow component.
Manipulation of fiber type recruitment.
In the present study, glycogen depletion of ST fibers was used to manipulate fiber recruitment towards a greater FT activation during subsequent moderate-intensity cycling. To evaluate whether this procedure was successful, we used single fiber metabolic measurements. During the control trial glycogen content decreased markedly in ST fibers, providing evidence of a large ST fiber recruitment. On the other hand, no net CP or glycogen utilization was observed in FT fibers during the control bout (Fig. 2), and the number of FT fibers having CP values below resting mean 1 SD was similar before and after exercise (Fig. 3). These observations support previous findings that exclusively ST fibers are active at 50% of V̇O2max when subjects have normal muscle glycogen levels (11,26). In contrast, the present data provide evidence for activity in both main fiber types during the depleted bout. Thus, mean CP content of ST and FT fibers decreased by 28 and 29% during the depleted exercise bout, and more than half of the individual FT fibers had end-exercise CP values below resting mean 1 SD (Figs. 2 and 3). Moreover, glycogen content of FT fibers decreased markedly in five of seven subjects during the depleted bout with average glycogen values being 32% lower in FT fibers after comparison before exercise (Fig. 2). Collectively, these data clearly show that previous exercise-induced glycogen depletion of ST fibers can be used as a model to increase the recruitment of FT fibers during moderate-intensity exercise, resulting in a partial shift in fiber type recruitment from ST to FT fibers.
Fiber type recruitment and O2 cost during moderate exercise.
During moderate exercise with glycogen depleted ST fibers, V̇O2 was higher than in the control bout from 2 to 20 min with total V̇O2 being 7% higher. This difference reflects a higher total energy expenditure in the depleted bout as anaerobic energy turnover was minor in both trials. Hence, no net muscle ATP breakdown or muscle lactate accumulation was observed in either of the two bouts. Moreover, blood lactate was unaltered during the depleted trial and slightly elevated in the control trial, whereas single fiber CP breakdown was higher in the depleted bout compared with control. To examine whether the higher V̇ O2 in the depleted trial was caused by the prolonged low-intensity exercise that was performed on the day before the main experiment, six subjects performed the glycogen depletion protocol followed by carbohydrate reloading. After this procedure, V̇O2, muscle CP breakdown, and lactate accumulation during subsequent moderate exercise were similar to the control trial (Fig. 4, Table 2), indicating that the previous exercise per se had no effect on energy turnover.
Factors such as muscle temperature, muscle pH, plasma catecholamines, substrate utilization, and energy cost of pulmonary and cardiac work could also influence V̇O2 (1,13,17). In the present study, muscle temperature, muscle pH, plasma catecholamines, and ventilation were observed to be similar in the two trials, whereas RER was lower and average heart rate was higher in the depleted bout compared with control (Table 1). Although some studies have seen no detectable effect of acute caloric restriction on work efficiency despite a larger fat oxidation during incremental cycling exercise (19), it is widely accepted that ATP production is about 10% lower with fatty acids than with pyruvate as fuel for mitochondrial respiration (17). It can be estimated that the fraction of fat oxidation in the depleted trial was ~25% higher compared with control (RER: 0.78 vs 0.86), which corresponds to a 2.5% higher V̇O2. Thus, up to one-third of the difference in V̇O2 between trials can be explained by a larger fat oxidation. Also O2 cost of cardiac work may have been different between trials as average heart rate was 11 bpm higher in the depleted bout. However, when using an O2 cost of heart muscle contraction of about ~0.2 mL·beat−1 (13), the higher heart rate can be calculated to elevate V̇O2 by only ~2 mL·min−1 or less than 2% of the total difference between trials.
It seems probable that a large fraction of the observed elevation in V̇O2 after previous glycogen depletion of ST fibers is caused by the greater recruitment of FT fibers in the depleted bout. Considering that the glycogen depletion protocol only resulted in a partial change in fiber type recruitment and that the exercise-induced V̇ O2 was more than 9% higher at the end of depleted bout compared with the control bout, the present data suggest that in vivo energy cost is significantly higher in FT than ST fibers during dynamic exercise. This suggestion is in agreement with the finding that the fraction of FT fibers correlates inversely with muscular efficiency during dynamic exercise (6,22) and the observation of a disproportional increase in V̇O2 during incremental exercise at intensities sufficiently high to involve FT fibers (26,30). The differences in exercise-induced V̇O2 per power output between moderate- and high-intensity exercise usually lies within a range of 5–10% (22,30). Nevertheless, it should be emphasized that in vivo differences in O2 consumption of FT and ST fibers may be even larger at high exercise intensities, as FT fibers may be more susceptible to metabolic stress (3,7,17). Intravenous infusion of curare has been used to cause partial neuromuscular blockade, preferentially in ST fibers, during dynamic exercise in humans (2,10). But to our knowledge, only one previous investigation has manipulated fiber type recruitment in humans during moderate dynamic exercise to study the effect on energy turnover (2). In the study by Asmussen et al. (2), two subjects cycled at ~40% V̇ O2max with and without infusion of curare that decreased handgrip strength by ~30%. With curarization, pulmonary V̇O2 was 0.11 L·min−1 or ~8% higher than in the control trial. Despite the limited number of subjects, the latter results provide further support that greater FT recruitment elevates O2 cost of dynamic contractions in human mixed-fiber skeletal muscles.
Fiber type recruitment and slow component O2 uptake.
Another interesting result of the present study is that V̇O2 increased by 5% from 3 to 20 min of moderate-intensity exercise after previous glycogen depletion of ST fibers, whereas a steady state V̇O2 was reached within the first minutes of the control and carbohydrate reloading trials. From 3 to 20 min of the depleted bout, V̇O2 was elevated for 11 of 12 subjects (range: 1–14%), and the rise in V̇O2 correlated with the fraction of FT fibers in the quadriceps muscle group (r2 = 0.47, N = 12, P < 0.05) but not to pulmonary V̇O2max. Both nonlinear and linear regression analyses revealed a slow phase V̇O2 response of about 0.12 L·min−1 during the depleted bout, whereas no slow component of V̇O2 was observed during the control and carbohydrate reloading trials. Thus, the sum of squared error was lowered only in the depleted bout when applying a two-component rather than a one-component exponential model (17 ± 4%, P < 0.05) and linear regression analyses revealed a positive slope of V̇O2 response from 3 to 20 min only in the depleted bout (6 mL·min−2, P < 0.05). During exercise in the depleted trial blood lactate, muscle lactate, and muscle pH remained at resting levels, and to our knowledge, this study is the first to observe a slow component of V̇O2 at an exercise intensity below the lactate threshold. Thus, the present results demonstrate that a slow component of V̇O2 can occur without muscular acidosis or blood lactate accumulation, thereby substantiating that other mechanisms play a role in the development of the slow phase V̇O2 response.
The observed increase in V̇O2 from 3 to 20 min of exercise cannot be explained by a change in substrate supply from carbohydrates to fatty acids, as RER increased during the depleted bout. Likewise, the observed increases in muscle temperature (~ 1°C), ventilation (7 L·min−1), and heart rate (14 bpm) from 3 to 20 min of exercise in the depleted bouts seem to have had minor influences on V̇O2, as similar increases were observed in the control trial without an alteration in V̇O2. When using an O2 cost of ventilatory and cardiac work of 1.8 mL·L−1 ventilation (1) and 0.2 mL·beat−1 (13), the observed increases from 3 to 20 min can be estimated to account for ~ 15 mL O2−1·min or less than 15% of the rise in V̇O2 from 3 to 20 min in the depleted trial. This is in accordance with results from the investigation by Poole et al. (21) demonstrating that a majority of the additional rise in pulmonary V̇O2 as dynamic exercise progresses can be attributed to the exercising legs. Also plasma epinephrine was elevated during the depleted trial, but it is questionable whether the observed increases had any effect on exercise V̇O2, as it has been shown that exercise-related V̇O2 is unaffected by infusion of epinephrine resulting in plasma catecholamine levels similar to or higher than in present study (9). A suggestion that is supported by the fact that plasma epinephrine also increased in the control trial without causing a V̇O2 slow component. Collectively, it appears as if substrate utilization and the observed increases in temperature, heart rate, ventilation, and plasma catecholamines had minor effects on the additional rise in V̇O2 in the depleted trial. Instead, a pronounced FT fiber recruitment was observed in the depleted trial in association with the V̇O2 slow component, whereas no FT fiber activation or V̇O2 slow component was observed in the control bout. Thus, the present study provides evidence of a significant role for active FT fibers in the production of the V̇O2 slow component, even at low exercise intensity with little metabolic stress.
An important question is whether the slow component of V̇O2 in the depleted bout is related to a gradual increase in FT fiber recruitment or to an increased O2 requirement of each of the already active FT fibers. Increases in muscle activation during cycle exercise at intensities causing a slow component of V̇O2 have been observed in some (23,24) but not all studies (25) using surface EMG or contrast shifts in magnetic resonance imaging. In the present study, some evidence exists that additional FT fibers were recruited over time in the depleted bout. That is, a net CP degradation tended to occur from 3 to 20 min of exercise (63 ± 5 and 50 ± 6 mmol·kg−1 d.w., respectively) and a higher fraction of FT fibers had CP values lower than resting levels after 20 min than after 3 min of exercise (56 vs 30%;Fig. 3). In addition, RER increased from 3 to 20 min in the depletion bout reflecting an elevated carbohydrate oxidation. As most ST fibers were depleted of glycogen, an increase in RER may indicate a greater recruitment of FT fibers at the end of exercise compared with the first 3 min. It may be speculated that some of the ST fibers that were almost empty of glycogen before the depleted bout (45%;Fig. 1) would be most active in the early stages of exercise. Such a scenario would explain the observation that the time delay of the V̇O2 slow component in the depleted bout is longer (~6 min) than usually observed during intense submaximal exercise (~3 min) (9,22,27). Another possibility is that V̇O2 in-creased over time in the fibers that were already active after 3 min, due to a gradual increase in the energy cost of activation and contraction coupling or a drop in mitochondrial P:O ratio (3,15,17). Such decreases in efficiency have mostly been reported to occur during severe exercise with major electrolyte and metabolite changes (3,17), although a recent study from our laboratory has shown that muscular efficiency also decreases during moderate knee-extensor exercise in a free flow condition but not with obstructed blood flow (15). Thus, further studies are needed to elucidate whether the energy cost of contracting fibers decreases during exercise with little metabolic stress, e.g., due to accumulation of intracellular Ca2+ in FT fibers with low mitochondrial density.
The present study demonstrated that previous exercise-induced ST fiber glycogen depletion can be used as a model to enhance FT fiber recruitment during subsequent moderate-intensity exercise. It was shown that previous glycogen depletion of ST fibers, not long-lasting previous exercise per se, elevates total energy requirement and causes a slow component of V̇O2 during dynamic exercise, without changes in muscle or blood lactate. Additionally, under control conditions no slow component of V̇O2 was present although pulmonary ventilation, heart rate, muscle temperature, and plasma catecholamines increased throughout exercise. Collectively, these results suggest that the energy cost is higher in active FT than ST skeletal muscle fibers during dynamic exercise in humans and substantiates that FT fiber recruitment plays a role in the development of the slow phase V̇O2 response.
We would like to thank the subjects involved in the study for their committed participation. We also thank Ingelise Kring, Merete Vannby, Winnie Taagerup, and Berit Sjöberg for excellent technical assistance. The practical aid of Dr. Erik A. Richter and assistance with mathematical modeling of O2 curves by Dr. Ansgar Sørensen is appreciated.
The study was supported by a grant from The Danish National Research Foundation (504-14). In addition, support was obtained from The Sports Research Council (Idrættens Forskningsråd).
1. Aaron, E. A., K. C. Seow, B. D. Johnson, and J. A. Dempsey. Oxygen cost of exercise hyperpnea: implications for performance. J. Appl. Physiol.
2. Asmussen, E., S. H. Johansen, M. Jørgensen, and M. Nielsen. On the nervous factors controlling respiration and circulation during exercise: experiments with curarization. Acta Physiol. Scand.
63: 343–350, 1965.
3. Barclay, C. J., J. K. Constable, and C. L. Gibbs. Energetics of fast- and slow-twitch muscles of the mouse. J. Physiol.
4. Barstow, T. J., A. M. Jones, P. H. Nguyen, and R. Casaburi. Influence of muscle fiber type and pedal frequency on oxygen uptake kinetics of heavy exercise. J. Appl. Physiol.
5. Brooke, M. H., and K. K. Kaiser. Three myosine adenosine triphosphatase systems: the nature of their pH lability and sulfhydryl dependence. J. Histochem. Cytochem.
6. Coyle, E. F., L. S. Sidossis, J. F. Horowitz, and J. D. Beltz. Cycling efficiency is related to percentage of Type I muscle fibers. Med. Sci. Sports Exerc.
7. Crow, M. T., and M. J. Kushmerick. Chemical energetics of slow- and fast-twitch muscles of the mouse. J Gen. Physiol.
8. Essén, B., and J. Henriksson. Glycogen content of individual muscle fibers in man. Acta Physiol. Scand.
9. Gaesser, G. A., S. A. Ward, V. C. Baum, and B. J. Whipp. Effects of infused epinephrine on slow phase O2
uptake kinetics during heavy exercise in humans. J. Appl. Physiol.
10. Galbo, H., M. Kjaer, and N. H. Secher. Cardiovascular, ventilatory and catecholamine responses to maximal dynamic exercise in partially curarized man. J. Physiol.
11. Gollnick, P. D., K. Piehl, and B. Saltin. Selective glycogen depletion pattern in human skeletal muscle fibres after exercise of varying intensity and at varying pedalling rates. J. Physiol. (Lond.)
. 241:45–57, 1974.
12. He, Z.-H., R. Bottinelli, M. A. Pellegrino, M. A. Ferenczi, and C. Reggiani. ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition. Biophys. J.
13. Kitamura, K., C. R. Jorgensen, F. L. Gobel, H. L. Taylor, and Y. Wang. Hemodynamic correlates of myocardial oxygen consumption during upright exercise. J. Appl. Physiol.
14. Krustrup, P., and J. Bangsbo. Muscle fibre recruitment affects energy requirement of constant load moderate intensity cycling. 30th European Muscle Conference, Pavia, Italy, 2001, 134 pp.
15. Krustrup, P., R. A. Ferguson, M. Kjaer, and J. Bangsbo. Heat production and ATP turnover during dynamic exercise in humans: higher efficiency of anaerobic than aerobic ATP resynthesis. J. Physiol.
16. Lowry, O. H., and J. V. Passonneau. A Flexible System of Enzymatic Analysis.
New York: Academic, 1972, pp. 237–249.
17. Nicholls, D. G., and S. J. Ferguson. Bioenergetics 2: Textbook of Biochemistry.
London: Academic Press, 1992, pp. 39–106.
18. Pedersen, P. K., J. B. Sorensen, K. Jensen, L. Johansen, and K. Levin. Muscle fiber type distribution and nonlinear V̇O2
-power output relationship in cycling. Med. Sci. Sports Exerc.
19. Poole, D. C., and L. C. Henson. Effect of acute caloric restriction on work efficiency. Am. J. Clin. Nutr.
20. Poole, D. C., L. B. Gladden, S. Kurdak, and M. C. Hogan. L-(+)-lactate infusion into working dog gastrocnemius: no evidence lactate per se mediates VO2
slow component. J. Appl. Physiol.
21. Poole, D. C., W. Schaffartzik, D. R. Knight, et al. Contribution of exercising legs to the slow component of oxygen uptake kinetics in humans. J. Appl. Physiol.
22. Pringle, J. S., J. H. Doust, H. Carter, K. Tolfrey, I. T. Campbell, and A. M. Jones. Oxygen uptake kinetics during moderate, heavy and severe intensity “sub-maximal” exercise in humans: the influence of muscle fiber type and capillarisation. Eur. J. Appl. Physiol.
23. Saunders, M. J., E. M. Evans, S. A. Arngrimsson, J. D. Allison, G. L. Warren, and K. J. Cureton. Muscle activation and the slow component rise in oxygen uptake during cycling. Med. Sci. Sports Exerc.
24. Shinohara, M., and T. Moritani. Increase in neuromuscular activity and oxygen uptake during heavy exercise. Ann. Physiol. Anthropol.
25. Tordi, N., S. Perrey, A. Harvey, and R. L. Hughson. Oxygen uptake kinetics during two bouts of heavy cycling separated by fatiguing sprint exercise in humans. J. Appl. Physiol.
26. Vøllestad, N. K., and P. C. Blom. Effect of varying exercise intensity on glycogen depletion in human muscle fibres. Acta Physiol. Scand.
27. Whipp, B. J., and K. Wasserman. Oxygen uptake for various intensities of constant-load exercise. J. Appl. Physiol.
28. Whipp, B. J., and K. Wasserman. Effect of anaerobiosis on the kinetics of O2
uptake during exercise. Fed. Proc.
29. Wibom, R., K. Söderlund, A. Lundin, and E. Hultman. A luminometric method for determination of ATP and phosphocreatine in single human skeletal muscle fibers. J Biolumin. Chemilumin.
30. Zoladz, J. A., A. C. Rademaker, and A. J. Sargeant. Non-linear relationship between O2
uptake and power output at high intensities of exercise in humans. J. Physiol.