In a previous study on subjects who regularly played soccer, rugby, or hockey, it was observed that drinking a carbohydrate-electrolyte (CHO-E) solution during prolonged intermittent high-intensity exercise improved endurance capacity by more than 2 min (31). Some investigators who have also reported that CHO ingestion delays fatigue have observed a reduction in the rate of muscle glycogen utilization (12,37,38,41). Others have reported that an enhanced exercise capacity was due to the maintenance of high blood glucose concentrations and a high rate of CHO oxidation late in exercise when muscle glycogen concentration is low (6,11,28,30). In our previously reported study, similar blood glucose concentrations were observed from 30 min through to the end of exercise, remaining at euglycemic levels throughout the CHO and CON trials (31). Therefore, we suggested that the difference in running times may have been as a consequence of different rates of glycogen utilization when the CHO-E solution was ingested.
Most prior studies investigating the ergogenic effect of carbohydrate ingestion have used prolonged, continuous submaximal cycling (6) or running (37,38). Others have monitored performance after a set period of continuous cycling (28,30) or intermittent cycling (12,29). However, few studies (18,21,34) have investigated the effect of CHO-E feedings on performance or muscle glycogen utilization during prolonged, intermittent high-intensity running. This type of exercise involves running and walking at various intensities of effort and is typical of the activity pattern in sports. Therefore, the aim of the present study was to examine the changes in muscle glycogen concentration before and after 90 min of the Loughborough Intermittent Shuttle Test (LIST), with and without the ingestion of a carbohydrate-electrolyte solution.
Subjects. Six, trained, healthy male games (soccer, hockey, or rugby) players (age 24.6 ± 2.2 yr; height 179.6 ± 1.9 cm; body mass 74.5 ± 2.0 kg; V̇O2max 56.3 ± 1.3 mL·kg−1·min−1; mean ± SEM) volunteered and gave their written informed consent to participate in this study, which had University Ethical Committee approval.
Preliminary measurements. Maximal oxygen uptake (V̇O2max) was estimated by means of a progressive shuttle run test (32), modified from the original protocol (22). From this estimate of V̇O2max, running speeds corresponding to 55% and 95% V̇O2max were calculated using the tables for predicted V̇O2max values (32). Subjects then performed the Loughborough Intermittent Shuttle Test (LIST) for 1 h to familiarize themselves with the required running speeds and experimental procedures.
The Loughborough Intermittent Shuttle Test (LIST). This comprised exercise periods of varying intensity for 15 min, separated by 3 min recovery (Fig. 1). The exercise periods consisted of running continuous 20 m shuttles at various speeds related to estimated individual maximal oxygen uptake (V̇O2max) in a fixed pattern (shown below) and was designed to be similar to the activity pattern typically recorded for soccer match play (33,40).
Exercise pattern during the LIST. 3 × 20 m at walking pace, 1 × 20 m at maximal running speed, 3 × 20 m at a running speed corresponding to 55% of individual V̇O2max, 3 × 20 m at a running speed corresponding to 95% of individual V̇O2max.
This exercise pattern was repeated approximately 11 times during each 15-min exercise period. Four 15-min exercise periods were performed by each subject during the familiarization, separated by 3-min recovery periods. During the main trials, this was increased to six 15-min exercise periods (Fig. 1).
Experimental design. All subjects completed two exercise trials separated by at least 7 d. The trials were randomized and counterbalanced to offset any training or order effects. On each occasion, they consumed either a 6.9% carbohydrate-electrolyte solution (Lucozade Sport, Smith-Kline Beecham, Coleford, Gloucestershire, U.K.) (CHO) or a noncarbohydrate placebo (CON) immediately before exercise (5 mL·kg−1 body mass (BM), and every 15 min thereafter (2 mL·kg−1 BM). The 6.9% CHO-E solution consisted of dextrose (1.9 g·L−1), fructose (1.9 g·L−1), maltose (0.5 g·L−1), higher saccharides (2.3 g·L−1), and electrolytes (sodium 55 mg·100−1 mL and potassium 25 mg·100−1 mL). The solutions ingested were of similar color, texture, and taste.
During the 2 d preceding each trial, subjects refrained from any strenuous physical activity and recorded their food intake in an effort to standardize muscle and liver glycogen stores. They were required to repeat exactly the same diet for the 2 d before the second trial.
Experimental protocol. Subjects reported to the laboratory on the day of each experiment after an overnight fast of approximately 10 h. Each subject then emptied his bladder before the measurement of nude body mass was made. Nude body mass was recorded to the nearest 50 g pre- and post-exercise. An indwelling catheter (Venflon, 16-18G, Ohmeda, Hatfield, Herts, U.K.) was inserted into an antecubital vein and was kept patent with frequent flushing with sterile saline. A resting muscle sample was then obtained from the vastus lateralis by needle biopsy. Each biopsy sample was taken from the central portion of the vastus lateralis muscle, mid-way between the hip and the knee. A resting 10-mL venous blood sample was then collected after subjects had remained standing for 15-20 min. A standardized warm-up consisting of jogging, stretching, and striding was then performed by each subject for 15 min. Then, immediately before starting exercise, subjects consumed 5 mL·kg−1 BM of the prescribed solution. Subjects then performed the 90-min intermittent high-intensity running test, which comprised six 15-min exercise periods separated by 3-min recovery (Fig. 1). During each 3-min rest period, a further 2 mL·kg−1 BM of the assigned fluid was ingested by each subject, and blood samples were collected. Blood samples were obtained after 30, 60, and 90 min, at the end of exercise.
The LIST was conducted in a sports hall, in which the ambient temperature was maintained between 16 and 18°C. Subjects were required to perform continuous 20-m shuttles, identified by cones and floor markings, at various speeds related to estimated individual maximal oxygen uptake (V̇O2max) values for a total of 90 min. The running and walking speeds during each 20 m of the LIST were dictated by an audio signal from a microcomputer (BBC Master Series) using software developed for this purpose. Sprint times were measured in one direction over the first 15 m using two infrared photoelectric cells (RS Components Ltd.), interfaced with the microcomputer. Heart rate was monitored by short-range telemetry system (Polar Electro sports testers PE3000, Kempele, Finland) every 15 s during exercise and the mean recorded for each 15 min. Subjective ratings of perceived exertion (4) were also obtained every 15 min.
Immediately upon cessation of the 90-min trial, a second muscle sample was obtained after the subject had been transferred to an examination couch adjacent to the running area. The subject's dry postexercise nude body mass was then obtained several minutes after exercise.
Blood sampling and analysis. Ten mL of blood was withdrawn at rest, and after 30, 60, and 90 min of exercise, of which 5 mL was dispensed into a lithium heparinized tube, and the remainder was dispensed into a plastic centrifuge tube and left to coagulate for 1 h. Serum was then obtained after centrifugation at 4°C for 15 min at 6000 rpm and was stored at −70°C before analysis for insulin (36) and sodium and potassium concentrations, measured by flame photometry. From the venous blood samples, duplicate 20-μL aliquots were deproteinized with 200 μL of 2.5% perchloric acid, centrifuged, and frozen at −20°C for subsequent analysis for blood lactate concentration using a modification of a method previously described (25) and for blood glucose using the glucose oxidase method (39). Hematocrit (Hct) and hemoglobin (Hb) concentrations were analyzed in triplicate by the procedures of microcentrifugation (Hawksley Ltd.) and cyanometHb (Boehringer Mannheim GmbH test combination, Mannheim, Germany), respectively. The results obtained were used to calculate changes in plasma volume (7). The remaining whole blood was centrifuged for 15 min at 6000 rpm at 4°C, and the plasma divided into aliquots and frozen at −20°C for later analysis of FFA using a commercially available kit (NEFA-C test, Wako, Osaka, Japan) and glycerol (20).
Muscle sampling and analysis. Muscle samples were obtained through separate incisions from the vastus lateralis muscle using the needle biopsy procedure (2), with suction being applied. All incisions were made through the skin and muscle fascia under local anesthetic (2-3 mL of 1% lidocaine) before the start of exercise while the subjects were lying supine on an examination couch. All muscle samples were snap-frozen in liquid nitrogen, freeze-dried and stored at −70°C. Subsequently, one part of the freeze-dried muscle tissue was analyzed enzymatically for glycogen, glucose, glucose 6-phosphate (G-6-P), lactate, ATP and phosphocreatine (PCr) using modifications of the methods described by Harris and coworkers (13) and Lowry and Passonneau (23). Glycogen was determined by hydrolysis in 1 M HCl and both the acid-soluble and acid-insoluble glycogen were determined (17). The glycogen concentration was also determined in pools of Type I and Type II muscle fibers dissected from the remaining piece of freeze-dried muscle tissue using methods that have been previously described (38). Mixed muscle metabolites were obtained for six subjects. Single fiber glycogen concentration was determined for five subjects during the CON trial, but the corresponding measurements from the CHO-E were possible in only three subjects. A high correlation (r = 0.94) was observed between the mean glycogen concentrations of Type I and Type II muscle fibers and the mixed muscle glycogen concentration from the same biopsy sample (38).
Statistical analyses. Muscle metabolites, heart rate, RPE, and blood biochemical responses in both trials were analyzed using a two-way (treatment by time) analysis of variance for repeated measures on two factors. Changes in muscle glycogen concentration for each trial were compared using Student's t-test for correlated data. Significant differences between means were identified using a Scheffe post hoc test. The level of significance was accepted at P < 0.05 and all results are reported as mean ± SEM.
Sprint times. Sprint times were similar between trials and over the duration of each trial. Average times for 15-m sprints during the CHO and CON trials were 2.44 ± 0.04 s and 2.42 ± 0.06 s, respectively.
Perceived rate of exertion and heart rate. There were no statistical differences in either ratings of perceived exertion or heart rate response between trials during 90 min of intermittent, high-intensity shuttle running, indicating that the physical stress imposed by the protocol was similar in both trials. Ratings of perceived exertion increased from initial values of 5 ± 1 to 7 ± 1 during the final 15 min of exercise in the CHO (P < 0.01) and CON (P < 0.05) trials. Mean heart rates were 169 ± 2 b·min−1 in CHO and 171 ± 2 b·min−1 in CON.
Blood glucose and serum insulin. Blood glucose concentrations were similar in each trial, although there was a tendency for values to be higher when fed CHO than when fasted (Fig. 2). In both trials, blood glucose were maintained within the normal range and after an initial rise after 30 min (P < 0.01) of exercise declined over the duration of exercise (P < 0.05) in the CON trial. Serum insulin values (Fig. 2) were higher after 30 min during the CHO trial (P < 0.05).
Plasma FFA and glycerol. There were no differences in plasma FFA or glycerol concentrations between trials during exercise. In both trials, plasma FFA concentration was suppressed after 30 min of exercise before steadily increasing after 60 and 90 min (Fig. 3). In the CHO trial, FFA concentration at the end of exercise was greater than at 30 min (P < 0.05). In the CON trial, FFA concentration at the end of 90-min exercise was greater than at rest (P < 0.05) and at 30 and 60 min during exercise (P < 0.01). Plasma glycerol concentrations increased throughout exercise under both conditions (Fig. 3). In the CHO trial, postexercise glycerol concentrations were significantly greater than at rest (P < 0.05).
Blood lactate. Thirty minutes after the start of exercise, blood lactate concentration was significantly higher in the CON than CHO trial (CON = 6.3 ± 1.1 mmol·L−1 vs CHO = 4.8 ± 0.8 mmol·L−1; P < 0.05). At this point in each trial, blood lactate concentrations reached a peak before steadily declining over the remainder of each trial. Similar concentrations were observed after 60 and 90 min, averaging 3.5 mmol·L−1 and 4.3 mmol·L−1, respectively.
Changes in plasma volume and body mass. There were no differences between trials in the changes in plasma volume from resting to the end of exercise (CHO, 1.8 ± 1.8%; CON, −2.3 ± 1.4%) or loss of body mass during each trial (CHO, 2.8 ± 0.5 kg; CON, 2.5 ± 0.3 kg). These changes were equivalent to a body mass loss of 3.7% and 3.3%, respectively, in the CHO and CON trials.
Muscle metabolites. Muscle glycogen concentration is shown in Figure 4. The pre- and post-exercise glycogen concentrations were not different between trials. However, total glycogen utilization was lower (P < 0.05) during the CHO [192.5 ± 26.3 mmol (kg·DM−1)] compared with the CON trial [245.3 ± 22.9 mmol (kg·DM−1)]. Mixed muscle metabolite concentrations of ATP, free glucose, PCr, G-6-P, and lactate are shown in Table 1. The concentration of PCr after 90 min of exercise was lower in the CON than the CHO trial (P < 0.05). Similar concentrations of ATP, free glucose, G-6-P, and lactate were observed between trials. However, in each trial, differences were observed between resting and postexercise metabolite concentrations of glycogen, glucose, G-6-P, and lactate (P < 0.05).
Single muscle fiber glycogen concentration. The concentration of muscle glycogen in Type I and Type II fibers before and after prolonged shuttle running with placebo ingestion is shown in Figure 5. There was a greater amount of glycogen utilized in Type II fibers compared with Type I (P < 0.01). The resting muscle glycogen concentration was higher in Type II fibers (P < 0.01). In addition, the glycogen concentration for three subjects was analyzed during the CHO trial, and the results of both trials are shown in Table 2. No statistical analysis was performed on these results because of the small sample size, although it was clear that glycogen utilization was lower in these three subjects during the CHO trial.
The main finding in this study was that the amount of muscle glycogen utilized during prolonged, intermittent, high-intensity exercise was reduced by 22% when a carbohydrate-electrolyte solution (CHO-E) was consumed immediately before and at frequent intervals during exercise. Preliminary evidence suggests that this reduced utilization of glycogen occurred in both Type I and Type II muscle fibers.
This apparent sparing of muscle glycogen with the ingestion of a CHO-E solution trial has also been reported in other studies during prolonged, continuous, submaximal running (37,38), cycling (3,9), and during intermittent exercise (12,41). A decrease in muscle glycogen utilization has also been observed when glucose was administered intravenously in humans (14) and rats (1,19).
There are several possible explanations for the decreased utilization of muscle glycogen in our study when a CHO-E solution is ingested during exercise. First, the ingested carbohydrate may have been utilized by the muscle, thereby sparing the endogenous store of muscle glycogen. Serum insulin concentration was higher in the CHO trial after 30 min, and blood glucose concentrations had a tendency to be higher following ingestion of the CHO-E drink. This would facilitate an increased glucose uptake by the exercising muscle (27,41) and reduce the contribution of intramuscular glycogen stores to energy metabolism (37,38,41).
Another explanation for these findings may be that glycolysis and entry of pyruvate into the mitochondria is better matched as a result of increased activation of the pyruvate dehydrogenase complex (PDH). It has been previously shown in resting humans that the activity of PDH is increased by hyperinsulinemia (24). Whether this is also the case during exercise remains to be elucidated. However, in support of a possible enhanced oxidation of pyruvate in the CHO trial, a higher insulin concentration was observed in parallel with a lower blood lactate concentration after 30 min in the CHO trial in the present study.
An alternative explanation for the apparent glycogen sparing is that glycogen resynthesis may have occurred in the Type II muscle fibers during the periods of low intensity exercise. Low-intensity exercise accounted for a total of 58 min, or 55%, of the total exercise duration. The elevated blood glucose and serum insulin levels would facilitate glycogen resynthesis in the Type II fibers, as has been previously reported (5,19). This would result in a reduced net glycogen use over the 90 min of exercise. Indeed, the preliminary findings from our study that glycogen sparing occurred in Type II fibers provides some evidence for glycogen resynthesis. Further research is required to elucidate whether the rate of glycogenolysis is reduced during this type of exercise when CHO is ingested.
Single fiber analysis showed that there was a decrease in glycogen concentration in both Type I and Type II fibers after 90 min of intermittent high-intensity running, which is in agreement with previous studies using intermittent cycling exercise (8,10,35). The greater amount of glycogen utilized in the Type II compared with the Type I fibers during the LIST emphasized the heavy demands of this type of exercise. Analysis of samples from three subjects shows that less muscle glycogen was utilized in both Type I and Type II fibers in the CHO trial. Differences between the two trials were 52.1 mmol (kg·DM−1) and 76.9 mmol (kg·DM−1) for the Type I and Type II fibers, respectively. Previous studies have shown that there may be a critical level of glycogen, below which, high-intensity exercise is impaired (15,16,26). Thus, it would be advantageous to reduce the amount of glycogen utilized in the Type II fibers during intermittent high-intensity exercise in order not to compromise performance.
In summary, the ingestion of a 6.9% carbohydrate-electrolyte solution providing 51 g CHO·h−1 during 90 min of intermittent high-intensity shuttle running resulted in a 22% reduction in the amount of muscle glycogen utilized, compared with drinking a noncarbohydrate placebo. This may explain the improvement in endurance capacity when soccer players ingested an identical carbohydrate-electrolyte solution immediately before and during a similar exercise trial (31).
The authors wish to record their appreciation of the excellent technical support for the single fiber analysis provided by S. Curtis, School of Biomedical Sciences, University of Nottingham Medical School, Nottingham NG7 2UH, U.K. This study was supported by SmithKline Beecham.
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