We examined the effect of glycogen-depleting exercise on subsequent muscle total creatine (TCr) accumulation and glycogen resynthesis during postexercise periods when the diet was supplemented with carbohydrate (CHO) or creatine (Cr) + CHO. Fourteen subjects performed one-legged cycling exercise to exhaustion. Muscle biopsies were taken from the exhausted (Ex) and nonexhausted (Nex) limbs after exercise and after 6 h and 5 days of recovery, during which CHO (CHO group, n = 7) or Cr + CHO (Cr+CHO group, n = 7) supplements were ingested. Muscle TCr concentration ([TCr]) was unchanged in both groups 6 h after supplementation commenced but had increased in the Ex (P < 0.001) and Nex limbs (P < 0.05) of the Cr+CHO group after 5 days. Greater TCr accumulation was achieved in the Ex limbs (P < 0.01) of this group. Glycogen was increased above nonexercised concentrations in the Ex limbs of both groups after 5 days, with the concentration being greater in the Cr+CHO group (P = 0.06). Thus a single bout of exercise enhanced muscle Cr accumulation, and this effect was restricted to the exercised muscle. However, exercise also diminished CHO-mediated insulin release, which may have attenuated insulin-mediated muscle Cr accumulation. Ingesting Cr with CHO also augmented glycogen supercompensation in the exercised muscle.
muscle total creatine (TCr) increases as a result of creatine (Cr) feeding (23, 25). This increase has been associated with an improvement in performance of maximal exercise of short duration (20) and an acceleration of phosphocreatine (PCr) resynthesis during recovery from maximal exercise (19). In these investigations it was observed that subjects with an initial TCr concentration ([TCr]) of close to or less than 120 mmol/kg dry matter demonstrated the most marked increase in muscle TCr accumulation during supplementation. Furthermore, it was specifically in these subjects that a marked improvement in exercise performance and an accelerated rate of PCr resynthesis was observed after supplementation (7, 19).
Insulin, at supraphysiological concentrations, has been demonstrated to increase Cr uptake in rat skeletal muscle cells ex vivo (24) and prevent Cr loss in vivo in radiation-damaged rat skeletal muscle (28). Recently, supraphysiological insulin concentrations ([insulin]) have also been shown to stimulate Cr uptake in a mouse myoblast cell line (37). In humans, whole body and muscle Cr retention was shown to be increased when large amounts of simple carbohydrates (CHO; ∼95% simple sugars) were ingested in conjunction with Cr (14, 17). It was suggested that this response was attributable to glucose-mediated insulin release, stimulating an increase in Na+-dependent muscle Cr transport. More recently, it has been demonstrated that high or supraphysiological circulating concentrations of insulin are also required to augment muscle Cr accumulation in humans (41).
Submaximal exercise performed immediately before Cr ingestion has also been suggested to enhance muscle Cr uptake (23). Five subjects performed 1 h of one-legged exercise in the morning of each day of Cr supplementation. Three of the five subjects ingested 30 g of Cr each day for 4 days, another ingested the same amount for 7 days, and a further subject ingested 20 g each day for 3.5 days. At the end of the supplementation period, the authors reported that [TCr] was significantly greater (∼9%) in the exercised leg than in the nonexercised leg. It was postulated that this effect may have been brought about by an exercise-induced increase in limb blood flow, and thereby Cr delivery, or by a change in the kinetics of muscle Cr transport. It should be noted, however, that the differences observed between limbs were relatively small and highly variable among individuals. Furthermore, the greatest exercise-induced increase in muscle Cr accumulation was observed in two vegetarian subjects. Preliminary evidence indicates that the magnitude of muscle Cr retention during Cr supplementation is, at least initially, markedly greater in vegetarians compared with nonvegetarians (15). It would appear therefore that the small number of subjects, the variation in the supplementation protocol, and the inclusion of vegetarians in the study by Harris et al. (23) has not provided clear insight into the effect of exercise on muscle Cr accumulation. Cr supplementation for 10 days, combined with exercise training, has been reported to produce improvements in high-intensity running performance in rats (6). Although no difference in muscle Cr concentration ([Cr]) was observed between rats that were trained and Cr supplemented and those that were untrained and Cr supplemented, it was suggested that the improvements in performance in the former group were attributable to changes in muscle Cr + PCr stores and an increase in muscle citrate synthase activity. It is necessary, therefore, to examine more closely the influence of exercise on muscle Cr accumulation.
In the study by Green et al. (14), it was observed that consumption of CHO with Cr resulted in a greater increase in body mass than when individuals consumed Cr alone. Further investigation of this regimen of supplementation (16) also revealed a significant correlation between the extent of muscle glycogen and Cr accumulation after Cr + CHO supplementation (r = 0.75,n = 8,P < 0.05), which was not evident when Cr or CHO alone was ingested. The availability of muscle glycogen is a principal determinant of endurance exercise performance (2). The accepted method of increasing muscle glycogen stores is by “glycogen loading,” which classically involves depletion of muscle glycogen, usually by exercise, followed by consumption of a high-CHO diet for several days (e.g., 3, 39). Methods such as these can increase muscle glycogen concentrations ([glycogen]) to between 150 and 200% of normal resting levels. This response has been attributable to an increase in glycogen synthase activity in the postexercise period and an exercise-induced increase in insulin sensitivity of the glycogen-depleted muscle (3). On the basis of the relationship we previously observed between muscle Cr and glycogen accumulation during Cr + CHO supplementation, it was of interest to us whether the glycogen supercompensation response could be augmented by Cr ingestion.
The principal purpose of the present study, therefore, was to examine a number of factors that have been proposed to increase muscle Cr accumulation in humans. By using a one-legged cycling model of exercise, we intended to determine whether exercise enhanced muscle Cr accumulation, and, if it did, whether this response was restricted to the exercised muscle and was influenced by the exercise-induced increase in muscle blood flow. In an attempt to stimulate insulin-mediated Cr transport, CHO was administered with Cr, which also provided us with an opportunity to further examine the relationship between muscle Cr and glycogen accumulation observed during Cr + CHO supplementation.
Fourteen healthy men [age 23 ± 1 (SE) yr, body mass index 22 ± 0.5 kg/m2] volunteered for the present study, which was approved by the University of Nottingham Medical School Ethics Committee. Before commencing the investigation, all subjects gave informed written consent, and routine physiological measurements and blood analyses were performed. All subjects were moderately active, and none was highly trained.
Protocol and treatment groups.
Subjects reported to the laboratory on the morning of the study after an overnight fast, having abstained from alcohol consumption and strenuous exercise for 48 h.
Then, subjects in pairs performed one-legged cycling exercise on a cycle ergometer, one subject on either side of the ergometer, each supporting himself with his contralateral leg (3). A cycling cadence of 70 rpm was maintained, and subjects worked against a load predetermined to raise heart rate to between 160 and 170 beats/min. Consumption of water was allowed ad libitum throughout the exercise. Subjects continued cycling until near fatigue, when they were allowed to rest for a maximum of 5 min. This was followed by short periods of cycling and rest until the required cadence could not be maintained for longer than 2 min, or until a subject chose not to continue. If one subject exercised longer than his partner did, an investigator continued cycling in the partner’s position. On exhaustion, subjects rested supine on a recovery bed, and a muscle biopsy sample (postexercise) was obtained from the vastus lateralis of the exhausted (Ex) and nonexhausted (Nex) limbs, using the needle biopsy technique (1). Further muscle samples were also obtained from each subject’s limbs 6 h postexhaustion and 5 days later. After the first muscle biopsy, a cannula was inserted into an antecubital vein of the subject’s arm, and 8-ml blood samples were obtained at 20-min intervals for a period of 100 min. The cannula was kept patent during the sampling period by using an isotonic saline drip.
After exercise, subjects were randomly allocated into two dietary treatment groups. The CHO group consisted of seven subjects (age 24 ± 2 yr, body mass index 21.3 ± 0.3 kg/m2). During the initial 6 h of recovery from exercise, each subject ingested a warm sugar-free drink, followed immediately by 500 ml of a commercially available CHO-containing solution (Lucozade, ∼18.5% wt/vol glucose and simple sugars; SmithKline Beecham, Coleford, UK) on three separate occasions, as described below.
The Cr+CHO group consisted of seven subjects (age 23 ± 2 yr, body mass index 22.5 ± 0.8 kg/m2). During the initial 6 h of recovery from exercise, each subject consumed 5 g of creatine monohydrate powder (Experimental and Applied Sciences, Golden, CO) dissolved in a warm, sugar-free drink, followed immediately by 500 ml of Lucozade on three separate occasions, as described below.
The first supplement was ingested immediately after collection of a postexercise blood sample (0 min), which occurred within 30 min of the end of the exercise and the postexercise biopsy sample. In the 6-h period of recovery from exercise, before further muscle sampling, subjects ingested two additional doses of their respective supplements at 2-h intervals (i.e., 2 and 4 h after the initial dose). In addition to this, all subjects consumed a small meal, containing ∼70 μg CHO per meal, 1 h after supplemental doses 2 and 3 (i.e., 3 and 5 h after the initial dose).
Subjects repeated ingestion of the supplements four times each day for another 4 days. In addition to the supplements, all subjects consumed a diet of high CHO content (CHO contributing >80% of daily energy intake), with which they had been supplied. During this period all subjects followed their normal daily activities, other than performance of strenuous exercise.
Muscle sample treatment and analysis.
After removal from the leg, each muscle sample was allowed to stand at room temperature for 1 min to allow PCr concentration ([PCr]), which is decreased by energy-utilizing processes associated with the biopsy procedure (40), to return to a normal level before being frozen in liquid nitrogen. For analysis, samples were freeze-dried, fat was extracted by using petroleum spirit, blood and visible connective tissue were removed, and the sample was powdered. Samples were then analyzed for [glycogen], ATP concentration ([ATP]), [PCr], and free [Cr] (22). Total [Cr] is reported as the sum of [PCr] and [Cr]. All [glycogen] and metabolite concentrations were corrected for nonmuscle constituents by using muscle [ATP] (23).
Blood sample treatment and analysis.
It has previously been observed that exhaustive exercise, such as that employed during “glycogen loading,” can result in a diminished insulin response to ingested glucose (5, 13, 27). To ascertain whether this occurred in the present investigation, the blood-sampling protocol was repeated during a separate visit to assess subjects’ nonexercised responses to the supplements ingested. Whole blood glucose concentration was measured by using an automated analyzer (Hemocue,Ängelholm, Sweden), and serum [insulin] was measured by using a commercially available radioimmunoassay diagnostic kit (Diagnostic Products, Los Angeles, CA).
Two-way analysis of variance for repeated measures was conducted between groups and between limbs within each group. Where appropriate, significant differences were located by appropriate post hoct-tests. Significance was declared atP < 0.05, and values in text and accompanying data are means ± SE.
Subjects reported compliance with all aspects of the protocol and did not report any ill effects. There was no significant body mass change in the CHO group after 5 days of supplementation; however, body mass of the Cr+CHO group increased significantly (presupplementation 72.8 ± 4.6 kg, postsupplementation 73.8 ± 4.6 kg;P < 0.05).
Muscle metabolite and substrate changes.
No change in muscle [ATP] was observed in limbs of either group throughout the study (Table 1).
No significant changes in muscle [TCr] were observed in the Ex and Nex limbs of either group 6 h after one-legged exercise (Fig.1) or in the limbs of the CHO group after 5 days (Fig. 1 A). There was a significant increase in muscle [TCr] in the Ex (23% increase; P < 0.001) and Nex (14% increase; P < 0.05) limbs of the Cr+CHO group after 5 days of supplementation, with the Ex limb having a 68% greater accumulation (P < 0.01; Fig. 1 B).
In accordance with the changes in muscle [TCr], no change in [PCr] or [Cr] was observed in limbs of the CHO group at any time. A significant increase in [PCr] was observed in the Nex limbs of the Cr+CHO group after 5 days of supplementation (Table 1), and there was also a significant increase in [Cr] in the Ex limbs of this group (allP < 0.05). No significant change in [PCr] was observed in the Ex leg after 5 days.
The exercise protocol almost completely depleted glycogen in the Ex limbs of the CHO group and Cr+CHO group (to 8 and 5% of the concentration measured in the Nex limb postexercise, respectively;P < 0.001; Fig.2). Muscle [glycogen] in the Nex limb after the exercise period was used as a measure of the initial [glycogen] in the Ex limb, as it has been previously shown that this one-legged cycling protocol does not affect muscle [glycogen] in the supporting leg (8).
Six hours after ingestion of the first supplement, glycogen had been restored in the Ex limb to 69 and 73% of the Nex [glycogen] in the CHO and Cr+CHO groups, respectively, but there was no difference between groups (Fig. 2). After 5 days of supplementation, [glycogen] had increased dramatically from the postexercise concentration measured in the Ex limb of the CHO and Cr+CHO groups (both P < 0.001; Fig.2). Furthermore, the magnitude of this increase was 23% greater in the Cr+CHO group (P = 0.06), such that a significant difference in [glycogen] between groups was very close to being achieved (P = 0.06).
The [glycogen] in the Nex limbs of both groups was unchanged 6 h after ingestion but was increased after 5 days of supplementation (CHO group, P < 0.01; Cr+CHO group, P < 0.05). However, there was no difference in concentration between groups (Fig.2).
Blood glucose and insulin changes.
There was no significant difference between groups in either blood glucose concentration (Table 2) or serum [insulin] responses (Fig. 3) at any point during the initial 100-min postsupplement ingestion. Comparison of these responses with the corresponding measurements made under nonexercised conditions showed that serum [insulin] in both groups in the exercised state were significantly lower during the first 40 min after ingestion of supplement (Fig. 3).
A major finding of the present study was the difference in [TCr] between the Ex and Nex limbs in the Cr+CHO group after 5 days of supplementation. This demonstrates that a single bout of exhaustive exercise before Cr supplementation can markedly augment skeletal muscle Cr accumulation and that this response is restricted to the exercised muscle, a finding which may be of practical importance. The present observation supports the indication of Harris et al. (23) that exercise can augment muscle Cr accumulation. In their investigation, the authors proposed that exercise increased muscle Cr uptake by increasing blood flow to the exercised muscle during the postexercise period or by increasing the kinetics of muscle Cr transport. The authors implied that increasing blood flow to the exercised muscle would maximize the exposure of that muscle to [Cr] above the Michaelis constant for its transport. However, although muscle blood flow was not measured in the present investigation, we believe that an increase in blood flow after exercise was unlikely to have been responsible for the greater Cr content in the Ex limb in the present study. This contention is supported, first, by the lack of any difference in [TCr] between limbs after 6 h of supplementation, when differences in limb blood flow would be expected to be at their greatest. Second, evidence demonstrating that the Michaelis constant of muscle Cr transport is substantially lower (20–110 μmol/l; 30, 36, 37) than the value of 500 μmol/l proposed by Fitch and Shields (12), and cited by Harris et al. (23), makes it unlikely that Cr availability will limit transport by using conventional regimens of Cr supplementation.
Cr transport into muscle cells is dependent on the presence of extracellular Na+ (10). A specific Cr transporter, which is highly expressed in skeletal muscle, has been identified and cloned (21, 33, 36). After 5 days of Cr + CHO supply, there was an 11% difference in the [TCr] between the Ex and Nex limbs in the present study. Given that both limbs were exposed to the same plasma [Cr] over the 5 days of supplementation, it is feasible that exercise may have enhanced Cr accumulation by increasing the maximal rate of Cr transport in the exercised limb. Indeed, exercise has been demonstrated to enhance the transport of other amino acids (4), which has been suggested to be only partly dependent on increased muscle blood flow (42). An exercise-induced augmentation of muscle Cr transport may have occurred through a number of as yet unknown mechanisms. For example, Odoom et al. (37) have suggested that an increase in the maximal rate of Cr transport may occur as a result of an allosteric effect on the transporter itself, by recruitment or synthesis of new transporters, or by production of changes in the forces driving Cr transport. In support of the latter suggestion, it has been proposed that the majority of Cr transport is achieved by a Na+-Cr cotransport system (30, 37), which makes use of the sarcolemmal Na+-K+pumps. It has been suggested that sarcolemmal Na+-K+pump total activity can be increased through either an increase in intrinsic pump activity or by a net gain in the number of pumps (9). Exercise training has been demonstrated to produce increases in the number of muscle Na+-K+pumps, which attenuates the loss of K+ from muscle during subsequent exercise (34, 35). Green and colleagues (18) have shown in humans that exercise training of sufficient duration and intensity (2 h/day, 65% maximal O2 uptake, 6 days) can upregulate Na+-K+pump concentration within the first week of training. Additionally, it has been demonstrated in rats that a single acute bout of running exercise can increase muscle concentration of some Na+-K+pump subunits in the sarcolemma and also elevate mRNA levels for additional Na+-K+pump subunits (43). It is therefore feasible that in the present study the single bout of exhaustive exercise produced an upregulation of muscle Na+-K+pump function in the Ex limb that facilitated muscle Cr transport. In support of this conclusion is the finding that pharmacological activation and inhibition of Na+-K+pump activity in mouse myoblast cells was paralleled by up- and downregulation of cellular Cr accumulation (37).
The Na+-K+-ATPase pump activity in skeletal muscle is also influenced by insulin (26,32). The abundance of some pump subunits in the muscle membrane has been shown to increase with acute exposure to insulin, most likely by recruitment from intracellular compartments. It is likely that this change contributes to the enhancement of muscle Cr accumulation that is observed when CHO is ingested with Cr (14). It remains to be seen whether any interaction exists between exercise- and insulin-induced increases in total pump activity.
TCr accumulation in the Ex muscle in the present study (28.6 ± 4.8 mmol/kg dry matter) was no greater when compared with the magnitude of accumulation previously observed in nonexercised subjects ingesting Cr alone (25 mmol/kg dry matter; 14). Furthermore, the magnitude of TCr accumulation in the Nex muscle in the Cr+CHO group (17.0 ± 4.7 mmol/kg dry matter), although significantly elevated above the initial concentration, was noticeably lower than that achieved previously in nonexercised individuals receiving the same supplements (14). Given the diminished insulin response to oral CHO observed after exhaustive exercise in the present study, these findings also indirectly support a role for insulin in promoting Cr transport. Specifically, the exercise-induced blunting of CHO-mediated insulin release observed in the present study (Fig. 3) would be expected to negatively affect insulin-mediated stimulation of muscle Cr transport, particularly because recent work has demonstrated that high physiological concentrations of insulin are required to stimulate muscle Cr accumulation in humans (41). This phenomenon is of practical importance in terms of optimizing muscle TCr accumulation during supplementation; i.e., individuals wishing to maximize muscle Cr transport by consuming CHO might refrain from performing exhaustive or prolonged exercise during the supplementation period.
This study found that combining Cr and CHO ingestion with prior glycogen-depleting exercise augmented glycogen resynthesis during recovery and, similar to muscle Cr accumulation, this response was restricted to the exercised muscle. This is the first documentation of this response in humans. Furthermore, this increase in [glycogen] was of a magnitude that one would consider sufficient to produce a significant improvement in endurance exercise performance. Green et al. (16) demonstrated a significantly greater peak [insulin] after the ingestion of Cr + CHO, compared with the ingestion of CHO alone and also showed a significant relationship between muscle Cr and glycogen accumulation (r = 0.75,n = 8,P < 0.05) after 5 days of supplementation in nonexercised individuals. In the present study, there was no significant difference in the glucose-stimulated insulin response between groups after exercise. However, the extent of glycogen depletion produced by the present study would be expected to stimulate insulin-independent glycogen resynthesis for a major part of the initial recovery period (38). Therefore, the effect of diminished circulating [insulin] observed after exercise might have, at least in terms of glycogen synthesis, been negligible during the early stage of resynthesis. However, the clear lack of an effect of Cr + CHO on glycogen accumulation in the Nex limb after 6 h and 5 days of recovery in the present study goes against an increase in insulin availability being responsible for the augmentation of glycogen resynthesis. Presumably, one could have expected to see an increase in [glycogen] in both limbs after Cr + CHO ingestion, albeit to a lesser extent in the Nex limb, had this mechanism been in operation.
It has recently been reported that muscle glycogen synthesis is modulated by muscle cell volume changes (31). Osmotically induced swelling of primary rat myotubes increased glycogen synthesis independently of changes in glucose uptake. Cr supplementation in humans has been shown to increase body mass (e.g., 20), in particular fat-free mass (11, 29), and has also been shown to increase body water content over the supplementation period (11). If muscle Cr accumulation can cause muscle swelling, it is possible that the greater accumulation of Cr in the Ex limb of the Cr+CHO group produced a greater muscle cell volume change than in the corresponding limb of the CHO group, thereby contributing to the higher [glycogen] in the Cr+CHO group after 5 days of supplementation. Such a response would also explain, at least in part, the lack of an effect of Cr + CHO on glycogen synthesis in the Nex limb, where Cr accumulation was markedly lower than previously observed after Cr + CHO ingestion (14).
In conclusion, exhaustive exercise enhanced subsequent Cr accumulation in exercised muscle through an as yet undetermined mechanism. This effect was restricted to the exercised muscle itself and did not appear to be due to an increase in muscle blood flow. Exhaustive exercise diminished glucose-stimulated insulin release, however, thereby attenuating any enhancement of Cr accumulation due to CHO ingestion. Finally, Cr + CHO supplementation after glycogen-depleting exercise augmented glycogen resynthesis in the exercised muscle, which might be considered sufficient to significantly improve endurance exercise performance.
This work was funded by grant support from Experimental and Applied Sciences, Golden, CO.
Address for reprint requests and other correspondence: T. Robinson, School of Biomedical Sciences (Floor E), Univ. of Nottingham Medical School, Queens Medical Centre, Nottingham, NG7 2UH, UK.
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