INTRODUCTION

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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.

	METHODS

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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.

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).

Statistical analysis. 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 hoc t-tests. Significance was declared at P < 0.05, and values in text and accompanying data are means ± SE.

	RESULTS

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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).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Muscle total creatine (TCr) concentrations. A: results from exhausted (Ex) and nonexhausted (Nex) limbs of carbohydrate (CHO) group post-1-legged exercise (Postex) and 6 h and 5 days after CHO supplementation. d.m., Dry matter. B: results from Ex and Nex limbs of creatine (Cr)+CHO group post-1-legged exercise and 6 h and 5 days after Cr + CHO supplementation. Values are means ± SE. Significantly different from Postex concentration: * P < 0.05, *** P < 0.001. Significant difference between Ex and Nex limbs after 5 days of supplementation, §§ P < 0.01.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Muscle glycogen concentrations in Ex and Nex limbs post-1-legged exercise and 6 h and 5 days after CHO or Cr + CHO supplementation.

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).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Serum insulin concentrations. A: results from CHO group subjects at rest and after exhaustive exercise in response to ingestion of 500-ml CHO solution (~90 g CHO). Significantly different from Postex concentration, * P < 0.05. B: results from Cr+CHO subjects at rest and after exhaustive exercise in response to ingestion of 5 g Cr monohydrate and 500-ml CHO solution. Significantly different from Postex concentration: * P < 0.05 and ** P < 0.01.

	DISCUSSION

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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.