Creatine supplementation does not affect human skeletal muscle glycogen content in the absence of prior exercise

J Appl Physiol 104: 508 512, 2008. First published November 21, 2007; doi:10.1152/japplphysiol.00787.2007. Creatine supplementation does not affect human skeletal muscle glycogen content in the absence of prior exercise Dean A. Sewell, 1 Tristan M. Robinson, 2 and Paul L. Greenhaff 3 1 School of Life Sciences, Heriot-Watt University, Riccarton, Edinburgh; 2 Company Nutritionist, H. J. Heinz, Wigan; and 3 School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom Submitted 19 July 2007; accepted in final form 19 November 2007 Sewell DA, Robinson TM, Greenhaff PL. Creatine supplementation does not affect human skeletal muscle glycogen content in the absence of prior exercise. J Appl Physiol 104: 508 512, 2008. First published November 21, 2007; doi:10.1152/japplphysiol.00787.2007. Due to the current lack of clarity, we examined whether 5 days of dietary creatine (Cr) supplementation per se can influence the glycogen content of human skeletal muscle. Six healthy male volunteers participated in the study, reporting to the laboratory on four occasions to exercise to the point of volitional exhaustion, each after 3 days of a controlled normal habitual dietary intake. After a familiarization visit, participants cycled to exhaustion in the absence of any supplementation (N), and then 2 wk later again they cycled to exhaustion after 5 days of supplementation with simple sugars (CHO). Finally, after a further 2 wk, they again cycled to exhaustion after 5 days of Cr supplementation. Muscle samples were taken at rest before exercise, at the time point of exhaustion in visit 1, and at subsequent visit time of exhaustion. There was a treatment effect on muscle total Cr content in Cr compared with N and CHO supplementation (P 0.01). Resting muscle glycogen content was elevated above N following CHO (P 0.05) but not after Cr. At exhaustion following N, glycogen content was no different from CHO and Cr measured at the same time point during exercise. Cr supplementation under conditions of controlled habitual dietary intake had no effect on muscle glycogen content at rest or after exhaustive exercise. We suggest that any Cr-associated increases in muscle glycogen storage are the result of an interaction between Cr supplementation and other mediators of muscle glycogen storage. phosphocreatine; carbohydrate; exercise FINDINGS THAT DIETARY CREATINE (Cr) supplementation increases muscle total Cr (TCr) content have led to a large number of investigations into the effects of Cr supplementation on, for example, muscle function and exercise performance with varying outcomes. One explanation for the lack of improvement in functional outcomes in some studies is a wide between-subject variation in the magnitude of muscle TCr accumulation as a result of supplementation (3, 9, 12). It has also been shown that any improvements in work output during maximal exercise (3) and postcontraction phosphocreatine (PCr) resynthesis (9) may well be dependent on the extent of muscle Cr accumulation (3). Subsequent investigations have addressed potential methods of improving and understanding the precise mechanisms of muscle Cr accumulation. Since findings from studies involving irradiated animals suggest Cr entry into muscle might be stimulated by insulin (e.g., Refs. 13, 16), we showed that the consumption of a large amount of carbohydrate at the time of Cr ingestion, when abstaining from strenuous exercise during the supplementation period, resulted in a significant increase in muscle Cr accumulation in healthy humans (8), which we proposed was insulin mediated. Indeed, it was later shown that the stimulatory effect of insulin on human muscle Cr accumulation occurs at physiologically high or supra-physiological insulin concentrations (26). Better management of blood glucose concentration has the potential to be of benefit and of therapeutic value in the treatment of metabolic disease states (reviewed in Ref. 29), and, for example, it was proposed by Op t Einde et al. (23) that Cr supplementation might have a role in improving glucoregulation. In that investigation, Cr supplementation appeared to prevent the decrease in muscle GLUT-4 protein content during a 2-wk period of immobilization and, subsequently, increased GLUT-4 content after 10 wk of Cr supplementation combined with rehabilitation training. In the same study, it was shown that muscle glycogen concentration was increased after 3 wk of rehabilitation training and Cr supplementation. Based on their results, the authors proposed that Cr supplementation might be of use in the treatment of diseases associated with peripheral insulin resistance. Having demonstrated that combined Cr and carbohydrate ingestion can augment muscle Cr accumulation (8), we have subsequently shown, using a one-legged exercise model, that postexercise muscle glycogen storage can be augmented by Cr and carbohydrate supplementation following exercise compared with carbohydrate ingestion alone (25). However, this effect was restricted to the exercised muscle. This highlighted for the first time a potential ergogenic effect of Cr supplementation on endurance-type exercise. Further support, and other factors responsible for this potential ergogenic effect, have been indicated by Nelson et al. (21), Derave et al. (5), van Loon et al. (28), and Cribb and Hayes (4); however, whether the effect on muscle glycogen storage can be achieved by Cr ingestion per se, and in the absence of prior glycogen-depleting exercise, remains equivocal. In the study by Op t Eijnde et al. (23), muscle glycogen concentration in subjects ingesting Cr was in the region of 650 mmol/kg dry weight after the initial 3 wk of rehabilitation training compared with around 520 mmol/kg dry weight in the placebo group. This prompted the authors to suggest that supplementation per se (and a standard diet; no dietary instruction was given to the subjects) coupled with a moderate resistance exercise program could produce similar glycogen storage effects to classic glycogen supercompensation protocols (2). Since the investigation examined individuals during a phase of rehabilitation from immobiliza- Address for reprint requests and other correspondence: D. A. Sewell, School of Life Sciences, Heriot-Watt Univ., Riccarton, Edinburgh EH14 4AS, UK (e-mail: d.a.sewell@hw.ac.uk). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 508 8750-7587/08 $8.00 Copyright 2008 the American Physiological Society http://www.jap.org

tion-induced muscle atrophy, similar responses may not necessarily be observed in normal individuals supplemented with Cr and ingesting a standard diet. van Loon et al. (28) reported an 18% increase in muscle glycogen content as a result of 5 days of concurrent Cr and carbohydrate supplementation, in the absence of an exercise intervention, compared with placebo (carbohydrate) ingestion. Nelson et al. (21) showed that performing a glycogen loading protocol (exhaustive exercise followed by a high carbohydrate diet for 3 days) after Cr loading resulted in a anticogen content 10% greater than when glycogen loading was before Cr loading; however, the Cr loading phase (20 g/day for 5 days) had no effect on muscle glycogen content. This latter lack of effect would add some support to the hypothesis that muscle has to be contracted for Cr to exert an effect on glycogen storage, as indicated in our previous work (25), where the augmentation of glycogen resynthesis due to prior glycogen-depleting exercise was restricted to exercised muscle. Ingesting Cr with or without protein during a period of immobilization (2 wk) and then resistance training (6 wk; Ref. 5) resulted in a higher muscle glycogen content ( 35%) compared with ingesting an isocaloric carbohydrate supplement, although this is another example of immobilization-induced muscle atrophy, and similar responses may not necessarily be observed in normal muscle supplemented with Cr. Indeed, Cr supplementation had no effect on glycogen content in the contralateral control leg that was not trained. Cribb and Hayes (4) showed that, after a 10-wk period of supervised exercise training and ingestion of Cr, carbohydrate, and protein, muscle glycogen content was higher in an experimental group consuming the supplement immediately pre- and posttraining compared with a group consuming the same supplement at other times. Thus, when accompanied by exercise training, supplementation appeared to have an effect on muscle glycogen content, but this was dependent on timing of the supplement ingestion in relation to the exercise bout. The interaction of Cr with other factors (i.e., exercise, together with carbohydrate and protein ingestion) complicates the interpretation of the data. There remains a need to further understand the effect of Cr supplementation on muscle glycogen content. To elucidate whether Cr ingestion per se can increase muscle glycogen storage, the present study examined the effect of dietary Cr supplementation on glycogen content in a group of nonexercising healthy male volunteers under conditions of controlled habitual dietary intake. We also examined muscle glycogen content at the point of exhaustion after prolonged submaximal exercise. For comparative purposes, the effects of 5 days of carbohydrate supplementation on glycogen storage were also investigated in the same subjects. CREATINE SUPPLEMENTATION AND MUSCLE GLYCOGEN CONTENT 509 After initial health screening and exercise testing, subjects reported to the laboratory on four separate occasions and performed cycling exercise to the point of exhaustion. Details of experimental conditions and protocols are outlined below. Experimental design. An overview of the study design is represented schematically in Fig. 1. Subjects made a dietary record for 3 days before visiting the laboratory for the first time. Familiarization with exhaustive exercise. To familiarize subjects with the exercise protocol, all subjects visited the laboratory in the morning, following an overnight fast, and performed cycling exercise to exhaustion (Fig. 1, Famil.). This visit was included to allow subjects to familiarize themselves with exercise of relatively high workloads to the point of exhaustion. It was anticipated that this would result in exhaustion during subsequent tests as a result of substrate depletion rather than for other reasons. Subjects performed 3 min of warm-up exercise on an electrically braked ergometer (Excalibur Sport, Lode NV Instrumenten, Groningen, The Netherlands) at a constant cadence of 70 rpm against a workload of 100 W. The electrical resistance of the cycle was then adjusted to produce a workload designed to elicit an oxygen consumption of 70% V O2 peak. Every 15 min during exercise, expired air samples were collected for measurement of oxygen consumption, and heart rate was recorded. In an effort to maintain euhydration, subjects ingested 200 ml of water at ambient temperature every 15 min throughout the exercise period. Subjects were verbally encouraged to continue cycling for as long as possible to the point of volitional exhaustion or until they could no longer maintain the required cadence. Supplementation conditions before exercise. During the 3 days before the next cycle to exhaustion (Fig. 1, EX1), each subject consumed his normal diet and recorded it in a food diary. This pattern of food consumption was subsequently repeated on the 3-day periods before the other test visits (Fig. 1, EX2 and EX3). For 5 days before the EX2 visit, subjects also supplemented their diets with four 500-ml servings of a carbohydrate drink containing 18.5% glucose and simple sugars (Lucozade, SmithKline Beecham, Coleford, UK) per day (at 0800, 1200, 1600, and 2000). For 5 days before the EX3 visit, subjects ingested 5gofCrmonohydrate (CrH 2O; Experimental and Applied Sciences) four times per day (as in EX2) in a warm, noncaffeinated drink. Subjects did not perform exercise (other than habitual walking activities) during the supplementation periods. Subjects nude weights METHODS Six healthy male subjects (means SE; age 26 3 yr; body mass index 24.1 0.8 kg/m 2 ) participated in this study, which was approved by the University of Nottingham Medical School Ethics Committee. Before commencing the investigation, all subjects gave their informed, written consent to participate. All subjects regularly performed strenuous exercise for 1 h or more on at least three occasions per week but were not highly trained. Mean peak oxygen consumption (V O2 peak) (determined by prestudy tests) of the subjects was 52.1 2.0 ml kg 1 min 1. Fig. 1. Overview of the experimental design. CHO, carbohydrate; Cr, creatine.

510 CREATINE SUPPLEMENTATION AND MUSCLE GLYCOGEN CONTENT were recorded on the day before the first day of supplementation and also on arrival at the laboratory on all experimental visits. Experimental protocol. On each test day, fasted subjects reported to the laboratory at 0800. Subjects were cannulated with an 18-gauge cannula inserted retrogradally into an antecubital vein of the left arm. Blood samples were obtained via a three-way tap, and the cannula was kept patent by isotonic saline (0.9% NaCl BP, Baxter Healthcare, Thetford, UK). During the three experimental visits (EX1, EX2, EX3), blood samples were obtained at rest, every 15 min during exercise, and 5 min after the end of exercise. After cannulation, the subject s leg was then prepared for muscle biopsy sampling. Three incisions (two in the case of EX1) were made through the skin of the central area of the ventral side of the thigh. Each incision was 5 mm long and spaced 3 cm apart from each other (11). A resting muscle sample (Rest) was obtained from the vastus lateralis using the percutaneous needle biopsy technique (1), by insertion through the uppermost of the incisions, whereon it was immediately frozen in liquid nitrogen. A sterile dressing, which was held in place using an adhesive elasticated bandage, was applied over the incisions. After 15 min of recovery from the muscle sampling procedure, the subject performed cycling exercise to exhaustion. Immediately at the point of exhaustion during EX1, the subject was supported in the saddle and a muscle sample was obtained from the leg through the previously prepared incision (TEX1 Normal). If during visits EX2 and EX3 they reached this same time point, each subject briefly stopped exercising, and another muscle sample was immediately taken by the same method. Muscle samples obtained at these times are termed TEX1 CHO or TEX1 Cr, depending on the supplementation condition preceding the exercise visit. After the removal of a TEX1 muscle sample, a new sterile dressing was applied to the biopsy area, and the subject continued cycling to the point of exhaustion. Muscle samples were obtained at exhaustion during test visits EX2 and EX3 and are termed EXH CHO and EXH Cr, respectively. All muscle samples were frozen in liquid nitrogen immediately after removal from the leg (time from stopping exercise to freezing was 12 2 s). Muscle and blood sample treatment and analysis. For analysis, each muscle sample was freeze-dried, fat was extracted using petroleum spirit, blood and visible connective tissue were removed, and the sample was powdered. Samples were then analyzed for glycogen, ATP, Cr, and PCr content (11). TCr is reported as the sum of PCr and Cr. All glycogen and metabolite contents were corrected for nonmuscle constituents by using muscle ATP content (12). On collection, blood samples were immediately analyzed for glucose concentrations using an automated analyzer (YSI 2300 STAT plus, Yellow Springs Instruments, Yellow Springs, OH). Statistical analysis. Analysis of variance was conducted between treatments using commercially available statistical analysis software (StatView, SAS Institute, Cary, NC). Where appropriate, significant differences were located by appropriate post hoc tests. Significance was declared at P 0.05, and values in text and accompanying data are means SE. RESULTS Body mass. No significant changes in body mass were seen following either carbohydrate or Cr supplementation (1.0 0.5 and 0.3 0.2 kg, respectively; n 6, P 0.05). Muscle glycogen content at rest and after exercise. After carbohydrate supplementation, resting muscle glycogen content was elevated above that determined after normal habitual dietary intake (P 0.05) but not after Cr supplementation (Fig. 2). Muscle glycogen content at TEX1 CHO and TEX1 Cr was no different from glycogen content at TEX1 Normal (Fig. 2). Similarly, muscle glycogen content at EXH CHO and EXH Cr was no different from that at TEX1 Normal (Fig. 2). Fig. 2. Muscle glycogen content at rest, during exercise, and immediately after exhaustion under normal, CHO, and Cr supplemented conditions. dm, dry mass. *Significantly different from normal, rest value (P 0.05). There was a trend for greater muscle glycogen utilization from the beginning of exercise to TEX1 after carbohydrate supplementation, although utilization did not differ from that of the whole exercise period under normal habitual dietary conditions [normal, 395 47 mmol/kg dry mass (dm); carbohydrate, 494 46 mmol/kg dm; Cr, 421 54 mmol/kg dm; P 0.05]. The workloads used in the exercise protocols produced an oxygen consumption of 73% V O2 peak. Muscle metabolite concentrations. Resting muscle TCr content increased after Cr supplementation (13.8 5 mmol/kg dm; P 0.01; Table 1). Resting muscle PCr content was unchanged from presupplemented conditions following Cr supplementation. Muscle PCr concentration at TEX1 Cr and EXH Cr was no different from that at TEX1 Normal (43.4 3.3 mmol/kg dm; data not shown). Resting muscle Cr concentration increased significantly from presupplemented conditions following Cr supplementation (9.4 3.4 mmol/kg dm; P 0.01; Table 1). Resting muscle ATP concentrations were similar on all occasions, being unchanged by dietary intervention (Table 1). ATP concentration immediately after exhaustion was not significantly different from that at rest during any experimental visit (data not shown). Blood glucose concentration during exercise. Concentrations of blood glucose were compared within and between treatments for exercise time points up to 60 min. After this time point, the number of samples contributing to the group s mean concentration decreased due to some subjects fatiguing earlier than others, which prevented valid statistical analysis. Blood glucose concentration did not change significantly from resting concentrations during the first 60 min of exercise following any dietary condition. No significant differences in blood glucose concentrations were observed at any time point (rest 60 min) between dietary conditions. Blood glucose concentrations 5 min postexhaustion were not significantly different from resting concentrations following any dietary condition (normal, 4.68 0.49 mm; CHO, 5.17 0.93 mm; Cr, 4.72 0.3 mm; P 0.05).

Table 1. Resting muscle ATP, TCr, PCr and Cr concentrations following normal, CHO, or Cr treatment conditions DISCUSSION Dietary Condition Normal CHO Cr TCr, mmol/kg dm 125.3 3.6 119.6 1.4 139.1 3.1* PCr, mmol/kg dm 76.7 3.2 73.8 5.9 81.1 3.6 Cr, mmol/kg dm 48.6 4.0 45.8 3.2 58.0 2.5* ATP, mmol/kg dm 24.0 1.4 25.9 0.5 26.0 0.7 Values are means SE. CHO, carbohydrate; Cr, creatine; TCr, total Cr; PCr, phosphocreatine; dm, dry mass. *Significant difference from normal and CHO conditions (P 0.01). The principal finding of this investigation is that 5 days of dietary Cr supplementation and controlled normal habitual dietary intake had no effect on muscle glycogen storage in healthy male volunteers. This lack of an effect on glycogen storage is not in concordance with some other reports, albeit recorded under different experimental conditions. After 2 wk of immobilization, Op t Eijnde et al. (23) demonstrated a higher muscle glycogen content in subjects ingesting Cr after 3 wk of rehabilitation training and a standard diet compared with a placebo group. Concomitant exercise training might therefore be an important factor, although the immobilization-induced muscle atrophy confounds the interpretation, and similar responses may not necessarily be observed in normal individuals supplemented with Cr. In a further study by the same group and using an immobilization-induced muscle atrophy model, Derave et al. (5) showed that, with the leg immobilized, Cr and protein supplementation in conjunction with resistance retraining increased muscle glycogen content compared with carbohydrate ingestion alone. Furthermore, similar to Robinson et al. (25), this effect was confined to the exercised (but previously immobilized) leg, supporting the hypothesis first proposed by Robinson et al. (25) that the ingestion of Cr, in the absence of prior exercise, does not increase muscle glycogen storage. In further support of this stance, Nelson et al. (21) found no effect of Cr supplementation per se on muscle glycogen content following a glycogen depleting/loading protocol (3 days before Cr supplementation). Despite this finding, in a subsequent Cr-loaded state, these authors demonstrated an augmented muscle glycogen content following a further glycogen depleting/loading protocol and concluded that muscle glycogen supercompensation is enhanced by prior Cr supplementation. In contrast to the current findings, van Loon et al. (28) showed that acute Cr and carbohydrate supplementation increases glycogen content without prior exercise or exercise training during supplementation. In a subject group with a relatively high aerobic capacity, the maintenance of normal dietary habits and physical activity is most likely to have consisted of a high carbohydrate diet and training compared with less active subjects or in immobilized muscle used by comparative studies. This would therefore support the notion that exercise is a key factor to accompany Cr supplementation and a high carbohydrate intake to achieve a positive effect on muscle glycogen content and possibly endurance exercise capacity. However, despite the previously reported increase in CREATINE SUPPLEMENTATION AND MUSCLE GLYCOGEN CONTENT 511 glycogen content as a result of Cr and carbohydrate supplementation (27), a measurable improvement in endurance exercise performance was not seen. Cribb and Hayes (4) explored the effect of timing of Cr (and carbohydrate and protein) supplementation relative to exercise during a 10-wk resistance exercise training program. Partly in support of our hypothesis, the authors showed that, when accompanied by exercise training, Cr supplementation appeared to have a positive effect on muscle glycogen storage, i.e., consuming the supplement immediately pre- and posttraining augmented muscle glycogen storage compared with a group consuming the supplement at least 5 h pre- or posttraining. However, the study did not include a subject group to control for exercise as an independent variable, and Cr was ingested along with carbohydrate and protein. Finally, further support for the suggestion that a combination of dietary Cr supplementation, high carbohydrate intake, and exercise can enhance muscle glycogen storage can be seen in the in the first report of this effect (25), where Cr and carbohydrate ingestion following one-legged exhaustive exercise resulted in an increase in glycogen content that was restricted to the exercised limb. The present investigation demonstrates that dietary Cr supplementation does not influence muscle glycogen content and supports previous proposals that dietary Cr-associated increases in muscle glycogen content are a result of an interaction between dietary supplementation and other mediators of muscle glucose transport, such as muscle contraction. The mechanisms behind such interactions are presently unclear; however, suggestions include cellular swelling due to Cr entry into muscle causing increased glycogen synthesis (17), increased muscle Na -K -ATPase pump concentration following exercise (7, 19, 20), and increased insulin-stimulated Na - K -ATPase pump activity (15, 18) enhancing Cr transport. If glucose-stimulated insulin release was a major factor in Crassociated increases in muscle glycogen content, it is possible that a longer period of Cr supplementation may be necessary to upregulate GLUT-4 expression sufficiently to influence glycogen synthesis; however, longer term, low-dose Cr supplementation appears not to alter muscle glycogen concentration (22, 28). Two weeks of Cr supplementation is sufficient to prevent the decline in muscle GLUT-4 protein content arising from a period of immobilization, but it may take up to 10 wk of supplementation to increase muscle GLUT-4 protein concentration above basal levels (23). The mechanism by which Cr might increase muscle sensitivity to circulating insulin remains of interest. A further potential mechanism worthy of investigation is the influence of the 5 -AMP-activated protein kinase cascade, which is indirectly activated by osmotic stress (6, 14) and can be stimulated by changes in the PCr-to-Cr ratio (24), as well as through changes in the AMP-to-ATP ratio (10). In designing the present study, we felt that any loss of experimental rigor created by the nonrandomization of treatment interventions would be outweighed by the control maintained by having subjects proceeding through the study at precise and relatively short intervals. This would avoid potential negative effects relating to subject training/detraining/ motivation that would be introduced into the study if a crossover design was employed that required long periods of time between treatment interventions to allow muscle Cr washout to occur. Nevertheless, we acknowledge that the lack of a cross-

512 CREATINE SUPPLEMENTATION AND MUSCLE GLYCOGEN CONTENT over design could be viewed as a limitation of the present study. In conclusion, the current investigation demonstrated that supplementation of a normal habitual diet with Cr had no effect on muscle glycogen content. We propose that Cr supplementation alone is insufficient to influence muscle glycogen synthesis and that the major factor influencing the potential for Cr to enhance muscle glycogen content is contraction in the form of glycogen-depleting exercise before Cr supplementation or training during supplementation. We suggest that combining Cr supplementation with exercise alongside a high carbohydrate intake will achieve the aim of enhancing skeletal muscle glycogen content and possibly endurance exercise capacity. ACKNOWLEDGMENTS The authors thank Liz Simpson, John Fox, and Julie Lambourne for technical assistance. GRANTS This work was supported by Experimental and Applied Sciences. REFERENCES 1. Bergstrom J. 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