Creatine Supplementation: Exploring the Role of the Creatine Kinase/Phosphocreatine System in Human Muscle

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1 SESSION 4: NUTRITIONAL ERGOGENIC AIDSCreatine Supplementation S79 Creatine Supplementation: Exploring the Role of the Creatine Kinase/Phosphocreatine System in Human Muscle Peter Hespel, Bert Op t Eijnde, Wim Derave, and Erik A. Richter Catalog Data Hespel, P., Op t Eijnde, B., Derave, W., and Richter, E.A. (2001). Creatine supplementation: Exploring the role of the creatine kinase/phosphocreatine system in human muscle. Can. J. Appl. Physiol. 26(Suppl.): Canadian Society for Exercise Physiology. Key words: exercise, skeletal muscle, phosphocreatine, muscle relaxation, muscle metabolism Mots-clés: exercice physique, muscle squelettique, phosphocréatine, relaxation musculaire, métabolisme musculaire Abstract/Résumé The effect of oral creatine supplementation on high-intensity exercise performance has been extensively studied over the past ten years and its ergogenic potential in young healthy subjects is now well documented. Recently, research has shifted from performance evaluation towards elucidating the mechanisms underlying enhanced muscle functional capacity after creatine supplementation. In this review, we attempt to summarise recent advances in the understanding of potential mechanisms of action of creatine supplementation at the level of skeletal muscle cells. By increasing intracellular creatine content, oral creatine ingestion conceivably stimulates operation of the creatine kinase (CK)/phosphocreatine (PCr) system, which in turn facilitates muscle relaxation. Furthermore, evidence is accumulating to suggest that creatine supplementation can beneficially impact on muscle protein and glycogen synthesis. Thus, muscle hypertrophy and glycogen supercompensation are candidate factors to explain the ergogenic potential of creatine ingestion. Additional P. Hespel, B. Op t Eijnde, and W. Derave are with the Exercise Physiology and Biomechanics Laboratory, Department of Kinesiology, Faculty of Physical Education and Physiotherapy at the Catholic University of Leuven, Leuven, Belgium; E.A. Richter is with the Copenhagen Muscle Research Centre, Department of Human Physiology, Institute of Exercise and Sports Sciences at the University of Copenhagen, Copenhagen, Denmark. S79

2 S80 Hespel, Op t Eijnde, Derave, and Richter issues discussed in this review are the fibre-type specificity of muscle creatine metabolism, the identification of responders versus non-responders to creatine intake, and the scientific background concerning potential side effects of creatine supplementation. L effet de la consommation de la créatine sur la performance au cours d un effort intense a été étudié au cours des dix dernières années ; son potentiel ergogène chez de jeunes sujets en bonne santé est maintenant bien connu. Dans le passé, de nombreuses études ont porté sur l évaluation de la performance ; de nos jours, les chercheurs s attardent aux mécanismes d amélioration de la capacité fonctionnelle consécutive à la consommation de créatine. Dans cet article, nous présentons les dernières percées concernant les mécanismes probables enclenchés dans les cellules musculaires par l action d un supplément de créatine. Il apparaît que la consommation de créatine augmente le contenu intracellulaire de créatine, laquelle active fort probablement le système créatine kinase (CK) / phosphocréatine (Pcr) et, de ce fait, favorise la relaxation musculaire. En outre, il y a de plus en plus d études attestant l effet bénéfique d un supplément de créatine sur la synthèse de glycogène et de protéines musculaires ; par conséquent, nous pouvons mettre de l avant l hypertrophie musculaire et la surcompensation glycogénique comme facteurs pouvant expliquer le potentiel ergogène de la créatine. Il est aussi fait mention de la spécificité du métabolisme de la créatine selon les types de fibres musculaires, de la caractérisation des répondeurs et des non-répondeurs en présence d un supplément de créatine et des bases scientifiques des effets secondaires causés par un supplément de créatine. Introduction Since Harris and his co-workers in 1992 showed that muscle total creatine content can be increased by oral creatine supplementation, over 100 studies have examined the effects of creatine supplementation on exercise performance. The consistency of findings has been obscured by data from many poorly designed performance studies. However, the majority of well-controlled studies show that oral creatine supplementation can enhance muscle force and power output during short all-out exercise in young healthy subjects, in particular if exercise bouts are interspersed with short (1 2 min) rest pauses (intermittent exercise). Because this matter has recently been well addressed in other review articles and Tarnopolsky, 1998; Terjung (Juhn and Tarnopolsky, 1998; Terjung et al., 2000) it will not be further addressed in detail here. Furthermore, for detailed information on the fundamentals of the cellular creatine kinase system we refer to other reviews et al., 1992; Terjung et al., 2000; Wyss and Kaddurah-Daouk, 2000; Persky and Brazeau, 2001) (Persky and Brazeau, 2001; Terjung et al., 2000; Wallimann et al., 1992; Wyss and Kaddurah-Daouk, 2000). The focus of today s creatine research in exercise physiology has shifted from evaluation of exercise performance impact, to elucidating the mechanisms underlying the reported beneficial effects of oral creatine loading on muscle functional capacity. There is no doubt that this research will lead to a better understanding of the role of the creatine kinase isoenzyme system in the regulation of energydependent processes in muscle cells, including energy metabolism during contractions, protein synthesis and ion balance. This line of research conceivably will soon elucidate previously unknown or poorly recognized functions of creatine kinase in muscle cells. A primary aim of the present paper is to discuss recent devel-

3 Creatine Supplementation S81 opments in this area, and where appropriate to indicate targets for future research. Furthermore, the booming interest for creatine in exercise physiology, also has prompted interest in the potential of creatine supplementation to treat and/or prevent muscle disuse atrophy, which is prevalent in many conditions such as musculoskeletal injuries, cardiopulmonary disease or other chronic diseases, and last but not least aging. In this respect we will summarize some recent findings that are relevant to the treatment and/or prevention of muscle disuse atrophy. Given the widespread use of creatine (worldwide creatine turnover today amounts to ~5 million kg on a yearly basis) we also found it appropriate to shortly elaborate on the scientific evidence regarding the side effects of creatine supplementation. This section may then serve to put the regular public bursts of misinformation concerning creatine side effects in their right scientific context. Finally, it is important to note that creatine supplementation is currently being extensively investigated as a potential therapeutic strategy to treat or prevent neuromuscular and neurological disorders et al., 1998; Klivenyi et al., 1999; Tarnopolsky and Beal, 2001) (Klivenyi et al., 1999; Matthews et al., 1998; Tarnopolsky and Beal, 2001). Because this clinical neurological area falls beyond the scope of this review, we will limit our approach to the effects of creatine supplementation on skeletal musculature in healthy subjects. Mechanisms of Action of Oral Creatine Supplementation ANAEROBIC ENERGY METABOLISM The starting-point for any mechanistic argumentation concerning the beneficial effects of creatine supplementation on muscle tissue is that creatine intake increases intracellular creatine content in muscle cells (Harris et al., 1992; Greenhaff et al., 1994; Casey et al., 1996; Hultman et al., 1996a; Vandenberghe et al., 1997; Vandenberghe et al., 1999) (Casey et al., 1996; Greenhaff et al., 1994; Harris et al., 1992; Hultman et al., 1996a; Vandenberghe et al., 1997, 1999), and thus in principle can impact on any cellular process that is linked to the action of the creatine kinase/phosphocreatine system. A primary function of phosphocreatine in muscle cells is to serve as a readily available anaerobic energy source fueling ATP resynthesis during high intensity exercise (Wallimann et al., 1992). Thus, from a theoretical point of view, increased muscle PCr content could facilitate muscle force and power production during any exercise mode involving PCr hydrolysis as an important rate-limiting pathway of ATP provision. This are generally maximal exercise bouts lasting ~30sec or less (Gaitanos et al., 1993; Hultman et al., 1996b). Contrary to such assumption, the ergogenic effect of short-term creatine supplementation in short maximal exercise bouts has been found to be small or even absent (for reviews see Juhn and Tarnopolsky, 1998; Terjung et al., 2000). However, apart from poor sensitivity and statistical power of many performance studies in this area, there are some readily available arguments to explain this small effect: (a) the increase of muscle PCr content due to creatine supplementation is relatively small and usually in the range of only 5 20%, and (b) the contribution of PCr hydrolysis to total ATP turnover during a 5 6sec sprint amounts to only 50% and decreases to ~25% during a 30sec sprint (Gaitanos et al., 1993). How-

4 S82 Hespel, Op t Eijnde, Derave, and Richter ever, during series of repeated short all-out exercise bouts with short rest pauses (1 2min) in between, progressive failure to re-activate ATP production through the glycogenolytic/glycolytic pathway 1995) (Spriet, 1995) causes the fraction of ATP production from PCr breakdown to increase. This may in part explain the higher ergogenic efficacy of creatine supplementation during intermittent sprint exercise, compared with single sprint exercise bouts and Tarnopolsky, 1998; Terjung (Juhn and Tarnopolsky, 1998; Terjung et al., 2000). MUSCLE RELAXATION Another important function of the creatine kinase reaction in muscle cells is to maintain a high ATP:ADP ratio during high rates of ATP turnover, which is critical to the amount of energy obtained per mmol of ATP hydrolysis. Sarcoplasmic reticulum (SR) Ca 2+ -ATPase activity is much more readily impaired by falling ATP:ADP ratio than myosin ATPase activity 1987; Kammermeier, 1993; Macdonald and Stephenson, 2001) (Kammermeier, 1987, 1993; Macdonald and Stephenson, 2001). This explains the tight structural and functional coupling between creatine kinase and SR Ca 2+ -ATPase proteins in the SR membrane of muscle cells (Rossi et al., 1990). Furthermore, recent in vitro studies in skinned human muscle fibers indicate that SR Ca 2+ -ATPase pumping accounts for a substantial fraction (40 50%) of total ATP utilization during contractile activity (Szentesi et al., 2001). Thus, SR Ca 2+ -ATPase conceivably is a primary site of action of creatine supplementation. Direct evidence to prove that SR Ca 2+ -ATPase activity is facilitated by creatine supplementation in skeletal muscle is lacking. However, it is well established that the rate of SR Ca 2+ -ATPase pumping is the primary rate-limiting factor for relaxation of human muscle cells 1985; Dux, 1993) (Dux, 1993; Gillis, 1985). Thus, shortening of muscle relaxation time after creatine supplementation may be interpreted as indirect evidence to prove enhanced SR Ca 2+ -ATPase activity. Indeed, we recently demonstrated that creatine intake shortened relaxation time either after maximal voluntary isometric contractions (Figure 1;Leemputte Van Leemputte et al., 1999) or after submaximal contractions induced by electrical stimulation in healthy volunteers (Hespel et al., in press). Consistent with this finding is impaired Ca 2+ handling, associated with lengthening of half-relaxation time, occurring in incubated medial gastrocnemius muscles from creatine kinase knock-out mice (Steeghs et al., 1997). Still the question then remains as to how facilitated muscle relaxation might contribute to enhanced muscle force and power output during sprint exercise. First, during fast repetitive concentric muscle contractions, typical to sprint exercise, recovery time from the previous contraction is critical to maximal force output during the next contraction and Leonard, 1997) (Herzog and Leonard, 1997). Second, it is reasonable to assume that shortening of relaxation time can increase the number of actomyosin activation cycles per unit of time and thereby power output. Indirect evidence to support our contention that facilitated muscle relaxation contributes to the ergogenic effect of creatine supplementation in sprint exercise comes from our studies with combined creatine and caffeine ingestion. We found caffeine administration not only to inhibit the ergogenic effect of creatine loading in maximal intermittent muscle contractions (Vandenberghe et al., 1996), but at the same time to negate the beneficial impact of

5 Creatine Supplementation S83 Figure 1. Muscle relaxation time (RT) for 12 consecutive maximal isometric contractions, interspersed with 10-s rest intervals, before (open symbols) and after (closed symbols) creatine (squares) and placebo (circles) intake. Values are means ± SE in ms of 8 observations. *P <.05 compared with creatine pretest values. Inset: mean RT over 12 contractions; illustrates test + group interaction, P < Figure is reproduced from reference Leemputte (Van Leemputte et al., 1999), with permission. creatine on muscle relaxation time (Hespel et al., in press). AEROBIC METABOLISM AND MUSCLE PCR RESYNTHESIS Another interesting issue is the potential of creatine supplementation to stimulate mitochondrial oxidative flux and thus aerobic ATP provision during exercise. In a recent study creatine supplementation was found to enhance oxygen uptake during a ~30min exercise bout alternating 3min episodes at 30% and 90% of maximal oxygen uptake and Mendez Marco, 2000) (Rico-Sanz and Mendez Marco, 2000). The increased oxygen uptake was tentatively explained by facilitated oxidative phosphorylation in slow-oxidative muscle, allowing a greater flux through the creatine kinase reaction and resulting in reduced adenine nucleotide degradation in these fibers. Free creatine plays an important role in transferring energy-rich phosphates from mitochondria to the sites of ATP consumption in muscle cells (actomyosin cross-bridges, energy dependent ion exchange proteins), the so-called creatine-shuttle and Savabi, 1990; Wallimann (Bessman and Savabi, 1990; Wallimann et al., 1992). In this context, creatine has been identified as a potent stimulator of cellular respiration (Saks et al., 1991). Consistent with this role of muscle free creatine to stimulate energy provision from aerobic metabolism in muscle cells,

6 S84 Hespel, Op t Eijnde, Derave, and Richter Greenhaff and co-workers recently showed that the mitochondrial utilization of acetyl groups after high intensity muscle contractions was increased by prior creatine supplementation (Jones et al., 2001). This was associated with an enhanced rate of PCr resynthesis, which is known to reflect the rate of postexercise ATP production through oxidative phosphorylation very well. Surprisingly, research with regard to the effect of creatine supplementation on postexercise PCr resynthesis have yielded inconsistent findings et al., 1994; Smith et al., 1998; Vandenberghe et al., 1999; Smith et al., 1999; Kreis et al., 1999; Jones et al., 2001) (Greenhaff et al., 1994; Jones et al., 2001; Kreis et al., 1999; Smith et al., 1998, 1999; Vandenberghe et al., 1999) with most studies not finding a positive effect et al., 1994; Smith et al., 1998; Vandenberghe et al., 1999; Smith et al., 1999; Kreis et al., 1999) (Greenhaff et al., 1994; Kreis et al., 1999; Smith et al., 1998, 1999; Vandenberghe et al., 1999). However, small changes of postexercise muscle PCr resynthesis may be very difficult to detect by 31 P-N.M.R. spectroscopy and Mossey, 1998) (Hochachka and Mossey, 1998) used by most of the latter studies. MUSCLE HYPERTROPHY AND PROTEIN EXPRESSION OF MUSCLE MYOGENIC FACTORS The first, yet debated and Morales, 1980) (Fry and Morales, 1980) evidence that creatine could possibly stimulate muscle protein synthesis comes from early in vitro studies showing that a high extracellular creatine concentration stimulated actin and myosin heavy chain synthesis in incubated embryonic skeletal muscle cells et al., 1972; Ingwall et al., 1974; Ingwall, 1976) (Ingwall, 1976; Ingwall et al., 1972, 1974). Stimulation of muscle hypertrophy by creatine intake in humans was reported for the first time more than 15 years ago in a study in patients suffering from gyrate atrophy of the choroid and retina, due to deficient creatine biosynthesis. Type II muscle fiber hypertrophy in these patients was found to occur as a side-effect of the supplementary creatine treatment (1.5g/day for 1 year; et al., 1981) (Sipilä et al., 1981)). A number of intervention studies from our and other groups in young healthy volunteers, recently have provided additional evidence to suggest that creatine intake in conjunction with heavy resistance training, indeed, can stimulate muscle hypertrophy et al., 1997; Kreider et al., 1998; Volek et al., 1999; Becque et al., 2000) (Becque et al., 2000; Kreider et al., 1998; Vandenberghe et al., 1997; Volek et al., 1999). Convincing evidence to support the potential anabolic action of creatine supplementation comes from our recent study in the context of rehabilitation of muscle disuse atrophy et al., 2001b) (Hespel et al., 2001). We immobilized the right leg of healthy subjects with a cast for a period of 2 weeks, where after they were enrolled in a rehabilitative resistance training program for 10 weeks. Subjects ingested creatine monohydrate (or placebo) at a rate of 20g/day during immobilization, decreasing the dose to 15-5g/day during the rehabilitation period. Creatine supplementation did not impact on the magnitude of disuse atrophy during the immobilization, yet facilitated the recovery of muscle volume (m. quadriceps) and knee-extensor torque during rehabilitation. Interestingly, the contralateral leg exhibited a ~10% increase in both quadriceps muscle volume and force output during the 12 weeks supplementation period. We cannot exclude that a fraction of the increasing muscle volume was due to intramuscular

7 Creatine Supplementation S85 water retention (Ziegenfuss et al., 1998), yet increased maximal isometric force after creatine supplementation definitely cannot be due to increased muscle water content. These findings can not be explained by a higher training workload because this was similar in the creatine and placebo group. Thus, even in the absence of any direct evidence that physiological concentrations of creatine can stimulate muscle protein synthesis, the cluster of findings mentioned above suggests that increased muscle creatine content can be associated with muscle hypertrophy and enhanced protein synthesis. The question then remains as to how creatine supplementation possibly could enhance protein synthesis in muscle cells. From a theoretical point of view, muscle cell swelling during episodes of creatine intake due to osmotic loading effected by creatine accumulation and Kilimann, 1993; Nash et al., 1994) (Guimbal and Kilimann, 1993; Nash et al., 1994), is a potential mechanism to stimulate protein synthesis et al., 1993) (Häussinger et al., 1993). However, the operation of such mechanism in the context of oral creatine supplementation in humans so far remains entirely speculative. Another possibility is that creatine supplementation enhances training-induced muscle hypertrophy by promoting the release of growthrelated hormones. Our recent work indicated that creatine does not significantly alter the circulating levels of growth hormone, testosterone nor cortisol following a heavy resistance training session t Eijnde and Hespel, 2001) (Op t Eijnde and Hespel, 2001). However, the effect on other growth stimulating hormones, like insulin-like growth factor-1 (IGF-1), has not been investigated to date. Interestingly, Dangott et al. et al., 1999) (Dangott et al., 1999) have recently shown that oral creatine supplementation in rats may induce increased satellite cell mitotic activity during compensatory hypertrophy, a process which is known to be governed by IGF-1 et al., 1999) (Barton-Davis et al., 1999). Future research will need to establish whether creatine is able to potentiate IGF-1-mediated satellite cell proliferation and thereby provide additional cell nuclei to the growing muscle. The only available evidence to prove that creatine supplementation can impact on the control of intracellular protein homeostasis in human muscle cells comes from our recent immobilization/rehabilitation study which showed creatine supplementation to alter the protein expression of myogenic transcription factors et al., 2001b) (Hespel et al., 2001). The myogenic transcription factors probably are involved in balancing catabolic versus anabolic processes in muscle cells et al., 1993; Loughna and Brownson, 1996; Marsh et al., 1997; Mozdziak et al., 1998; Adams et al., 1999) (Adams et al., 1999; Hughes et al., 1993; Loughna and Brownson, 1996; Marsh et al., 1997; Mozdziak et al., 1998). We found rehabilitative exercise training in combination with creatine intake not only to induce muscle fiber hypertrophy but at the same time to increase protein expression of MRF4 and to prevent the training-induced increase in myogenin protein expression et al., 2001b) (Hespel et al., 2001). In addition, the change in mixed muscle MRF4 protein content was positively correlated (r = 0.73, Figure 2) with the concomitant change of the average muscle fiber size. These findings thus suggest that MRF4 in particular may play an important role in regulating muscle hypertrophy due to resistance training. Whether there is any causal link between the stimulation of satellite cell proliferation et al., 1999) (Dangott et al., 1999) and altered myogenin and MRF4 expression on the on hand, and creatine-induced muscle hypertrophy

8 S86 Hespel, Op t Eijnde, Derave, and Richter Figure 2. Correlation between the individual changes (R10 minus PRE) in Western Blot quantification of MRF4 protein expression (arbitrary units) and average muscle fiber size ( m 2 ) from the start (PRE) to end (R10) of the study. Muscle samples were taken from the m. vastus lateralis before and after two weeks of immobilization followed by 10 weeks of rehabilitation training of the right leg. During immobilization and rehabilitation subjects, ingested creatine monohydrate or placebo. Figure is reproduced from Hespel et al. (2001). on the other hand, remains to be established. INITIAL MUSCLE CREATINE CONTENT Responders Versus Non-Responders Creatine studies (too) often discriminate between responders and non-responders to explain divergent findings. For sure such clear-cut distinction between responders and non-responders is inappropriate and a response continuum is probably more close to reality. Still it is clear that the response to creatine supplementation can be quite variable amongst individuals. A simple example of this response variability is body weight: we have often experienced that creatine intake at a rate of ~20g/day in some individuals can increase body weight by more than 4 kg within a week, whilst others exhibit unchanged body weight. Literature indicates that in a healthy population initial muscle creatine content probably is an important determinant of responsiveness to creatine supplementation. Harris and his co-workers already showed in their early work that subjects with low initial creatine levels exhibit the greatest increment of muscle creatine content upon creatine loading (Harris et al., 1992). YOUNGER VERSUS OLDER INDIVIDUALS

9 Creatine Supplementation S87 It has also been suggested that older people might be particularly prone to the beneficial impact of creatine supplementation on muscle functional capacity (Tarnopolsky, 2001) because they not only have decreased muscle mass and strength et al., 1991; Frontera et al., 2000) (Frontera et al., 1991, 2000) but in addition exhibit decreased muscle creatine content. However, available literature data are scarce and are inconsistent in showing an age-associated decline of muscle creatine content et al., 1980; McCully et al., 1991; Conley et al., 2000; Kent-Braun and Ng, 2000) (Conley et al., 2000; Kent-Braun and Ng, 2000; McCully et al., 1991; Möller et al., 1980). In fact published literature rather indicates that older people respond badly to creatine supplementation. In contrast with young et al., 1997; Kreider et al., 1998; Volek et al., 1999) (Kreider et al., 1998; Vandenberghe et al., 1997; Volek et al., 1999), resistance training in combination with creatine intake did not result in increased muscle functional capacity et al., 1998; Rawson et al., 1999) (Bermon et al., 1998; Rawson et al., 1999). We recently performed a doubleblind placebo-controlled intervention trial in males 55 to 75 years old (n = 24). Subjects received 5g/day of creatine monohydrate for a period of 6 months and meanwhile participated in a supervised fitness training program (endurance training + moderate resistance training) at a rate of 2 3 sessions (1h) per week. Muscle biopsies showed initial muscle total creatine contents to be about 20% higher (144 ± 3mmol kg -1 d.w.) than commonly observed in young male volunteers ( mmol kg -1 d.w.). Hence, muscle total creatine content was not increased by 6 months of creatine supplementation. Furthermore, the training program increased V O 2 max (+12%) and maximal isometric force of the knee (Figure 3) and arm extensor muscles to the same degree in the placebo and the creatine group. Thus, in contrast to the prevailing opinion, older people do not appear to benefit from creatine supplementation, possibly because of the high prevalence of elevated muscle Figure 3. Mean maximal isometric torque of the knee-extensors (90, 110, and 130 ) after 3 (POST-3m) and 6 (POST-6m) months of fitness training in combination with creatine (black bars) or placebo (open bars) intake. Values represent means ± SE of 23 observations., p <.05 compared with corresponding PRE value.

10 S88 Hespel, Op t Eijnde, Derave, and Richter creatine levels in this population. FIBER TYPE DISTRIBUTION It is well established that fast-twitch muscle fibers (type II) have higher creatine contents than slow-twitch muscle fibers (type I). The difference is most explicit in rodent muscles et al., 1999; Murphy et al., 2001; Op t Eijnde et al., 2001a) (Murphy et al., 2001; Op t Eijnde et al., 2001a; Willott et al., 1999) but also exists in human muscles et al., 1996; Constantin-Teodosiu et al., 1996; Rico-Sanz et al., 1999) (Casey et al., 1996; Constantin-Teodosiu et al., 1996; Rico-Sanz et al., 1999). Thus creatine content in mixed human muscle biopsies conceivably at least partly reflects fiber type distribution, in that the proportion of slow oxidative muscle fibers is higher in individuals with low mixed muscle creatine content than in individuals with high creatine contents. Along the same line of reasoning one could assume that persons with more type I fibers relative to type II fibers are the better responders to creatine supplementation. If such a hypothesis proves to be valid, then the response to creatine supplementation conceivably depends on the fiber type distribution of the muscle (group) considered. In fact, recent studies provide clear evidence to suggest that type I fibers, indeed, are more liable to the effects of oral creatine supplementation than type II fibers. Murphy et al. recently examined the gene expression and cellular localization of the creatine transporter protein in rat skeletal muscles et al., 2001) (Murphy et al., 2001). They showed that soleus and red gastrocnemius muscles, despite lower creatine content, had a greater total creatine transporter protein content than white gastrocnemius muscle. In addition, experiments in isolated mice muscles have shown that creatine transport in soleus and extensor digitorum longus (EDL) exhibits similar V max (~75nmol h -1 g w.w.) but K m is lower in soleus (~75 M) than in EDL (160 M). This implies that within the range of plasma creatine concentrations established by oral creatine intake (Harris et al., 1992) creatine uptake conceivably is most prominent in slow oxidative muscle tissue. Consistent with this line of reasoning is our finding that creatine supplementation in rats increased total creatine content in soleus muscles, less in red gastrocnemius and not in white gastrocnemius t Eijnde et al., 2001a) (Op t Eijnde et al., 2001a; see Figure 4-A). Furthermore, creatine supplementation decreased half-relaxation time of isometric twitch and increased fatigue resistance in incubated rat soleus but not in EDL et al., 2001) (McGuire et al., 2001). Accordingly, we found that the decrease of relaxation time of the knee-extensor muscles caused by creatine supplementation in humans was closely correlated with initial relaxation time. The slower initial relaxation time, the more it was shortened by creatine intake (Figure 1). It is well established that relaxation rate is slower in slow-oxidative muscle fibers than in fast-glycolytic fibers 1985; Dux, 1993; McGuire et al., 2001) (Dux, 1993; Gillis, 1985; McGuire et al., 2001). Thus the above relationship might simply indicate that individuals (or muscles) with a higher fraction of slow-twitch fibers, indeed, respond better to creatine supplementation than individuals (or muscles) with a more fast-twitch muscle profile. Furthermore, a recent 31 P-NMR study showed net PCr breakdown and H + accumulation to be reduced after creatine supplementation during low intensity isometric muscle contractions involving mainly type I fibers, but not during high intensity contractions

11 Creatine Supplementation S89 A B Figure 4. Muscle total creatine (Panel A) en glycogen (Panel B) content in various hindlimb muscles of rats (n = 13) after 5 days of creatine (5%; black bars) or unsupplemented feeding (open bars). *P <.05 compared with corresponding control value. Figure is drawn from data published in t Eijnde (Op t Eijnde et al., 2001a). requiring more extensive type II fiber recruitment 2000) (Rico-Sanz, 2000). This is compatible with in vitro findings showing creatine to have the potential to stimulate mitochondrial oxidative function in type I fibers but not in type II fibers et al., 2001) (Kuznetsov et al., 2001). Still it must be mentioned that one study found similar increases of muscle PCr concentration in pools of type I and type II fibers dissected from mixed human muscle biopsies et al., 1996) (Casey et al., 1996). However, free creatine content was not measured in this study and the type II fiber pool obviously represented a mixture of type IIa and type IIx/b fibers. Clearly it is worthwhile to further explore the relationship between fiber type distribution and responsiveness to creatine supplementation in humans, and if possible to find non-

12 S90 Hespel, Op t Eijnde, Derave, and Richter invasive parameters indicating responsiveness. Interaction of Carbohydrate and Creatine Metabolism Phosphocreatine (PCr) and muscle glycogen are the primary energy sources fueling high-intensity exercise. In addition to their plain energy storage function, phosphocreatine and glycogen also serve as important energy buffers for ATP utilization during exercise (for reviews on this issue see et al., 1984; Wallimann et al., 1999) Meyer et al., 1984; Wallimann et al., 1999). Clear evidence for the temporal buffer function of phosphocreatine comes from a recent NMR study by Chung and co-workers et al., 1998) (Chung et al., 1998), who investigated the millisecond fluctuations of muscle metabolites during single twitch contractions. They observed ATP to be constant during the entire twitch duration, whereas a rapid transient decrease in PCr concentration was observed with half-time decrease/recovery rates as fast as 8 and 14ms, respectively. Given the inertia of oxidative metabolism, this precipitous recovery of PCr (restoration of initial concentration within 50msec from the onset of the twitch contraction) presumably can only originate from glycogenolysis. These findings have led to the glycogen shunt model, as described by Shulman and Rothman and Rothman, 2001) (2001), indicating that the rapid bursts of ATP consumption during every single muscle contraction may be covered by continued breakdown and resynthesis of both phosphocreatine and glycogen. Given the pivotal role of phosphocreatine and glycogen in the energy homeostasis of the working muscle, a mutual control of both energy systems may be anticipated. Therefore, in the following paragraphs, an attempt is made to summarize arguments supporting the existence of important cross-links between creatine and glycogen metabolism in muscle. EFFECTS OF CREATINE ON CARBOHYDRATE METABOLISM In order to investigate the sensitivity of muscle carbohydrate/glycogen metabolism to altered creatine availability, one can either artificially lower or increase muscle creatine content. In the former case (creatine depletion), it might be expected that carbohydrate storage might try to compensate for the loss of creatine availability. Accordingly, rats fed -guanidinoproprionic acid (GPA), a competitive inhibitor of cellular creatine uptake, which decreased muscle phosphocreatine content by ~90%, exhibited a doubling of muscle glycogen content et al., 1993) (Ren et al., 1993). These data must, however, be interpreted with care because the GPA treatment also caused a substantial decrease of muscle ATP content and thus the increase of muscle glycogen may simply represent an adaptation of a dying cell. By analogy, glycogen content was increased by ~60% in muscle of transgenic mice deficient of the M-CK enzyme and net glycogen breakdown during 3 min of intense muscle contractions was greater in the CK-mutant mice than in controls Deursen et al., 1993) (Van Deursen et al., 1993). Along the same line of reasoning, one might expect a higher than normal creatine content to cause a downregulation of carbohydrate availability. However, based upon some recent findings, the opposite seems to be true (i.e., creatine loading facilitates muscle carbohydrate accumulation). Robinson et al. (1999) have

13 Creatine Supplementation S91 evaluated the effect of postexercise carbohydrate loading in conjunction with creatine supplementation. As a result, muscle glycogen supercompensation is augmented significantly (Robinson et al., 1999). Meanwhile, the potential of creatine supplementation to boost postexercise muscle glycogen accumulation has been confirmed repeatedly et al., 2001; Op t Eijnde et al., 2001b) (Nelson et al., 2001; Op t Eijnde et al., 2001b). Furthermore, a recent rat study in our laboratories showed creatine supplement per se (5% creatine added to the normal chow) is capable of increasing muscle glycogen content t Eijnde et al., 2001a) (Op t Eijnde et al., 2001a). Interestingly, the increased glycogen as well as creatine content was most prominent in soleus muscle (+40%), which predominantly consists of slow-twitch fibers, less in the red gastrocnemius muscle (+15%) with a mixed fiber type distribution, and non-significant (+10%) in the white gastrocnemius, which expresses mainly type IIb fibers (see figure 4-B). Thus, similar to creatine depletion, creatine loading also appears to promote glycogen accumulation in muscle and this process appears to be most prominent in slow-twitch muscle fibers. By which mechanism could creatine supplementation stimulate glycogen synthesis in muscle cells? Some authors have proposed that cell swelling caused by an increase in osmolarity during creatine accumulation might trigger glycogen synthesis et al., 1999; Nelson et al., 2001) (Nelson et al., 2001; Robinson et al., 1999). However, evidence for stimulation of glycogen synthesis by cell swelling in human muscle is lacking, and is limited to studies on cultured myotubes exposed to supraphysiological osmotic challenges et al., 1996) (Low et al., 1996). A more likely explanation is that creatine supplementation promotes insulin-induced glycogen synthesis in muscle cells either by stimulating pancreatic insulin secretion or by improving insulin sensitivity of glycogen synthesis at the muscle level. Support for the former hypothesis comes from early in vitro work on isolated pancreas and islet preparations, showing insulin-releasing effects of supraphysiological concentrations of creatine and related guanidino-acetate compounds et al., 1970; Marco et al., 1976) (Alsever et al., 1970; Marco et al., 1976). However, later in vivo experiments have failed to demonstrate hyperinsulinemia following an oral creatine ingestion in either humans et al., 1996a; Robinson et al., 1999) (Green et al., 1996a; Robinson et al., 1999) or rats t Eijnde et al., 2001a) (Op t Eijnde et al., 2001a). Therefore, despite positive evidence in vitro, creatine-induced glycogen synthesis is unlikely to result from increased pancreatic insulin secretion. The alternative hypothesis remains that improved muscle insulin sensitivity may generate the glycogen supercompensation effect of creatine supplementation. No data showing enhanced insulin signaling of creatine supplementation have, however, been published. Still, support for such an assumption comes from a recent study by our laboratories t Eijnde et al., 2001b) (Op t Eijnde et al., 2001b). We investigated the effect of oral creatine supplementation on GLUT4 expression in the quadriceps muscle of healthy subjects during 2 weeks of leg immobilization followed by 10 weeks of rehabilitative resistance training t Eijnde et al., 2001b) (Op t Eijnde et al., 2001b). Creatine supplementation not only prevented the immobilization-induced fall of muscle GLUT4 content, but in addition increased muscle GLUT4 to ~40% above the baseline value by the end of the rehabilitation period (see Figure 5). Since GLUT4 is an important determinant of insulin-stimulated glucose uptake and storage in muscle, enhancement of glycogen accumulation with creatine loading may possibly be caused by upregulation of GLUT4 expression. However, this hypothesis is not supported by our recent rat experiments,

14 S92 Hespel, Op t Eijnde, Derave, and Richter Figure 5. Effect of creatine supplementation on muscle GLUT-4 protein content during immobilization and subsequent rehabilitation training. Values are means ± SEM (n = 8) and are expressed relative to the baseline value that was set to be equal to 1. Muscle samples were taken from the m. vastus lateralis before and after two weeks of immobilization and after 3 and 10 weeks of rehabilitation training of the right leg. During immobilization and rehabilitation subjects, ingested creatine monohydrate (closed symbols) or placebo (open symbols). *Indicates a significant treatment effect compared with placebo, p <.05. Indicates a significant time effect compared with the pre-immobilization value. Figure is reproduced from t Eijnde et al., 2001b) (Op t Eijnde et al., 2001b). showing that glycogen accumulation during creatine supplementation was not associated with increased expression of GLUT4 t Eijnde et al., 2001a) (Op t Eijnde et al., 2001a). EFFECTS OF CARBOHYDRATES ON CREATINE METABOLISM The CK-PCr system does not seem to be very sensitive to altered carbohydrate availability. Within a broad physiological range of glycogen concentrations, neither glycogen depletion nor glycogen supercompensation appears to change resting PCr levels nor PCr breakdown during muscle contractions et al., 1990; Bangsbo et al., 1992) (Bangsbo et al., 1992; Ren et al., 1990). Even the dramatic limitation of muscle glycogen availability for energy provision occurring in patients with McArdle s disease, who are unable to degrade glycogen due to deficiency of the muscle phosphorylase enzyme, does not seem to consistently affect muscle PCr/ Cr storage and/or utilization and Haller, 1992; Sahlin et al., 1995; Nielsen et al., 2001) (Bertocci and Haller, 1992; Nielsen et al., 2001; Sahlin et al., 1995). Thus, unlike glycogen, the muscle total creatine content is much less prone to variation, or in other words, is maintained in a more narrow concentration range than glycogen, conceivably because it is immediately linked to cellular ATP provision and thus is critical to cell survival.

15 Creatine Supplementation S93 Still, creatine retention in muscle during oral creatine supplementation depends on carbohydrate intake because of insulin stimulation of creatine transport. In an attempt to improve creatine retention following supplementation in humans, Green et al. et al., 1996a) (1996a) observed a much lower urinary creatine excretion when simple carbohydrates were co-ingested with creatine, compared with creatine alone. Subsequent studies by these researchers confirmed and strengthened the finding that carbohydrate ingestion augments muscle creatine uptake by virtue of the insulin-releasing effects of carbohydrate ingestion et al., 1996b; Steenge et al., 2000) (Green et al., 1996b; Steenge et al., 2000). There are at least two mechanisms by which insulin could enhance creatine accumulation in muscle. First, insulin might enhance creatine delivery to muscle cells by its vasodilatory action on muscle vascular beds 1994; Rattigan et al., 1997) (Baron, 1994; Rattigan et al., 1997). Secondly, most and Andrew, 1972; Haughland and Chang, 1975; Odoom et al., 1996) (Haughland and Chang, 1975; Koszalka and Andrew, 1972; Odoom et al., 1996) but not all et al., 1999) (Willott et al., 1999) studies indicate that insulin might also directly stimulate transsarcolemmic creatine transport. In this respect, Steenge et al. evaluated muscle creatine accumulation and limb blood flow during euglycemic insulin clamps establishing high to supraphysiological plasma insulin levels. The experiment showed incremental creatine accumulation with increasing circulating insulin levels, whereas insulin-stimulated limb blood flow was not dose-dependent et al., 1998) (Steenge et al., 1998). These findings would thus favor the explanation of direct transport-mediated, rather than indirect flow-mediated, enhancement of muscle creatine uptake by insulin in humans. The potential of creatine to stimulate oxidative phosphorylation, on the one hand, and to stimulate muscle glycogen accumulation, on the other hand, creates a logical context for endurance performance enhancement by creatine supplementation. By increasing intracellular creatine content, creatine supplementation appears to stimulate oxidative metabolism in a resting muscle postexercise (Jones et al., 2001). However, creatine supplementation might not be able to stimulate oxidative phosphorylation in muscle cells operating at maximal aerobic power during contractions. According to the prevailing opinion increased muscle glycogen content after creatine loading should result in greater endurance capacity, yet wellcontrolled and adequate performance studies remain to be done in this area. Side-Effects As for any new ergogenic substance the incidence of side-effects is an issue of primary importance. We limit our discussion here to side-effects that thus far have been substantiated by controlled research findings and not by anecdotal reports. Even so it must be emphasized that controlled long-term safety data (>1 year) in humans are lacking and thus that the absence of scientific evidence for adverse side-effects to date does not prove product safety. RENAL DISEASE The kidney certainly is a site of primary concern. In healthy individuals both creatine and creatinine, the end-products of oral creatine, are easily filtered by the kidney with no apparent harmful effects on renal function et al., 1997; Poortmans

16 S94 Hespel, Op t Eijnde, Derave, and Richter and Francaux, 1999; Mihic et al., 2000) (Poortmans et al., 1997; Poortmans and Francaux, 1999; Mihic et al., 2000). However, creatine intake in a patient with pre-existing interstitial nephritis 1999) (Koshy, 1999) or focal glomerular necrosis and Kalra, 1998) (Pritchard and Kalra, 1998) was accompanied by a further deterioration of renal function. In none of these cases a causal relationship was proven between the degree of deterioration of renal function and the intake of creatine. However, a recent study in an animal model of cystic renal disease showed that creatine supplementation at a rate similar to human dosage regimens, slightly exacerbated disease progression et al., 2001) (Edmunds et al., 2001). Thus, available evidence indicates that individuals with compromised renal function or persons at increased risk for renal dysfunction probably must be advised against oral creatine supplementation especially at high doses. BODY WEIGHT The most common side-effect of creatine supplementation is increased body weight. The rapid increase of body weight seen in some, but not all individuals during the early stage of high-dose creatine loading (~20g/day) is probably largely accounted for by intramuscular water retention due to the osmotic swelling associated with muscle creatine uptake et al., 1998; Mihic et al., 2000) (Mihic et al., 2000; Ziegenfuss et al., 1998). Urinary output, indeed, is decreased during the initial days of creatine ingestion et al., 1996a) (Hultman et al., 1996a). However, if creatine supplementation is continued in conjunction with heavy-resistance training, muscle hypertrophy probably comes into prominence to explain increased body weight. Commensurate with this idea is our observation that after an episode of heavy resistance training in conjunction with creatine ingestion, cessation of creatine intake causes a much more rapid fall of muscle creatine content than decrease of fat-free mass et al., 1997) (Vandenberghe et al., 1997). CELLULAR CREATINE HOMEOSTASIS Another important issue is the impact of long-term creatine supplementation on cellular creatine homeostasis. Studies in both animals and humans have demonstrated that dietary creatine supplementation suppresses endogenous creatine synthesis in the kidney, presumably by downregulation of the amidinotransferase enzyme, the rate-limiting step in creatine biosynthesis et al., 1948; Walker, 1960; Walker, 1979) (Hoberman et al., 1948; Walker, 1960, 1979). However, this process appears to be rapidly reversible and there are no indications that cessation of creatine supplementation in humans is associated with a rebound effect on muscle creatine content. In addition, given that only about 2% of the muscle creatine store is degraded to form creatinine on a daily basis it is unlikely that a provisional suppression of creatine biosynthesis, in the presence of a normal dietary creatine intake (~1g/day), could result in a significant reduction of muscle creatine content. Longitudinal intervention studies in humans indicate that cessation of creatine supplementation causes muscle creatine content to return to normal values within a time window of 4 5 weeks with no undershoot or incidence of adverse sideeffects et al., 1997) (Vandenberghe et al., 1997). Another concern is the potential impact of chronic creatine administration on muscle creatine transport capacity

17 Creatine Supplementation S95 and on the functional characteristics of the intracellular creatine kinase isoenzyme system. Creatine is transported into the muscle cell by a high affinity creatine transporter protein and Kilimann, 1993; Nash et al., 1994) (Guimbal and Kilimann, 1993; Nash et al., 1994), different isoforms of which have been identified and Wallimann, 1998) (Guerrero-Ontiveros and Wallimann, 1998). The rate of creatine transport into muscle cells appears to be closely related to the sarcolemmic abundance of the creatine transporter et al., 2000) (Tran et al., 2000). Studies in rats have shown that muscle total creatine transporter content can be reduced by chronic creatine supplementation and Wallimann, 1998) (Guerrero-Ontiveros and Wallimann, 1998). However, the creatine dose administered exceeded human dosages by at least a factor 10, and there are at present no literature data to suggest downregulation of membrane creatine transport can also occur in skeletal musculature of humans ingesting creatine at normal rates. The subcellular organization of the muscle creatine transport system, which involves different isoforms with conceivably different functions and/or sites of action, and the impact of creatine supplementation here on is an important target for future research et al., 2000) (Walzel et al., 2000). Furthermore, evidence is accumulating to indicate that the creatine kinase isoenzyme system plays a pivotal role in modulating most, if not all energy-dependent cellular processes and Savabi, 1990; Wyss et al., 1992; Wallimann et al., 1992; Ponticos et al., 1998; Wallimann et al., 1998; Dolder et al., 2001) (Bessman and Savabi, 1990; Dolder et al., 2001; Ponticos et al., 1998; Wallimann et al., 1992, 1998; Wyss et al., 1992). Thus it is critical to explore in detail the effects of chronic creatine intake on the cellular distribution and activity of the different creatine kinases present in the mitochondria and cytoplasm of muscle cells. CANCER The French food security agency Agence Française de Sécurité Sanitaire des Aliments (A.F.S.S.A.) recently reported creatine to be a potential carcinogenic substance (January 2001, However, their statement was based on invalid extrapolations from in vitro observations (creatine was exposed together with amino acids and sugars at temperatures of 150 to 250 C) to oral creatine supplementation in humans. Thus, the Council for Responsible Nutrition (Washinton DC, USA) immediately disclaimed the French creatine report. In addition, the A.F.S.S.A. conclusions are in conflict with an earlier safety report issued by the European Commision ( September 2000, europa.eu.int/comm/food/fs/sc/scf/out70-en.pdf). There is no scientific evidence to indicate that creatine supplementation in humans at rates currently being considered (2-20g/day) might be carcinogenic. In contrast, several animal studies have pointed out the potential of creatine to inhibit tumor growthet al., 1993; Kristensen et al., 1999) (Kristensen et al., 1999; Miller et al., 1993). Although thousands of athletes worldwide have been consuming creatine in ever increasing numbers since 1992, almost no incidences with clinically significant side-effects have been reported. It must be stressed however, that clinically controlled double-blinded longterm studies on the safety of creatine supplementation involving a large number of subjects are still missing, with some smaller studies just starting to appear and Francaux, 2000) (Poortmans and Francaux, 2000). Our recent finding that creatine supplementation can alter the protein expression of myogenic transcription