Does caffeine alter muscle carbohydrate and fat metabolism during ...

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Does caffeine alter muscle carbohydrate and fat metabolism during exercise? Terry E. Graham, Danielle S. Battram, Flemming Dela, Ahmed El-Sohemy, and Farah S.L. Thong

Abstract: Caffeine, an adenosine receptor antagonist, has been studied for decades as a putative ergogenic aid. In the past 2 decades, the information has overwhelmingly demonstrated that it indeed is a powerful ergogenic aid, and frequently theories have been proposed that this is due to alterations in fat and carbohydrate metabolism. While caffeine certainly mobilizes fatty acids from adipose tissue, rarely have measures of the respiratory exchange ratio indicated an increase in fat oxidation. However, this is a difficult measure to perform accurately during exercise, and small changes could be physiologically important. The few studies examining human muscle metabolism directly have also supported the fact that there is no change in fat or carbohydrate metabolism, but these usually have had a small sample size. We combined the data from muscle biopsy analyses of several similar studies to generate a sample size of 16–44, depending on the measure. We examined muscle glycogen, citrate, acetyl-CoA, glucose-6-phosphate, and cyclic adenosine monophosphate (cAMP) in resting samples and in those obtained after 10–15 min of exercise at 70%–85% maximal oxygen consumption. Exercise decreased (p < 0.05) glycogen and increased (p < 0.05) citrate, acetyl-CoA, and glucose-6-phosphate. The only effects of caffeine were to increase (p < 0.05) citrate in resting muscle and cAMP in exercise. There is very little evidence to support the hypothesis that caffeine has ergogenic effects as a result of enhanced fat oxidation. Individuals may, however, respond differently to the effects of caffeine, and there is growing evidence that this could be explained by common genetic variations. Key words: methylxanthines, ergogenic aid, SNPs, adenosine, muscle glycogen, endurance. Re´sume´ : Depuis des de´cennies, la cafe´ine, un antagoniste du re´cepteur de l’ade´nosine, fait l’objet d’e´tudes en tant que facteur ergoge`ne potentiel. Au cours des deux dernie`res de´cennies, les e´tudes de´montrent fortement le puissant agent ergoge`ne qu’est le cafe´ et, souvent, on a e´labore´ des the´ories pour expliquer son effet par son action sur le me´tabolisme des graisses et des sucres. Meˆme si la cafe´ine mobilise, on le sait, les acides gras du tissu adipeux, il y a tre`s peu d’e´tudes portant sur la mesure du ratio d’e´changes gazeux pour de´montrer l’augmentation de l’oxydation des graisses. C’est cependant une mesure difficile a` effectuer au cours d’un effort physique d’autant plus que de le´ge`res variations ont un impact physiologique important. Les quelques e´tudes qui ont analyse´ directement le me´tabolisme musculaire chez l’humain rapportent qu’il n’y a aucune modification du me´tabolisme des graisses et des sucres, mais leur e´chantillon est plutoˆt de faible taille. Nous combinons les observations issues de l’analyse des biopsies musculaires re´alise´es dans des e´tudes semblables pour obtenir des e´chantillons dont l’effectif varie entre 16 et 44 spe´cimens selon le type de mesure. Nous analysons le contenu musculaire de glycoge`ne, de citrate, d’ace´tyl-CoA, de glucose 6-phosphate et de cAMP dans des e´chantillons pre´leve´s au repos et apre`s 10 a` 15 min d’effort physique re´alise´ a` une intensite´ suscitant 70 a` 85 % du consommation d’oxygene maximale. L’exercice physique diminue (p < 0,05) la teneur en glycoge`ne et augmente (p < 0,05) les concentrations de citrate, d’ace´tyl-CoA et de glucose 6-phosphate. Les seuls effets suscite´s par la cafe´ine sont l’augmentation (p < 0,05) de la concentration de citrate dans le muscle au repos et de la cAMP au cours de l’exercice physique. Il y a tre`s peu d’e´vidence a` l’appui de l’hypothe`se selon laquelle les effets ergoge`nes de la cafe´ine sont dus a` l’augmentation de l’oxydation des graisses. Ne´anmoins, des individus peuvent re´agir diffe´remment aux effets de la cafe´ine; d’ailleurs, il y a de plus en plus d’e´tudes sugge´rant que la cause serait naturelle et d’origine ge´ne´tique.

Received 25 July 2008. Accepted 25 September 2008. Published on the NRC Research Press Web site at apnm.nrc.ca on 6 December 2008. T.E. Graham.1 Human Health and Nutritional Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada. D.S. Battram. Brescia University College, The University of Western Ontario, London, ON N6G 1H2, Canada. F. Dela. Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. A. El-Sohemy. Department of Nutritional Sciences, University of Toronto, Toronto, ON M5S 3E2, Canada. F.S.L. Thong. SHI Consulting Inc., 162 Cumberland Street, Suite 310, Toronto, ON M5R 3N5, Canada. 1Corresponding

author (e-mail: [email protected]).

Appl. Physiol. Nutr. Metab. 33: 1311–1318 (2008)

doi:10.1139/H08-129

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Mots-cle´s : me´thylxanthines, facteur ergoge`ne, polymorphismes mononucle´otidiques (SNPs), ade´nosine, glycoge`ne musculaire, endurance. [Traduit par la Re´daction]

______________________________________________________________________________________ Introduction Caffeine (trimethylxanthine) is a naturally occurring compound found in approximately 60 different plants. In Canada, coffee is the most common dietary source of caffeine (Graham and Moisey 2005), but it is also found in colas and, in the last decade, a number of new products have been introduced that contain caffeine or guarana extract. Tea contains both caffeine and theophylline (a dimethylxanthine). The global popularity of caffeine is reflected in international trade, where coffee beans are second only to oil in importance. Caffeine and the 3 dimethylxanthines (theophylline, theobromine, and paraxathine) are biologically active, and the putative impact of the methylxanthines on metabolism has received considerable attention. There is no botanical source for paraxanthine, which may account for it being studied less often. Theobromine is the least active of the dimethylxanthines, and theophylline is biologically active (Graham and Moisey 2005) and is used pharmacologically. Among the many actions of the methylxanthines are the stimulation of the central nervous system (CNS), the increase in circulating epinephrine, and the mobilization of nonesterified fatty acids (NEFAs). These effects may at least partly account for the fact that caffeine has often been incorporated in weight loss supplements and used as a potential ergogenic aid. This review will address the latter and will consider the evidence that caffeine alters fat and carbohydrate metabolism in skeletal muscle. We will also address how caffeine may mediate effects on tissue peripheral to the CNS and, finally, consider whether there are individual differences in response to caffeine (that is, if some people are ‘‘responders’’ and others ‘‘nonresponders’’).

Caffeine: what is the fundamental mechanism for action? Potentially, caffeine could have a number of actions that affect skeletal muscle. It can inhibit adenosine receptors, it can increase sympathetic activity, and it can have direct intracellular actions. Two to 3 cups of coffee will result in plasma caffeine levels of 20–40 mmol
L–1 (as will ingestion of approximately 5 mg
kg–1 of caffeine), and this is accompanied by lower levels of the dimethylxanthines. Of the dimethylxanthines, paraxanthine is the most abundant, usually reaching 5–8 mmol
L–1. Caffeine in such biological concentrations (5–50 mmol
L–1) is an antagonist to adenosine receptors. However, the receptors have several isoforms (A1, A2a, A2b, and A3), and caffeine is believed to antagonize all except the A3 form (Daly and Fredholm 2004). The receptors are associated with intracellular pathways that influence cyclic adenosine monophosphate (cAMP) production, phospholipase C, and mitogen-activated protein kinases (MAPKs) (Schulte and Fredholm 2003). Thus, the action of caffeine on a given tissue depends on its complement of re-

ceptor isoforms. It is particularly important that the A1 and A2 adenosine receptors have opposite actions. A1 adenosine receptors are associated with Gi protein input into adenylate cyclase and decreases in intracellular cAMP, while A2 adenosine receptors are associated with Gs protein and increases in cAMP. The A1 receptor is also associated with Gbg subunits of the heterotrimeric G protein, which can have separate actions on the Ga subunit, which mediates the inhibition of adenylate cyclase. This Gbg subunit affects calcium release, potassium channels, and voltage-sensitive calcium channels. ‘‘Functionally’’ various studies (Challis et al. 1984; Han et al. 1998; Vergauwen et al. 1994, 1997) have suggested that caffeine’s effects on skeletal muscle are related to the antagonism of A1 receptors and the subsequent elevation of cAMP. Until recently, only A2 receptors had demonstrated this (Lynge and Hellsten 2000). However, Thong et al. (2007) have now shown that A1 receptors exist in the plasma membrane of rat soleus muscle. There is also the possibility that some of the actions of caffeine are secondary to increased sympathetic stimulation. The sympathetic nervous system is stimulated when the CNS is exposed to caffeine. There are a number of reports that caffeine increases circulating levels of epinephrine (Robertson et al. 1981; Van Soeren et al. 1993; Arciero et al. 1995; Graham and Spriet 1995), and Graham et al. (2000) reported an increase in norepinephrine ‘‘spillover’’ from the leg of humans at rest and in exercise. In addition, caffeine may also have direct intracellular effects, as it is also known to cross cell membranes. Thong et al. (2002) found that caffeine ingestion resulted in a 50% decrease in leg (rested or postexercise) glucose uptake, and this was accompanied by a decrease in glycogen synthase (fractional velocity reduced by 17% and I form reduced by 35%). Rush and Spriet (2001) reported that physiological concentrations of caffeine could inhibit glycogen phosphorylase, and Chesley et al. (1998) found a tendency for a subgroup (glycogen sparers) to have a decreased glycogen phosphorylase mole fraction during exercise after ingesting caffeine. While the evidence that glycogen sparing is not strong (see Caffeine: possible individual differences?), these studies do present the possibility that caffeine could have a direct action on metabolic enzymes, independent of the A1 receptor. Adenosine receptors are ubiquitous, occurring throughout the nervous system, and in the vascular endothelium, heart, liver, adipose tissue, and muscle (Reppert et al. 1991; Dixon et al. 1996; Fredholm et al. 1999). Thus, the actions that result from caffeine depend on which type of receptors it blocks and in which tissue the receptors are located. It is unlikely that any 1 tissue dominates the response, but, rather, it is likely a combination of the actions of the various tissues. The effects of caffeine on muscle could be, in part, secondary to initial effects on other tissues. As reviewed by #

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Tarnopolsky (2008) in this issue, there are a number of studies that lead to the conclusion that while caffeine clearly affects the CNS, the responses are not vital to the ergogenic aspects of caffeine. This review restricts the CNS consideration to how this could affect metabolism. Mohr et al. (1998) found that when people with tetraplegia ingested caffeine and then had their lower limb muscles electrically stimulated, their muscle endurance was enhanced and there was an elevation in their circulating NEFAs. It is highly likely that the elevation in NEFAs is not the cause of the ergogenic effect. The mobilization of the NEFAs could be due to caffeine directly antagonizing A1 receptors in adipose tissue, increased sympathetic nervous system stimulation of adipose tissue, or the actions of the elevation in epinephrine on adipose beta adrenergic receptors. The antagonism of A1 receptors in adipose tissue by caffeine is the most likely mechanism, as there are very low levels of norepinephrine and epinephrine in people with tetraplegia, which do not increase when caffeine is ingested (Van Soeren et al. 1996; Mohr et al. 1998; Battram et al. 2007a). Recently, Johansson et al. (2007) studied adipocytes from rodents with the A1 receptor knocked out, and concluded that only the A1 receptor mediated the antilipolytic actions of adenosine. Thus, we concluded that the mobilization of NEFAs is due to a direct action of caffeine on the A1 receptors of adipocytes. However, one should not make the generalization that catecholamines are not critical to any of caffeine’s actions. It has been repeatedly shown that caffeine causes insulin resistance in resting humans, which is likely due to actions on skeletal muscle. However, if one administers caffeine in combination with a beta-adrenergic blocker to able bodied subjects (Thong and Graham 2002), no insulin resistance is observed. A similar result is found when caffeine is ingested by persons with tetraplegia (Battram et al. 2007a). Thus, some actions of caffeine require at least permissive levels of catecholamines.

Caffeine: fat and carbohydrate metabolism There can no longer be any doubt that caffeine is a potent ergogenic aid (Tarnopolsky 1994; Graham 2001b), and it has been commonly proposed to enhance exercise capacity by promoting fat oxidation and inhibiting carbohydrate oxidation via feedback mechanisms in the active muscle. This action has been suggested to result in a reduced dependence on muscle glycogen stores, and the glycogen sparing then promotes increased endurance. This theory was proposed by Costill and coworkers (Costill et al. 1978; Ivy et al. 1979; Essig et al. 1980) more than a quarter of a century ago and, at the time of their classic studies, there were many reasons to support this hypothesis. The decade of investigations preceding their work had convincingly shown that muscle glycogen was a critical metabolic store for endurance exercise. Furthermore, the work pioneered by Randle and coworkers (1970) led to the concept that muscle oxidation of fats and carbohydrates was regulated by feedback of the tricarboxylic acid (TCA) intermediate, citrate onto glycolysis. This was later expanded and challenged by several scientists (Dyck et al. 1993; Duan and Winder 1994; Watt et al. 2002) to include acetyl-CoA and other putative factors that could inhibit glycolysis and increase glucose-6-phosphate (G-6-P), which in turn could decrease glucose uptake. It has also

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been demonstrated that caffeine mobilizes NEFAs from adipose tissue and increases circulating levels of epinephrine. Thus, when Costill et al. (Costill et al. 1978; Ivy et al. 1979; Essig et al. 1980) reported that caffeine had an ergogenic effect on endurance exercise while muscle glycogen was used more slowly and muscle triglycerides were degraded more extensively, the glycogen-sparing postulation had considerable support. The applied physiology field abounds with descriptive studies of caffeine and exercise, but has suffered from a lack of indepth investigations. Most scientists have not even measured plasma caffeine or epinephrine concentrations, nor have muscle biopsies been taken in most investigations. For example, we know of no studies since the report by Essig et al. (1980) in which human subjects consumed caffeine and intramuscular triglycerides were directly measured. Three studies (Essig et al. 1980; Erickson et al. 1987; Spriet et al. 1992) reported that caffeine resulted in glycogen sparing but, more recently, a number of investigations (Jackman et al. 1996; Graham et al. 2000; Greer et al. 2000; Laurent et al. 2000) failed to confirm this. Very seldom have there been determinations of metabolic intermediates that were proposed to be increased by fat oxidation and to inhibit carbohydrate catabolism. A number of such studies have been conducted (Graham et al. 2000, Greer et al. 2000, Spriet et al. 1992) and, in each case, the sample size has been small (6–8 subjects). This can result in a type 2 statistical error. For example, the variability of glycogen within a single biopsy is approximately 10% (Adamo and Graham 1998) and, thus, with a small number of subjects, it is conceivable that differences between treatments may not be detected. To re-examine this possibility, we pooled the data from these investigations; the subjects ingested 5 or 9 mg
kg–1 of caffeine, and protocols had biopsies at rest and at either 10 or 15 min of exercise (70% or 85% maximal oxygen consumption (VO2 max)). Chesley and coworkers (1998) published their individual data, so we were able to include these, as well as the resting data from the study by Thong et al. (2002). The result is a large dataset for glycogen (n = 44 and 37 for rest and exercise, respectively), citrate (n = 16 and 18 for rest and exercise, respectively), acetyl-CoA (n = 21 and 25 for rest and exercise, respectively), G-6-P (n = 24 and 16 for rest and exercise, respectively), and cAMP (n = 24 and 16 for rest and exercise, respectively). For the muscle glycogen, we were also able to include the data from Chesley et al. (1998), as the individual data were presented in that report. Muscle glycogen data that reflect no evidence of sparing during exercise have been previously presented (Graham 2001a) (Fig. 1). While the differences in the change in glycogen (155 ± 16 and 129 ± 14 mmol glucosyl units
kg–1 dry weight) are in the direction favouring glycogen sparing, statistical analysis (p = 0.22) does not approach significance, despite a sample size of 37. While muscle citrate and acetyl-CoA increased during exercise, and caffeine resulted in higher citrate concentrations at rest, there was no caffeine effect during exercise (Figs. 2 and 3). Muscle G-6-P increased during exercise (Fig. 4), but there was no effect of caffeine. While there was no effect of caffeine on muscle cAMP at rest, there was an increase in cAMP during exercise when caffeine had been ingested (Fig. 5). Table 1 summarizes the changes from rest #

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Fig. 1. Muscle glycogen concentrations at rest and after 10–15 min of exercise (Ex) at 70%–85% maximal oxygen consumption. The bars represent the mean (+ 1 standard error of the mean (SEM)) for 37 subjects. The filled bars represent the resting state and the open bars represent exercise. For both treatments, 10–15 min of exercise decreased (p < 0.05) muscle glycogen, and there was no effect of caffeine ingestion. dwt, dry weight; PL, placebo; CAF, caffeine; Ex, exercise. *, p < 0.05 vs. rest.

Fig. 3. Muscle acetyl-CoA concentrations at rest and after 10– 15 min of exercise at 70%–85% maximal oxygen consumption. The bars represent the mean (+ 1 SEM) for 19 subjects. The filled bars represent the resting state and the open bars represent exercise. For both treatments, 10–15 min of exercise increased (p < 0.05) muscle acetyl-CoA, and there was no effect of caffeine ingestion. dwt, dry weight; PL, placebo; CAF, caffeine; Ex, exercise. *, p < 0.05 vs. rest.

Fig. 2. Muscle citrate concentrations at rest and after 10–15 min of exercise at 70%–85% maximal oxygen consumption. The bars represent the mean (+ 1 SEM) for 15 subjects. The filled bars represent the resting state and the open bars represent exercise. For both treatments, 10–15 min of exercise increased (p < 0.05) muscle citrate. Citrate also increased (p < 0.05) in resting muscle following caffeine ingestion. dwt, dry weight; PL, placebo; CAF, caffeine; Ex, exercise. *, p < 0.05 vs. rest; {, p < 0.05 vs. PL.

Fig. 4. Muscle glucose-6-phosphate concentrations at rest and after 10–15 min of exercise at 70%–85% maximal oxygen consumption. The bars represent the mean (+ 1 SEM) for 12 subjects. The filled bars represent the resting state and the open bars represent exercise. For both treatments, 10–15 min of exercise increased (p < 0.05) muscle glucose-6-phosphate, and there was no effect of caffeine ingestion. dwt, dry weight; PL, placebo; CAF, caffeine; Ex, exercise. *, p < 0.05 vs. rest.

to exercise for the 2 conditions, and a comparison of the D values failed to show any effects of caffeine for any of the parameters. The finding that caffeine increased cAMP concentration during exercise is compatible with the caffeine antagonism of A1 receptors in skeletal muscle. This analysis does not support the theory that caffeine results in increased fat oxidation or an inhibition of carbohydrate catabolism. Graham (2001a) examined the VO2, respiratory exchange ratio (RER), and intramuscular triglyceride data of Essig et al. (1980), and concluded that it is possible that the intramuscular triglyceride data are not quantitatively accurate, as the magnitude of reduction in

intramuscular triglycerides was well in excess of what could be accounted for by the metabolic rate and RER. Only a few investigations have employed isotopically labelled substrates (Raguso et al. 1996; Roy et al. 2001) or leg arterio-venous measures (Graham et al. 2000) to assess how caffeine influences leg metabolism during exercise. Raguso et al. (1996) used stable isotopes to examine the impact of theophylline on fat metabolism during 60 min of exercise at 75% VO2 max. They found no impact on the whole-body RER or on the rate of disappearance of NEFAs. Similarly, Roy et al. (2001), using labeled glucose infusion during 1 h of exercise (65% VO2 max), found that caffeine ingestion did not affect total fat or carbohydrate oxidation, #

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1315 Table 1. The change from rest to exercise in selected intramuscular metabolic intermediates. Measurement Glycogen (mmol glucosyl units
kg–1 dry wt) cAMP (umol
kg–1 dry wt) Acetyl-CoA (mmol
kg–1 dry wt) Citrate (mmol
kg–1 dry wt) Glucose-6-phosphate (mmol
kg–1 dry wt)

Placebo –155.52±16.21 0.56±0.37 16.20±2.58 0.44±0.08 1.38±0.22

Caffeine –128.96±14.22 1.04±0.52 13.61±3.11 0.42±0.08 1.72±0.41

Note: wt, weight; cAMP, cyclic adenosine monophosphate.

Fig. 5. Muscle cyclic adenosine monophosphate (cAMP) concentrations at rest and after 10–15 min of exercise at 70%–85% maximal oxygen consumption. The bars represent the mean (+ 1 SEM) for 13 subjects. The filled bars represent the resting state and the open bars represent exercise. The only significant (p < 0.05) effect was that after 10–15 min of exercise, muscle cAMP was increased when caffeine had been ingested prior to the exercise. dwt, dry weight; PL, placebo; CAF, caffeine; Ex, exercise. *, p < 0.05 vs. rest.

nor was there an impact on the rate of appearance or disappearance of glucose. Graham et al. (2000) used direct Fick determinations of metabolic exchange across the legs of men during 1 h of exercise at 60% VO2 max. They found that caffeine ingestion did not alter the uptake of either NEFAs or glucose, and muscle biopsy analysis did not show any indication of glycogen sparing. Thus, from a number of perspectives, there is very little evidence that the ergogenic aspects of caffeine are the result of shifts in carbohydrate and fat metabolism.

Caffeine: possible individual differences? Chesley et al. (1998) suggested that there may be some who spare and some who do not spare glycogen during exercise following caffeine ingestion. These 2 subgroups were generated after the measurement of muscle glycogen was made. They suggested that increased fat oxidation resulted in better energy balance and less glycogen catabolism. Exercise endurance was not measured, so it is not known whether only the glycogen-sparing group has an endurance benefit, and the authors did not retest the subjects to examine whether the response was reproducible.

In recent years, single-nucleotide polymorphisms (SNPs) have been shown to account for differences in subgroups of a population in response to caffeine. For example, Cornelis and colleagues (2006) demonstrated an enhanced risk of acute nonfatal myocardial infarction with coffee consumption in subjects characterized as slow metabolizers of caffeine (carrier of the –163C allele for the cytochrome P450 1A2 (CYP1A2) enzyme), compared with fast metabolizers of caffeine (homozygous for the –163C allele). Recent work by Battram et al. (2005, 2007b) suggests that some people may respond differently to the effects of caffeine on carbohydrate metabolism than the general population. Either caffeine ingestion or an epinephrine infusion was employed to reduce glucose disposal during an hyperinsulinemic-euglycemic clamp. In the 2 studies, 25% of the subjects (n = 6 in total) responded to both caffeine and epinephrine, with an increase in whole-body insulinmediated glucose disposal rather than the expected decrease. While the sample size is small, it is possible that an inherent difference may have existed between our caffeine responders (a decrease in glucose disposal) and nonresponders (an increase in glucose disposal). Adenosine is well known to be a vasodilator because of the effects of stimulation on vascular smooth muscle adenosine receptors, and caffeine inhibits this effect. Martin and colleagues (2006) reported a similar uncharacteristic response in a subset of subjects, with respect to both adenosine- and epinephrine-induced increases in forearm vascular conductance. While both groups of subjects had the same vasodilatory response to the handgrip exercise, in the nonresponders (45% of the subjects), infusion of adenosine resulted in only a 25%–50% response in conductance to adenosine or the nonspecific beta-adrenergic agonist isopreterenol, compared with the responders. In addition, nitric oxide synthase inhibition (L-arginine anolog) blunted the response in the responders, but had no effect on the nonresponders. One possible explanation for this is that the 2 groups differed in the structure or density of the various adenosine receptors. These various findings suggest that there could be subsets of the population that are either less responsive to adenosine and (or) caffeine or, indeed, respond in an opposite manner. In a preliminary exploration to determine whether this could be due to common genetic variations, we took blood samples from 19 subjects who took part in the 2 studies by Battram et al. (2005, 2007b). Of the subjects, 6 had responded to caffeine and to epinephrine in a manner opposite to what was predicted from the literature (i.e., an increase in glucose disposal) and to what was demonstrated by the remaining 13 subjects. No commonalities could be found to #

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Appl. Physiol. Nutr. Metab. Vol. 33, 2008 Table 2. Summary of preliminary findings of genetic polymorphisms for the positive (n = 6) and negative (n = 13) caffeine responders. Protein CYP1A2

Adrenergic b2 receptor

A2a receptor

A1 receptor

A1 receptor

A1 receptor

SNP 743 C . /C A . /A C . /A 16 A . /A A . /G G . /G 1976 C . /C C . /T T . /T 90 G . /G G . /T T . /T 451/471/756 G . /G G . /A A . /A 444/799 G . /G G . /A A . /A

Positive responders 2 2 2 0 4 2 2 2 2 3 3 0 2 2 2 2 2 2

Negative responders 0 5 8 1 3 8 1 3 8 10 2 1 1 4 8 8 4 1

Note: The adrenergic b2 receptor Arg16Gly polymorphism was detected using an allelic discrimination assay with real-time polymerase chain reaction (PCR) (ABI 7000 Sequence Detection System), in which allele-specific primers and probes were purchased from Applied Biosystems (ABI No. C_2084764_20 (adrenergic b2 receptor)). The –163A > C polymorphism (rs762551) of the CYP1A2 gene and the adenosin (A)2a receptor 1976T > C polymorphism (rs5751876) were detected by real-time PCR or restriction fragment length polymorphism (RFLP)-PCR, as described elsewhere (Cornelis et al. 2006). Allelic discrimination was performed using the ABI 7000 Sequence Detection System. PCR conditions were set at 95 8C for the initial denaturation, followed by 40 cycles at 95 8C for 15 s and 60 8C for 1 min. The mixture was then was genotyped by PCR-RFLP. Each 10 mL reaction contained 10 ng of DNA and TaqMan Universal PCR Master Mix (Applied Biosystems), with the addition of 100 nmol
L–1 of each probe and 800 nmol
L–1 of each primer. PCR conditions were set at 50 8C for 2 min, 95 8C for 10 min, and 40 cycles at 95 8C for 15 s and 60 8C for 1 min. SNP, single-nucleotide polymorphism.

account for the different responses among these subjects with respect to body composition, fitness level, habitual caffeine use, resting glycogen concentrations, or blood pressure. Therefore we investigated whether these differences could be explained by SNPs in CYP1A2, the adrenergic b2 receptor, the A2a receptor, or the A1 receptor. c2 analysis of our subjects revealed no relationship between the response to caffeine and epinephrine on glucose disposal and CYP1A2, the adrenergic b2 receptor, or either adenosine receptor genotype (Table 2), suggesting that the ability to metabolize caffeine and adrenergic and adenosine receptor genotypes does not explain the opposing responses to caffeine and epinephrine in our subjects. It must be noted, however, that our sample size was small (n = 6 and n = 13), and therefore may have resulted in a type 2 statistical error. Further work is needed in this area.

Summary Not only is caffeine of interest from an applied and sport science perspective as an ergogenic aid, it is also a very useful research tool to study human metabolism and the putative roles of adenosine in metabolic regulation. It is now clearly documented that caffeine is a powerful ergogenic aid, and it is known to be an adenosine receptor antagonist. Among the physiological actions of caffeine are the stimulation of the CNS and the sympathetic nervous system, in-

creased arousal, and mobilization of NEFAs. However, there is little evidence to support the fact that these effects are critical to the ergogenic actions of caffeine. Recently, there have been a few studies that have demonstrated that some people may not respond to caffeine in the same manner as the general population. This opens up the strong possibility that SNPs in genes that code for receptors or postreceptor signaling proteins could play a very important role.

References Adamo, K.B., and Graham, T.E. 1998. Comparison of traditional measurements with macroglycogen and proglycogen analysis of muscle glycogen. J. Appl. Physiol. 84: 908–913. PMID:9480951. Arciero, P.J., Gardner, A.W., Calles-Escandon, J., Benowitz, N.L., and Poehlman, E.T. 1995. Effects of caffeine ingestion on NE kinetics, fat oxidation, and energy expenditure in younger and older men. Am. J. Physiol. 268: E1192–E1198. PMID:7611396. Battram, D.S., Graham, T.E., Richter, E.A., and Dela, F. 2005. The effect of caffeine on glucose kinetics in humans – influence of adrenaline. J. Physiol. 569: 347–355. doi:10.1113/jphysiol.2005. 097444. PMID:16150793. Battram, D.S., Bugaresti, J., Gusba, J., and Graham, T.E. 2007a. Acute caffeine ingestion does not impair glucose tolerance in persons with tetraplegia. J. Appl. Physiol. 102: 374–381. doi:10. 1152/japplphysiol.00901.2006. PMID:17068214. Battram, D.S., Graham, T.E., and Dela, F. 2007b. Caffeine’s impairment of insulin-mediated glucose disposal cannot be solely #

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Graham et al. attributed to adrenaline in humans.. J. Physiol. 583(Pt 3): 1069– 1077. PMID:17656440. Challis, R.A.J., Budohoski, L., McManus, B., and Newsholme, E.A. 1984. Effects of an adenosine-receptor antagonist on insulin-resistance in soleus muscle form obese Zucker rats. Biochem. J. 221: 915–917. PMID:6383352. Chesley, A., Howlett, R.A., Heigenhauser, J.F., Hultman, E., and Spriet, L.L. 1998. Regulation of muscle glycogenolytic flux during intense aerobic exercise following caffeine ingestion. Am. J. Physiol. 275: R596–R603. PMID:9688698. Cornelis, M.C., El-Sohemy, A., Kabagambe, E.K., and Campos, H. 2006. Coffee, CYP1A2 genotype, and risk of myocardial infarction. JAMA, 295: 1135–1141. doi:10.1001/jama.295.10.1135. PMID:16522833. Costill, D.L., Dalsky, G.P., and Fink, W.J. 1978. Effects of caffeine ingestion on metabolism and exercise performance. Med. Sci. Sports, 10: 155–158. PMID:723503. Daly, J.W., and Fredholm, B.B. 2004. Mechanisms of action of caffeine on the nervous system. In Coffee, tea, chocolate and the brain. CRC Press, Boca Raton, Fla. pp. 1–11. Dixon, A.K., Gubitz, A.K., Sirinathsinghji, D.J.S., Richardson, P.J., and Freeman, T.C. 1996. Tissue distribution of adenosine receptor mRNAs in the rat. Br. J. Pharmacol. 118: 1461–1468. PMID:8832073. Duan, C., and Winder, W.W. 1994. Effect of endurance training on activators of glycolysis in muscle during exercise. J. Appl. Physiol. 76: 846–852. PMID:8175598. Dyck, D.J., Putman, C.T., Heigenhauser, G.J.F., Hultman, E., and Spriet, L.L. 1993. Regulation of fat-carbohydrate interaction in skeletal muscle during intense aerobic cycling. Am. J. Physiol. 265: E852–E859. PMID:8279540. Erickson, M.A., Schwarzkopf, R.J., and McKenzie, R.D. 1987. Effects of caffeine, fructose, and glucose ingestion on muscle glycogen utilization during exercise. Med. Sci. Sports Exerc. 19: 579–583. PMID:3431374. Essig, D., Costill, D.L., and Van Handel, P.J. 1980. Effects of caffeine ingestion on utilization of muscle glycogen and lipid during leg ergometer cycling. Int. J. Sports Med. 1: 86–90. Fredholm, B.B., Battig, K., Holmen, J., Nehlig, A., and Zvartau, E.E. 1999. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol. Rev. 51: 83–133. PMID:10049999. Graham, T.E. 2001a. Caffeine, coffee and ephedrine: impact on exercise performance and metabolism. Can. J. Appl. Physiol. 26(Suppl): S103–S119. PMID:11897887. Graham, T.E. 2001b. Caffeine and exercise: metabolism, endurance and performance. Sports Med. 31: 785–807. doi:10.2165/0000 7256-200131110-00002. PMID:11583104. Graham, T., and Moisey, L. 2005. Caffeine, creatine and food/drug synergy: applications to human health. In Food-drug synergy and safety. CRC Press, Boca Raton, Fla. pp. 375–409. Graham, T.E., and Spriet, L.L. 1995. Metabolic, catecholamine, and exercise performance responses to various doses of caffeine. J. Appl. Physiol. 78: 867–874. PMID:7775331. Graham, T.E., Helge, J.W., MacLean, D.A., Kiens, B., and Richter, E.A. 2000. caffeine ingestion does not alter carbohydrate or fat metabolism in human skeletal muscle during exercise. J. Physiol. 529: 837–847. doi:10.1111/j.1469-7793.2000.00837.x. PMID:11118510. Greer, F., Friars, D., and Graham, T.E. 2000. Comparison of caffeine and theophylline ingestion: exercise metabolism and endurance. J. Appl. Physiol. 89: 1837–1844. PMID:11053334. Han, D.-H., Hansen, P.A., Nolte, L.A., and Holloszy, J.O. 1998. Removal of adenosine decreases the responsiveness of muscle

1317 glucose transport to insulin and contractions. Diabetes, 47: 1671–1675. doi:10.2337/diabetes.47.11.1671. PMID:9792534. Ivy, J.L., Costill, D.L., Fink, W.J., and Lower, R.W. 1979. Influence of caffeine and carbohydrate feedings on endurance performance. Med. Sci. Sports, 11: 6–11. PMID:481158. Jackman, M., Wendling, P., Friars, D., and Graham, T.E. 1996. Metabolic, catecholamine, and endurance responses to caffeine during intense exercise. J. Appl. Physiol. 81: 1658–1663. PMID:8904583. Johansson, S.M., Yang, J.N., Lindgren, E., and Fredholm, B.B. 2007. Eliminating the antiliipolytic adenosine A1 receptor does not lead to compensatory changes in the antilipolytic actions of PGE2 and nicotinic acid. Acta Physiol (Oxf). 190: 87–96. PMID:17428236. Laurent, D., Schneider, K.E., Prusaczyk, W.K., Frankin, C., Vogel, S.M., Krssak, M., et al. 2000. Effects of caffeine on muscle glycogen utilization and the neuroendocrine axis during exercise. J. Clin. Endocrinol. Metab. 85: 2170–2175. doi:10.1210/jc.85.6.2170. PMID:10852448. Lynge, J., and Hellsten, Y. 2000. Distribution of adenosine A1, A2A and A2B receptors in human skeletal muscle. Acta Physiol. Scand. 169: 283–290. doi:10.1046/j.1365-201x.2000.00742.x. PMID:10951119. Martin, E.A., Nicholson, W.T., Eisenach, J.H., Charkoudian, N., and Joyner, M.J. 2006. Bimodal distribution of vasodilator responsiveness to adenosine due to difference in nitric oxide contribution: implications for exercise hyperemia. J. Appl. Physiol. 101: 492–499. doi:10.1152/japplphysiol.00684.2005. PMID:16614358. Mohr, T., van Soeren, M., Graham, T.E., and Kjaer, M. 1998. Caffeine ingestion and metabolic responses of tetraplegic humans during electrical cycling. J. Appl. Physiol. 85: 979–985. PMID:9729573. Raguso, C.A., Coggan, A.R., Sidossis, L.S., Gastaldelli, A., and Wolfe, R.R. 1996. Effect of theophylline on substrate metabolism during exercise. Metabolism 45: 1153–1160. doi:10. 1016/S0026-0495(96)90016-5. PMID:8781304. Randle, P.J., England, P.J., and Denton, R.M. 1970. Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart. Biochem. J. 117: 677–695. PMID:5449122. Reppert, S.M., Weaver, D.R., Stehle, J.H., and Rivkees, S.A. 1991. Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord. Mol. Endocrinol. 5: 1037–1048. PMID:1658635. Robertson, D., Wade, D., Workman, R., Woosley, R.L., and Oates, J.A. 1981. Tolerance to the humoral and hemodynamic effects of caffeine in man. J. Clin. Invest. 67: 1111–1117. doi:10.1172/ JCI110124. PMID:7009653. Roy, B., Bosman, M., and Tarnopolsky, M.A. 2001. An acute dose of caffeine does not alter glucose kinetics during prolonged dynamic exercise in trained endurance athletes.. Eur. J. Appl. Physiol. 85(3–4): 280–286. PMID:11560082. Rush, J.W.E., and Spriet, L.L. 2001. Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine nucleotides and caffeine. J. Appl. Physiol. 91: 2071–2078. PMID:11641346. Schulte, G., and Fredholm, B. 2003. Signalling from adenosine receptors to mitogen-activated protein kinases.. Cell Signal. 15: 813–827. PMID:12834807. Spriet, L.L., MacLean, D.A., Dyck, D .J., Hultman, E., Cederblad, G., and Graham, T.E. 1992. Caffeine ingestion and muscle metabolism during prolonged exercise in humans. Am. J. Physiol. 262: E891–E898. PMID:1616022. Tarnopolsky, M.A. 1994. Caffeine and endurance performance. Sports Med. 18: 109–125. doi:10.2165/00007256-19941802000004. PMID:9132918. #

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1318 Tarnopolsky, M.A. 2008. Effect of caffeine on the neuromuscular system — potential as an ergogenic aid. Appl. Physiol. Nutr. Metab. 33: this issue. Thong, F.S., and Graham, T.E. 2002. Caffeine-induced impairment of glucose tolerance is abolished by beta-adrenergic receptor blockade in humans. J. Appl. Physiol. 92: 2347–2352. PMID: 12015346. Thong, F.S.L., Derave, W., Kiens, B., Graham, T.E., Urso, B. Wojtaszewski, J.F.P., et al. 2002. Caffeine-induced impairment of insulin action but not insulin signaling in human skeletal muscle is reduced by exercise. Diabetes, 51: 583–590. PMID:11872654. Thong, F., Lally, J., Dyck, D., Greer, F., Bonen, A., and Graham, T. 2007. Activation of the A1 adenosine receptor increases insulin-stimulated glucose transport in isolated rat soleus muscle. Appl. Physiol. Nutr. Metab. 32: 701–710. PMID:17622285. Van Soeren, M.H., Sathasivam, P., Spriet, L.L., and Graham, T.E. 1993. Caffeine metabolism and epinephrine responses during ex-

Appl. Physiol. Nutr. Metab. Vol. 33, 2008 ercise in users and nonusers. J. Appl. Physiol. 75: 805–812. PMID:8226485. Van Soeren, M., Mohr, T., Kjaer, M., and Graham, T.E. 1996. Acute effects of caffeine ingestion at rest in humans with impared epinephrine responses. J. Appl. Physiol. 80: 999–1005. PMID:8964766. Vergauwen, L., Hespel, P., and Richter, E.A. 1994. Adenosine receptors mediate synergistic stimulation of glucose uptake and transport by insulin and by contractions in rat skeletal muscle. J. Clin. Invest. 93: 974–981. doi:10.1172/JCI117104. PMID:8132783. Vergauwen, L., Richter, E.A., and Hespel, P. 1997. Adenosine exerts a glycogen-sparing action in contracting rat skeletal muscle. Am. J. Physiol. 272: E762–E768. PMID:9176173. Watt, M.J., Heigenhauser, F.J.G., Dyck, J.D., and Spriet, L.L. 2002. Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercsie in man. J. Physiol. 541: 969–978. doi:10.1113/jphysiol.2002.018820. PMID:12068055.

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