FOURTH OXFORD WORKSHOP Editor: Gil Hardy, PhD, FRCS Glutamine: An Anaplerotic Precursor Joanna L. Bowtell, MD, and Mark Bruce, MD From the Sport and Exercise Science Research Centre, South Bank University, London, UK; and the Department of Physical Education, Sports Science and Recreation Management, Loughborough University, Loughborough, Leicestershire, UK There is an up to four-fold increase in the concentration of the tricarboxylic acid (TCA) cycle interme- diates at the start of exercise. The rate of TCA cycle flux and, hence, oxidative may be limited by the concentration of the intermediates in the cycle. The dramatic decline in intramuscular glutamate at the start of exercise, in tandem with increased intramuscular alanine, suggests that glutamate is an important anaplerotic precursor. We hypothesized that oral glutamine might enhance the exercise-induced TCA cycle intermediate pool expansion. Indeed, a greater increase in the sum of muscle citrate, malate, fumarate, and succinate concentrations (ϳ85% total TCA intermediate pool) occurred at the start of exercise after ingestion of glutamine rather than of placebo or ornithine ␣-ketoglutarate. However, neither endurance capacity nor the degree of phosphocreatine depletion or lactate accumulation was altered. This suggests that TCA cycle intermediates do not limit flux through the cycle or that more intense exercise is required to show the limitation. Nutrition 2002;18:222Ð224. ©Elsevier Science Inc. 2002 KEY WORDS: glutamine, tricarboxylic acid cycle, tricarboxylic acid cycle intermediates, anaplerosis, exercise

INTRODUCTION cologically4Ð7 and nutritionally,8,9 and the effect on oxidative energy delivery has been investigated. The tricarboxylic acid (TCA) cycle occupies a position of prime At the start of exercise, the capacity of the TCA cycle to importance in the pathways of oxidative metabolism. Acetyl units oxidize acetyl units is outstripped by the rate of pyruvate produc- derived from the breakdown of glucosyl units, fatty acids, and tion from . Excessive pyruvate accumulation is averted amino acids are oxidized during their passage through the TCA by the reduction of pyruvate to lactate and the formation of alanine cycle, generating carbon dioxide and the reducing equivalents and 2-oxoglutarate (2-OG) by the near-equilibrium ALAT reac- tion. Indeed, intramuscular glutamate decreases by almost 60% NADH2 and FADH. The majority of adenosine triphosphate (ATP) produced via oxidative pathways is derived from the po- and alanine increases by almost 50% over this period, suggesting tential energy of these electrons harnessed by oxidative phosphor- that the ALAT reaction contributes significantly to TCAi ylation. The eight intermediates of the TCA cycle (TCAi) are not expansion.3,10,11 consumed by the oxidation of acetyl units but are recycled. How- Dicholoracetate (DCA) is a potent activator of the pyruvate ever, the concentrations of TCAi are in constant flux due to the dehydrogenase complex through inhibition of pyruvate dehydro- drainage of intermediates taking part in other reactions such as genase complex kinase. When infused at rest, DCA caused a synthesis of amino acids and the influx of TCAi via anaplerotic depletion of TCAi, presumably due to the observed diversion of reactions such as alanine aminotransferase (ALAT) and glutamate pyruvate to acetyl coenzyme A and acetylcarnitine, thus decreas- 4,5 dehydrogenase. ing pyruvate availability for anaplerosis. This supports a pivotal Gibala et al.1 calculated from measurements of leg oxygen role for pyruvate availability in determining TCAi pool size. uptake that the rate of ATP turnover and, hence, TCA cycle flux However, when DCA was infused before exercise, TCAi pool size increase approximately 100-fold during intense exercise. This is was not different between DCA and control conditions after 1 min 5 paralleled by an up to four-fold increase in skeletal muscle TCAi of exercise. The enhanced glycolytic flux at the onset of exercise content during the first few minutes of contraction, in an exercise seems to ensure that pyruvate availability is sufficient to support intensity-dependent fashion. This expansion of the TCAi pool may anaplerosis. be necessary to facilitate the increase in TCA cycle flux and, At the onset of exercise, there is a shortfall in ATP production hence, oxidative energy delivery.2,3 Alternatively, rather than play- from oxidative pathways that is met by substrate-level phosphor- ing a functionally important role, increased TCAi concentration ylation of adenosine diphosphate from glycogenolysis and phos- may merely reflect increases in pyruvate availability at the start of phocreatine breakdown. Therefore, the extent of phosphocreatine (PCr) breakdown and lactate accumulation during this transitional exercise.1,4 To distinguish between these opposing hypotheses, the phase are indicative of the magnitude of the mismatch between extent of TCAi expansion has been manipulated pharma- ATP demand and ATP production through oxidative phosphory- lation.12 Interestingly, DCA infusion before exercise appears to cause a relative sparing of PCr stores during the transition from rest to exercise.5Ð7 Oxidative energy delivery thus appears to be Work cited from the authors’ laboratories was supported in part by The enhanced by increasing acetyl group availability without a further Nuffield Foundation, University of Nottingham, Loughborough University, expansion of TCAi pool. This apparent dissociation between ox- and Laboratoires Jacques Logeais, Paris. idative energy delivery and TCAi pool size suggests that TCAi Correspondence to: Joanna L. Bowtell, MD, Sport and Exercise Science pool size is not normally limiting for oxidative energy delivery. Research Centre, South Bank University, 103 Borough Road, London, SE1 However, ideally, TCAi pool size should be altered in isolation 0AA, UK. E-mail: [email protected] to allow a cleaner dissection of the contributing factors. We

Nutrition 18:222Ð224, 2002 0899-9007/02/$22.00 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved. PII S0899-9007(01)00795-X Nutrition Volume 18, Number 3, 2002 Glutamine and Anaplerosis 223 hypothesized that the exercise-induced expansion of the TCAi crease observed after 2 min of exercise at 65% maximum oxygen pool could be enhanced by increasing the availability of glutamate consumption (ϳ40 mM/kg dw).6 Third, exercise at 70% maximum for anaplerotic reactions. Graham et al.13 did not find such an oxygen consumption may not have been sufficiently intense to enhancement with infusion of monosodium glutamate; however, show any enhancement in TCA cycle flux with the augmented transport of glutamate into skeletal muscle is poor.14 In contrast, TCAi expansion. Fourth, TCA cycle flux may be limited by the glutamine (GLN) is readily taken up into skeletal muscle via the availability of substrate; therefore, any further expansion of the high-capacity, sodium-dependent amino acid transport system Nm, TCAi pool could not facilitate increased TCA cycle flux without resulting in an increased intramuscular glutamine content.15 The increased availability of acetyl units, a hypothesis supported by the required to catalyze the conversion of glutamine to 2-OG DCA studies.4Ð7 Fifth, TCAi concentration in the muscle homog- (glutaminase; EC 3.5.1.2)16 and (EC enate may not accurately reflect changes in the mitochondrial 1.4.1.2)17 or ALAT (EC 2.6.1.2)18 or glutamine transaminase (EC compartment. Until methodologic advances allow the measure- 16,19,20 ␻ 16,19,20 2.6.1.15) and -amidase (EC 3.5.1.3) should exist in ment of mitochondrial and cytosolic TCAi concentrations, the human skeletal muscle. Thus, it is feasible that carbon derived latter question mark will remain. from GLN could enter the TCA cycle at the level of 2-OG. Indeed, Research in the 1960s first correlated fatigue during prolonged when glutamine was infused after exhaustive exercise, muscle moderate- to high-intensity exercise with a depletion of skeletal 21 glycogen storage was increased. There appeared to be an in- muscle glycogen stores.23 However, the precise biochemical crease in the availability of carbon units for incorporation into mechanism of this association is not fully understood. Depletion of glycogen, presumably due to 2-OG feeding into the TCA cycle. TCAi content might provide the link causing a reduction in TCA This result was recently replicated when GLN was ingested after cycle flux and, hence, oxidative energy delivery.24,25 Many of the exhaustive exercise.22 We hypothesized that ingestion of GLN anaplerotic reactions depend on pyruvate; thus, glycogen depletion before exercise would result in augmented TCAi expansion and, hence, increased oxidative energy delivery during the transition is likely to result in decreased influx of TCAi. On the other side of from rest to exercise. the equation, the rate of TCAi depletion might be increased in the 25 The protocol adopted was to deplete glycogen in subjects by glycogen-depleted state. Branched-chain amino acid (BCAA) prior exercise and a low-carbohydrate diet, so that the glycogen transamination, the first step in the oxidation of BCAA, relies availability would be identically low in all trials. Subjects were heavily on 2-OG as the amino group acceptor, resulting in the then provided with a drink of a placebo or two small anaplerotic formation of keto acid and glutamate. In conditions of plentiful precursors (ornithine ␣-ketoglutarate or GLN, both at 0.125 g/kg), pyruvate availability, 2-OG is recycled by the ALAT reaction. and then the effects of bicycle exercise at 70% maximum oxygen However, in the glycogen-depleted and hyperammonemic state, consumption were studied with appropriate muscle biopsies.9 Oral glutamine synthesis is favored,26 presumably resulting in a net loss GLN supported a greater expansion of TCAi concentration during of TCAi. The rate of BCAA oxidation is inversely proportional to the first 10 min of exercise. Plasma alanine concentration was carbohydrate availability: it is decreased with carbohydrate sup- higher in the GLN than in the placebo trial after 10 min of exercise, plementation27,28 and enhanced in the glycogen-depleted state.28 suggesting that the enhanced expansion of TCAi could, at least in Gibala et al.29 found that, during exercise under conditions ex- part, be attributed to increased flux through the ALAT reaction. pected to promote BCAA oxidation (glycogen depleted with Interestingly, there was no difference between treatments in the BCAA ingestion), the expansion of the TCAi pool size at the start intramuscular glutamate content, although resting intramuscular of exercise is not impaired.29 However, if glutamate limits anaple- GLN content was elevated in the GLN trial 1 h after ingestion of rosis at the start of exercise, this perhaps is not surprising because the supplement. During the first 10 min of exercise, the decline in there was no difference between trials in intramuscular glutamate intramuscular GLN and glutamate was approximately four-fold content. At fatigue, TCAi concentration is decreased relative to greater than the increase in the four measured TCAi (citrate, peak concentrations, although still elevated relative to the resting malate, fumarate, and succinate, 85% total TCAi pool) in the GLN TCAi concentration.24 Also, acetylcarnitine concentration at fa- trial and approximately three-fold greater in the placebo and orni- tigue was unchanged relative to values at 5 min of exercise, thine ␣-ketoglutarate trials. There are two possible explanations suggesting that the capacity to oxidize acetyl units rather than for this discrepancy: 1) drainage of the TCAi taking part in substrate availability per se was compromised at fatigue.24 ancillary reactions, e.g., amino acid synthesis; and 2) glutamate We investigated the involvement of TCAi depletion in the use in reactions by which there is no net production of TCAi. The etiology of fatigue. We hypothesized that, if we could improve the formation of aspartate, catalyzed by aspartate aminotransferase, is maintenance of TCAi pool throughout exercise, the onset of fa- one such reaction where the TCAi, oxaloacetate, acts as the amino tigue could be delayed.8 Unfortunately, despite increasing TCAi at group acceptor for glutamate, resulting in the formation of the 10 min of exercise with ingestion of glutamine 1 h before exercise, TCAi, 2-OG. Plasma aspartate concentration was elevated during this single bolus dose was not sufficient to maintain TCAi through- the GLN trial and thus may account for some of the “missing” out exercise. TCAi concentration was similar in all trials at fatigue, glutamate. Nonetheless, it is clear that GLN or glutamate carbon but there was no significant delay in the onset of fatigue. Most enters the TCA cycle, presumably at the level of 2-OG. Therefore, significantly, there was no association between measured peak glutamate and not pyruvate availability likely limits the exercise- TCAi achieved and time taken to reach fatigue. induced TCAi expansion. Despite the augmented TCAi pool expansion, there was no sparing of PCr stores or attenuation of muscle lactate production, suggesting that there was no enhancement of TCA cycle flux. This may be explained in a number of ways. First, expansion of TCAi CONCLUSIONS pool may not be functionally important. Second, any sparing of PCr was no longer evident after 10 min of exercise. In the studies The TCAi pool size expands at the start of exercise. Ingestion of where DCA was infused, reduced PCr breakdown was evident GLN before exercise results in an augmented expansion of the after 2 min of exercise but not after 10 min of exercise.6 Steady- TCAi pool but does not appear to facilitate increased TCA cycle state oxygen consumption is normally achieved 4 to 5 min after flux. The increase in measured peak TCAi pool size did not extend any change in exercise intensity. It is likely that, after this initial endurance capacity. Evidence from studies in which DCA has been period, resynthesis of PCr stores occurs, thus masking any earlier infused suggests that the limitation to oxidative energy delivery, at sparing effect. Certainly, the extent of PCr depletion in our study least at the start of exercise, may reside at the level of acetyl unit (ϳ20 mM/kg dw) was considerably less than the observed de- availability. 224 Bowtell and Bruce Nutrition Volume 18, Number 3, 2002

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