THE IMPORTANCE OF GLUTAMATE IN SKELETAL MUSCLE METABOLISM UNDER DIFFERENT EXERCISE CONDITIONS. A Thesis. Presented to. The Faculty of Graduate Studies

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1 THE IMPORTANCE OF GLUTAMATE IN SKELETAL MUSCLE METABOLISM UNDER DIFFERENT EXERCISE CONDITIONS A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by MARINA MOURTZAKIS In partial fulfilment of requirements for the degree of Doctor of Philosophy February, 2004 Marina Mourtzakis, 2004

2 ABSTRACT THE IMPORTANCE OF GLUTAMATE IN SKELETAL MUSCLE METABOLISM UNDER DIFFERENT EXERCISE CONDITIONS Marina Mourtzakis University of Guelph, 2004 Advisor: Dr. T. E. Graham The tricarboxylic acid (TCA) cycle provides a central process for not only fat and carbohydrate pathways to merge but also for amino acids to integrate and play a putative role in regulating oxidative metabolism. Muscle glutamate is a central amino acid for all transamination reactions that integrate with the TCA cycle. However, at the onset of exercise muscle glutamate decreases despite a constant uptake from the circulation. This thesis investigated whether: 1) glutamate affects metabolic homeostasis in skeletal muscle and potential tissue interactions, 2) alterations in glutamate availability impose changes on oxidative metabolism, and 3) whether the interactions between glutamate and other amino acids impact on oxidative metabolism. The first study examined whether glutamate affects oxidative metabolism during prolonged exercise since pyruvate flux decreases under these conditions. Muscle biopsies and direct Fick measurements revealed that the largest net exchanges in glutamate and alanine occurred in the first hour of exercise while glutamate was consistently taken up for the rest of exercise. Pyruvate flux remained constant implying that it may not affect glutamate metabolism. The second study examined whether increased glutamate availability via glutamate ingestion would perturb oxidative metabolism. With increased circulating glutamate, pulmonary VO 2 was elevated during moderate cycling and the proportions of nitrogen sources released were

3 altered. The final study isolated endurance training to the quadriceps muscle of one thigh for 5 weeks while the other thigh remained untrained. Following training, each thigh underwent maximal work tests. Direct Fick measurements and muscle biopsies were obtained from each thigh at rest and maximal work rate during control and glutamate conditions. Muscle glutamate was not different between thighs at rest or during exercise under either conditions. However, glutamate uptake was increased with glutamate infusion compared to control. Training enhanced VO 2peak, attenuated the increase in fumarate and malate, and lessened the decrease in 2-oxoglutarate. In contrast, glutamate administration resulted in ~20% and ~50% decreased muscle VO 2peak and 2-oxoglutarate levels, respectively, while enhancing fumarate and malate concentrations in the trained thigh only. Collectively, these results suggest that alterations in glutamate availability can impact the regulation of oxidative energy provision in human skeletal muscle.

4 ACKNOWLEDGEMENTS This thesis dissertation could not have been accomplished without the support and guidance that I received from several individuals. First, I would like to thank my advisor, Dr. Terry Graham, for providing me with an exceptional learning environment and excellent academic experiences. These experiences and his insightful advice have helped me develop as a scientist. More importantly, he has always had confidence in my ideas and his enthusiasm for science has been inspiring. I thank him for his support and direction. He has been an excellent teacher as well as a wonderful colleague and friend. I would also like to thank Drs. David Wasserman (External), Brian McBride (Internal/External), Arend Bonen (Chair), and Martin Gibala for agreeing to be a part of my examining committee and for the stimulating discussions that emerged. I particularly would like to thank my advisory committee, Drs. Lawrence Spriet & Martin Gibala. I have been fortunate to have their expertise and guidance throughout my graduate studies. They challenged me when I needed to be challenged and encouraged me when I needed support. They have also been great teachers, colleagues and friends. I am also grateful for the discussions I had with several other faculty during my tenure at Guelph. As I spent several months in Denmark for the completion of 2 studies, there are several individuals that I would like to acknowledge. First, I express my sincerest gratitude to Dr. Henriette Pilegaard, who is an incredibly inspiring and caring individual. I enjoyed our collaborative work and fruitful discussions. Most importantly, I am thankful for our friendship. I am also very grateful to Dr. Bengt Saltin for the honourable opportunity to i

5 work with him and his team at the Copenhagen Muscle Research Centre. He has been a stimulating teacher and his words of wisdom will always stay with me. Exceptional thanks to Drs. José Alonso-González and Gerrit van Hall for the interesting and insightful discussions. We were also very fortunate to have dedicated subjects that participated in our studies as they were devoted individuals that underwent a number of demanding tasks with enthusiasm. I sincerely thank you. There are several fellow lab-mates past and present that I would like thank for their support. In particular, I would like to thank Danielle, Lindsay & Premila for their continual support and friendship. As well, Jane, Farah, Shannon, Sherry, Krista and Bec whose encouraging words and friendships have been wonderful. To my close friends Effie & Patrick, Liz, and Laura who have been incredible friends and have been through many joyous events and life obstacles with me. Their unconditional support and their encouragement have been my strength I thank you. To my family Mom, Dad, Stelios I finally did it! You have taught me that I can achieve my goals through perseverence and dedication. Thank you especially for your infinite support - you have always been there for me with your words of encouragement and your love. And to Chris, I thank you for your unconditional love and support. You have helped me achieve some balance in my life and your faith in me has shaped my confidence. ii

6 LIST OF TABLES Table 2.1 Summary of prolonged exercise (>60min) studies examining amino acid metabolism in humans. 22 Table 2.2 Summary of studies examining the effects of training on amino acid metabolism in humans. 31 Table 3.1 Pulmonary VO 2, blood flow, muscle VO 2 and RER during prolonged exercise and recovery. 68 Table 3.2 Leg glucose clearance, leg FFA clearance and lactate flux. 73 Table 3.3 Catecholamine, insulin, and glucagon levels during prolonged exercise and recovery. 75 Table 3.4a Muscle amino acids (mmol/kgdw) during prolonged exercise and recovery. 80 Table 3.4b Arterial (A) and venous (V) amino acids (um) during prolonged exercise and recovery. 84 Table 3.5 Pyruvate and acetyl-coa during prolonged exercise and recovery. 89 Table 4.1 Oxygen consumption and RER data are depicted during exercise. 115 Table 4.2a Summary of amino acids (um) for rest trials. 124 Table 4.2b Summary of the plasma amino acids (um) for the exercise trials. 126 Table 4.3a Rest trial data for glucose, insulin, C-peptide, FFA and glycerol. 131 Table 4.3b Exercise trial data for lactate, glucose, insulin, C-peptide, FFA and glycerol. 132 iii

7 Table 5.1 Oxygen parameters for different work rates and training states under control and glutamate conditions. 153 Table 5.2a Arterial and venous amino acids at rest and peak work rate for each thigh under different control and glutamate conditions. 156 Table 5.2b Muscle amino acids (mmol/kgdw) at rest and peak work rate in both thighs under control and glutamate conditions. 167 Table 5.3 Arterial lactate and glucose concentrations as well as lactate and glucose fluxes in both thighs under control and exercise conditions. 173 Table 5.4 Arterial hormone levels at rest and peak work rate for thighs under control and glutamate conditions. 175 iv

8 LIST OF FIGURES Figure 2.1 Amino acid dynamics between skeletal muscle and circulation. 7 Figure 2.2 This diagram depicts the integration of glutamate with reactions that are involved in the TCA cycle in skeletal muscle. 11 Figure 2.3 Whole body amino acid metabolism during dynamic exercise. 17 Figure 3.1 The experimental protocol used. 62 Figure 3.2 Whole body and muscle energy expenditure. 69 Figure 3.3 Glucose and FFA uptake during prolonged exercise and recovery. 71 Figure 3.4 Arterial and venous glucose and FFA. 72 Figure 3.5 Glutamate data. 76 Figure 3.6 Alanine data. 77 Figure 3.7 Glutamine data. 78 Figure 3.8 BCAA data. 79 Figure 3.9 Ammonia data. 86 Figure 3.10 Nitrogen distribution of glutamate, glutamine, alanine, BCAA and ammonia. 87 Figure 3.11 Pyruvate dehydrogenase during prolonged exercise and recovery. 90 Figure 4.1 Experimental design. 112 Figure 4.2 Plasma glutamate response to MSG ingestion. 117 Figure 4.3 Plasma aspartate responses to MSG ingestion. 118 Figure 4.4 Plasma taurine responses to MSG ingestion. 119 Figure 4.5 Plasma alanine responses to MSG ingestion. 120 Figure 4.6 Plasma glutamine responses to MSG ingestion. 122 v

9 Figure 4.7 Plasma BCAA responses to MSG ingestion. 123 Figure 4.8 Plasma ammonia responses to MSG ingestion during the exercise trials. 128 Figure 4.9 Nitrogen distribution of glutamate, alanine, glutamine and ammonia following MSG ingestion. 130 Figure 5.1 The experimental protocol. 148 Figure 5.2 Summary of glutamate metabolism. 155 Figure 5.3 Summary of alanine metabolism. 159 Figure 5.4 Summary of glutamine metabolism. 160 Figure 5.5 Summary of BCAA metabolism. 161 Figure 5.6 Summary of ammonia metabolism. 162 Figure 5.7 Total amount uptake/release of key nitrogen carriers during exercise (~12min). 165 Figure 5.8 Summary of TCAI metabolism. 170 Figure 5.9 Muscle lactate and phosphocreatine. 172 Figure 6.1 Whole body amino acid metabolism. 196 Figure 6.2 Glutamate in muscle metabolism. 200 vi

10 TABLE OF CONTENTS Abstract Acknowledgements i List of Tables iii List of Figures v GENERAL INTRODUCTION & RATIONALE General Introduction Rationale of Studies 3 REVIEW OF LITERATURE General Overview A General Introduction to Amino Acid Metabolism Factors that Regulate Amino Acid Metabolism The Effects of Physical Activity on Amino Acid Metabolism The Effects of Short-Term Exercise on Amino Acid Metabolism Prolonged Exercise Effects on Amino Acid Dynamics Training Effects on Amino Acid Metabolism Ammonia Production in Relation to Amino Acid Metabolism The Relationship of Amino Acids with the TCA cycle During Exercise Glutamate Metabolism Glutamate and Insulin Glutamate Transport in Skeletal Muscle Glutamate Metabolism: Interactions with Nitrogen-based Metabolites and the TCA cycle Approaches to Studying Glutamate Metabolism Methodological Tools in Studying Glutamate Metabolism Summary 56 STUDY I GLUTAMATE METABOLISM AND PROLONGED EXERCISE Introduction 57 vii

11 3.1 Methods Experimental Protocol Blood Analysis Muscle Analysis Statistics and Calculations Results Pulmonary VO 2, Respiratory Exchange Ratio (RER), blood flow, and muscle VO Glucose, FFA, and Lactate Analysis Catecholamine, insulin and glucagon analysis Muscle and Plasma Amino Acid, Plasma Ammonia and Nitrogen Distribution Analysis PDHa, glycogen, pyruvate, and acetyl-coa Discussion Potential Role of Glutamate in Substrate Utilization Glutamate and Oxidative Metabolism Glutamate Interactions with Other Amino Acids and Nitrogen Balance 100 STUDY II THE EFFECTS OF GLUTAMATE SUPPLEMENTATION ON REST AND EXERCISE METABOLISM Introduction Methods Experimental Protocol Analyses Statistics Results Respiratory and Electrolyte Data Plasma Amino Acids Ammonia Lactate, Glucose, Insulin, C-peptide Glycerol, Free fatty acids Discussion 133 STUDY III TRAINING AND GLUTAMATE SUPPLEMENTATION: IMPLICATIONS ON OXIDATIVE METABOLISM Introduction Methods Experimental Protocol Blood Analyses 150 viii

12 5.1.3 Muscle Mass Measurements Statistics and Calculations Results Training Effects on Muscle Mass and Work Rate Cardiovascular Responses Plasma Amino Acids and Ammonia Concentrations Amino Acid Flux, Ammonia Flux and Total Uptake/Release Calculations Muscle Amino Acids Tricarboxylic Acid Intermediates Phosphocreatine and Lactate Data Glucose and Hormone Data Discussion Training Effects on Amino Acid Metabolism and the TCA Cycle Effects of Glutamate Administration on Amino Acid and Ammonia Metabolism Glutamate Supplementation and Oxidative Metabolism Implications of Glutama te on Whole Body Metabolism Methodological Considerations 192 GENERAL DISCUSSION Nitrogen Balance: Glutamate & Its Interactions with other Amino Acids Glutamate and Tissue Communication The Role of Glutamate in Oxidative Metabolism via Energy Homeostasis & Supply Concluding Remarks Future Directions 210 REFERENCES 212 APPENDIX A ADENINE NUCLEOTIDES AND PHOSPHOCREATINE 231 APPENDIX B ELECTROLYTES 232 APPENDIX C DETAILED PROTOCOL FOR STUDY III 233 APPENDIX D ADDITIONAL CARDIOVASCULAR MEASUREMENTS 234 ix

13 CHAPTER 1 GENERAL INTRODUCTION & RATIONALE 1.0 General Introduction Amino acid metabolism involves very intricate and complex processes that interact with several pathways at various levels of metabolism. Each amino acid has specific roles that can vary between different tissues. While the free amino acid pool is generally smaller in magnitude when compared to compounds such as glucose, lactate and free fatty acids involved in substrate metabolism, amino acids may be key in the regulation of oxidative metabolism. Skeletal muscle consists of a dynamic pool of metabolizable amino acids that are fundamental to structural integrity as well as energy processes. Glutamate is one of the critical amino acids because it is integral to all amino acid transamination reactions within skeletal muscle, and therefore, can affect a number of substrates, other amino acids, metabolites and the tricarboxylic acid (TCA) cycle intermediates. However, our understanding of the role of glutamate, particularly in skeletal muscle, is limited. Glutamate is one of the most abundant amino acids in various tissues (Brosnan, 2000) and its molecular structure has unique features that may be important for trafficking amino groups within and across different tissues (Young & Ajami, 2000). These characteristic properties of glutamate may explain its abundance and centrality to numerous amino acid reactions in skeletal muscle. It is possible that glutamate may have several roles in muscle metabolism especially during periods of increased metabolic demand (ie. exercise). Glutamate is particularly interesting because of its interactions with the TCA cycle via the alanine aminotransferase (AAT) reaction (pyruvate + 1

14 glutamate alanine + 2-oxoglutarate). This link with the TCA cycle suggests that glutamate may have a regulatory function in substrate utilization and/or TCA cycle flux. The premise of this thesis is to investigate the role of glutamate in oxidative metabolism through its associations with the TCA cycle, which is vital to energy production. Glutamate is integral to the TCA cycle via several transamination reactions. The AAT reaction provides glutamate with an association to energy metabolism and is also tied to carbohydrate oxidation since pyruvate is one of its substrates. Therefore, three studies were conducted to examine glutamate s putative role in the regulation of metabolic homeostasis along with its interactions with other amino acids. Glutamate was also examined as a potential signal for modulating interorgan communication. In the first study, glutamate s effects on energy metabolism were examined along with its interactions with other amino-carriers. This study focused on the regulatory potential of certain amino acids in controlling the shift in substrate utilization from carbohydrate to fat during prolonged exercise. Particular attention was drawn to glutamate and its possible roles in energy metabolism through the AAT reaction as well as its potential role in tissue communication. The second study manipulated circulating glutamate concentrations to examine the potential effects of glutamate on whole body oxygen consumption and its interactions with other amino acids at rest and during exercise. In the third study, glutamate administration was combined with endurance training to directly measure potential changes in the TCA cycle and oxidative metabolism. Thus, 2

15 these studies specifically examine the potential role(s) of glutamate in different regulatory aspects of metabolism. 1.1 Rationale of Studies Since glutamate is central to all transamination reactions that interlink with the TCA cycle, glutamate may have an essential role in the regulation of energy metabolism. These near-equilibrium, transamination reactions involve interactions with several key amino acids such as branched-chain amino acids (BCAA), glutamine, and alanine. Thus, glutamate may co-ordinate the regulation of oxidative metabolism by adjusting these near-equilibrium reactions rapidly depending on the metabolic demands of the muscle. Glutamate is also one of the few amino acids to be taken up by muscle, and hence, may be key to nitrogen balance. Therefore, this thesis entails three studies that were designed to explore these issues under different exercise conditions and enhance our understanding of glutamate s role in substrate and oxidative metabolism as well as nitrogen balance. The key objectives of this thesis were to investigate: a) the potential changes in glutamate concentrations in skeletal muscle during prolonged, low intensity exercise, b) the potential relationship between glutamate and other amino acids as well as pyruvate flux during prolonged exercise when carbohydrate availability becomes compromised, c) the interrelationship between glutamate and other amino acids by increasing glutamate availability during rest and exercise, 3

16 d) the effects of enhanced glutamate availability on oxidative metabolism, e) the role of glutamate in oxidative metabolism through endurance training, f) the possible differential effects of increased glutamate availability on oxidative capacity in the untrained and trained muscle, and g) the role of glutamate on associated changes in nitrogen distribution under different exercise protocols. 4

17 Chapter 2 REVIEW OF LITERATURE 2.0 General Overview Various amino acids have specific roles in developing and maintaining muscle integrity and function. Understanding the metabolic roles of amino acids in the healthy individual is complex and we are faced with further challenges in understanding the functions of amino acids in clinical conditions such as burns, sepsis, and cancer-associated cachexia. Amino acid metabolism comprises a wide array of intricate biological processes that involve 20 physiologically important amino acids. The vast range of intracellular functions of amino acids include maintenance of adenine nucleotide concentrations (Aragón & Lowenstein, 1980), de novo synthesis of purine and pyrimidine bases (Labow et al., 1999; Rennie, 1996), transfering reducing equivalents into mitochondrial spaces (Rennie, 1996), maintaining anaplerotic processes of the tricarboxylic acid (TCA) cycle (Gibala et al., 1997a, 1998, 2001), detection of changes in cell volume (Häussinger & von Dahl, 1995; Hediger, 1999; Hue & Gaussin, 1995; Low et al., 1997), provision of fuel for the immune system (Robinson et al., 1999;Yoo et al., 1997), neurological function (Daikhin & Yudkoff, 2000; Hertz et al., 2000; Zhou & Norenberg, 1999), acidbase regulation as well as producing precursors for gluconeogenesis and ureagenesis (Charlton et al., 1996; Halseth et al., 1998; Nair et al., 1987; Olde Damink et al., 2002; Wasserman et al., 1991). Although amino acids are essential to the functioning of cells, there are large gaps in the literature in understanding their mechanistic capabilities of regulating metabolic processes. 5

18 While the focus of this thesis is on glutamate metabolism, a general overview of amino acid metabolism will introduce the key functions of various amino acids across different tissues. It is important to have a general understanding of this intricate web that integrates glutamate with other amino acids as well as energy homeostasis and regulation, hormonal status and interorgan communication. Following the dynamic illustration of the various processes involving amino acids, a more focused review will discuss the actions and potential roles of amino acids in oxidative metabolism during exercise. With this general introduction in amino acid metabolism, glutamate and its putative role in energy metabolism and interorgan communication will be explored. 2.1 A General Introduction to Amino Acid Metabolism The vast majority of amino acids are found as part of proteins and very few (~1% of whole body amino acids) are contained in free amino acid pools. The free amino acid pools alter in size and composition with protein turnover and amino acid transport. These processes portray a dynamic illustration of amino acid metabolism whereby amino acids are constantly building and breaking down proteins as well as flowing into and out of intracellular free pools (Figure 2.1). Collectively, these alterations result in continual fluctuations in the free amino acid pools that influence intermediary metabolism. Regulation of amino acid uptake from the circulation into various tissues in addition to the size of the amino acid pools is important in understanding their putative roles in oxidation and protein metabolism in diverse tissues. 6

19 Figure 2.1 Amino acid dynamics between skeletal muscle and circulation. 7

20 Quantitatively, the liver and the gut are the most important tissues for amino acid metabolism (Newsholme & Leech, 1983; Newsholme et al., 2003). The liver is especially key in protecting the essential amino acids (EAA) from high rates of degradation (Newsholme & Leech, 1983; Wasserman & Cherrington, 1996). By regulating plasma concentrations of amino acids, the liver can help supply specific amino acids to different tissues for protein synthesis (Newsholme & Leech, 1983; Wasserman & Cherrington, 1996). Furthermore, it is also a primary site for metabolizing ammonia, often produced by amino-based catabolic processes, and utilizing glutamine and alanine for gluconeogenesis (Halseth et al., 1998; Newsholme et al., 2003; Wasserman & Cherrington, 1996). Tissue interaction is different during exercise as compared to rest due to increases in metabolic demands (Halseth et al., 1998; Wasserman et al., 1991; Wasserman & Cherrington, 1996). At times of high metabolic demand, the gut is key to releasing a number of amino acids, particularly aspartate, glutamate, asparagine, alanine and glutamine, via proteolysis to be used as substrates in the liver and most of the amino acids are taken up by the muscle for further use (Newsholme & Leech, 1983; Wasserman & Cherrington, 1996). Moreover, some amino acids such as aspartate and glutamate, are metabolized almost completely by splanchnic metabolism, and thereby small amounts actually enter the blood stream following protein ingestion (Fuller & Reeds, 1998; Newsholme & Leech, 1983). Thus, the capacity for which glutamate and other amino acids are utilized by the splanchnic tissues could have implications on the availability of these amino acids for other tissues to use. This could ultimately shape the role of such amino acids in different processes. 8

21 Amino acids are required for diverse purposes in various tissues for proper functioning. Amino acids such as glutamate and glutamine are important in maintaining acid-base balance in renal circulation and function (Newsholme et al., 2003; Welbourne et al., 2001). Glutamate receptors have also been identified in pancreatic â-cells where glutamate may have a putative role in the activation of insulin release (Maechler et al., 2002; Molnár et al., 1995). The brain, in addition to other neurological tissues, can oxidize glutamate (Daikhin & Yudkoff, 2000; Hertz et al., 2000), is sensitive to certain amino acids, (Newsholme & Leech, 1983; Newsholme et al., 2003; Tanaka, 2000) and is especially sensitive to changes in ammonia concentrations (Graham, 1994; Newsholme & Leech, 1983). Furthermore, glutamate can also act as a neurotransmitter and stimulate the activation of neuronal tissues (Kunishima et al., 2000; Malomouzh et al., 2003). Interestingly, the inactivation of glutamate is carried out by glial cells that take up glutamate and convert it to glutamine (Newsholme & Leech, 1983; Tanaka, 2000). Glutamate, as such, appears to be integral for the proper functioning of numerous tissues while its availability is regulated predominantly by splanchnic metabolism. Although amino acids are important in gut, liver, renal and neuronal metabolism (Newsholme & Leech, 1983; Rennie, 1996), the dynamic free pool of amino acids are also essential to skeletal muscle metabolism and interorgan relationships especially during increased metabolic demands. In the past, physiologists considered the amino acid pool in skeletal muscle as a static depot of amino acids for building various proteins such as enzymes and receptors. Skeletal muscle encompasses the largest free amino acid pool 9

22 in the body (Rennie, 1996; Rennie & Tipton, 2000; Wagenmakers, 1998a). Yet, there are only 6 amino acids that are metabolized within skeletal muscle: BCAA (the branchedchain amino acids which are valine, leucine, isoleucine), alanine, glutamate, and aspartate. Alanine and glutamine comprise about 60% of the release of total amino acids from skeletal muscle while glutamate is the predominant amino acid that is taken up during rest and exercise metabolism. Fluctuations in certain amino acid concentrations within the skeletal muscle free amino acid pool have significant modulatory effects on protein synthesis and degradation, nitrogen balance as well as intermediary metabolism (Rennie, 1996; Wagenmakers, 1998a, 1999b). Skeletal muscle can account for up to one half of all protein turnover in the body were ~15% of amino acids are found in their free form (Rennie & Tipton, 2000). Amino acids released from protein breakdown have a large impact on the characteristics of the free amino acid pool; for instance, the BCAA account for about 20% of the amino acids released from protein (Rennie & Tipton, 2000). In addition to protein turnover, changes to the amino acid pool are also affected by the transport of amino acids into and out of the muscle tissue. Because the free amino acid pool in skeletal muscle is relatively small in magnitude compared to substrates such as glycogen and intramuscular triglycerides, slight fluctuations in the size and composition of the pool may have large impacts on the regulation of intermediary metabolism. Intermediary metabolism of particular amino acids, including glutamate, BCAA, glutamine and alanine, may be important in oxidative metabolism since they are closely linked to the TCA cycle, which is the principal source of energy (Figure 2.2). 10

23 Figure 2.2 This diagram depicts the integration of glutamate with reactions that are involved in the TCA cycle in skeletal muscle. 11

24 In the average, healthy individual, 17% of body mass is comprised of protein and ~0.3% is free amino acids. Over half of these proportions of protein and free amino acids are present intracellularly in skeletal muscle (Rennie & Tipton, 2000; Wagenmakers, 1998a, 1999b). Quantitatively, amino acid metabolism may not appear to be important in comparison to the magnitude of other substrates such as carbohydrates and fats, that are metabolized in humans. However, this small amino acid pool participates in transamination and transdeamination reactions of oxidative metabolism within muscle and may play important regulatory roles (Gibala, 2001). It is only in recent investigations that the potential, regulatory characteristics of these small amino acid pools have been revealed. Glutamate distinctly interacts with all of the metabolizable amino acids and integrates them with the TCA cycle suggesting that it may have a central role in regulating oxidative metabolism Factors that Regulate Amino Acid Metabolism The size and composition of amino acid pools for different tissues is quite variable and may reflect the role that free amino acids have in regulating several metabolic processes. There are numerous ways to modulate changes in the free amino acid pools of different tissues which primarily include amino acid transport, hormonal alterations, dietary intake, and physical activity. In part, the regulation of amino acid metabolism can be exerted at the membrane level of a given tissue by amino acid transporters (Low et al., 1994). Amino acid transporters are found in a number of tissues but are particularly essential in the intestines for absorption 12

25 of dietary amino acids (Rennie & Tipton, 2000; Wagenmakers, 1998a, 1999b). Although they have rarely been studied in skeletal muscle, amino acid transporters are important for absorption of amino acids into intracellular spaces that are going against a concentration gradient (Wagenmakers, 1999b). Transport generally operates at rates that are directly proportional to intracellular and extracellular concentrations. Most of these transporters have a low affinity but are sufficiently active so as not to limit any metabolic processes (Rennie & Tipton, 2000). However, the transport of glutamate is an exception because this transporter is thought to be saturated at concentrations similar to plasma concentrations, and has a low maximum capacity which could have implications in its role in skeletal muscle (Rennie et al., 1996; Rennie & Tipton, 2000). Hormones are normally considered to have a regulatory effect on diverse realms of metabolism. Insulin affects certain amino acid transporters (Rennie, 1996) and it has also been shown to attenuate protein breakdown (Abu-Lebdeh & Nair, 1996; Wolfe, 2000). In insulin-dependent (Type 1) diabetics, insulin deprivation results in an overall catabolic state while the replacement of insulin decreases protein breakdown as well as protein synthesis (Abu-Lebdeh & Nair, 1996; Millward, 1995). It is now evident that insulin will enhance protein synthesis as long as there are sufficient amino acids available to be used as substrates (Rennie et al., 2002; Tessari et al., 1996; Wolfe, 2000). However, the specific amino acids responsible for these effects have not been studied extensively. Glucagon, on the other hand, has displayed proteolytic features (Battezzati et al., 2000; Charlton et al., 1996; Nair et al., 1987), and activates gluconeogenesis from glutamine and alanine, which are released from the muscle (Wasserman & Cherrington, 1991). 13

26 Glucagon has also been shown, in canines, to attenuate ileal glutamate release into the circulation (Weber, Jr. et al., 1981). It appears that changes in circulating glutamate concentrations may instigate glucagon and inhibit glutamate release from the gut. With the little evidence that does exist, it seems that glucagon and insulin both have effects on protein metabolism and ultimately amino acid metabolism which may result in disparate effects on different tissues. Another regulator of amino acid metabolism is dietary consumption of proteins which following absorption can result in fluctuations in plasma amino acid concentrations. A variety of studies have examined the dietary protein requirements for diverse human needs ranging from the healthy, active individual (Biolo et al., 1999; Fielding & Parkington, 2002; Millward, 1994, 1997, 1998; Reeds, 2000; Tarnopolsky, 1999; Tipton et al., 1999, 2001; Wagenmakers, 1999a) to the hypercatabolic patient (Biolo et al., 1997, 2000; Malmezat et al., 1998; Mangialardi et al., 2000; Reynolds et al., 1988). For protein synthesis to take place, all of the required amino acids must be present in the cell. There are a few amino acids, the EAA, that are required in the diet because the body does not synthesize them at a sufficient rate to meet the normal daily requirements of protein synthesis in the body (Newsholme & Leech, 1983). The EAA consist of histidine, isoleucine, leucine, valine, lysine, methionine, phenylalanine, threonine, and tryptophan; additionally, glutamine and cysteine can be conditionally essential depending on the health status of the individual. Furthermore, glutamate ingestion has also been studied and the majority of glutamate ingested is sequestered by the gut and little actually reaches the blood circulation (Fuller & Reeds, 1998; Newsholme & Leech, 1983). It is possible 14

27 that the splanchnic tissues may require glutamate to a greater extent than other tissues and thus only release glutamate under certain circumstances where changes in metabolic demands have occurred in the body. In general, the scope of the nutritional research explores the optimal ways to manipulate dietary intake of specific amino acids to increase their availability and promote protein synthesis (Millward, 1995; Rennie et al., 2002; Rennie & Tipton, 2000; Tessari et al., 1996; Wolfe, 2000). Therefore, while there are several diverse functions for various amino acids, there is a breadth of modulatory factors involved in the regulation of amino acid metabolism. More importantly, many amino acids may have potential roles in interorgan communication and regulation of oxidative metabolism that have not been explored. 2.2 The Effects of Physical Activity on Amino Acid Metabolism There are many whole body effects that occur with the onset of exercise. Regardless of the intensity or duration of the exercise challenge, increased metabolic demand will usually generate alterations in hormonal status, cardiovascular effects, as well as many other metabolic adjustments; the intensity and duration of the exercise bout normally determine the extent of these changes as well as factors such as level of training. The commencement of physical activity also affects a number of tissues in addition to skeletal muscle, including the heart, liver and gut. The responses to exercise from these tissues are designed to maintain a constant supply of amino acids to muscle to modulate energy metabolism while striving to maintain steady-state. To understand the grand scheme of amino acid interactions, skeletal muscle metabolism will be discussed in context with amino acid interplay that occurs with splanchnic tissues during exercise. 15

28 There is little known about the interorgan communication between kidneys, liver, gut, heart, and skeletal muscle. However, during exercise and rest, the gastrointestinal tract is extremely proteolytic and displays the highest protein turnover rates in the body for the mobilization of amino acids to be used as substrates in liver and muscle metabolism (Wasserman & Cherrington, 1996). The majority of amino acids released by the gut are taken up by the liver for various processes, including gluconeogenesis and ureagenesis (see Figure 2.3), and few enter the systemic circulation (Wasserman et al., 1991; Wasserman & Cherrington, 1996). The intricate relationship of the splanchnic tissues with blood circulation and skeletal muscle can be seen in the various associations with specific amino acids released by the gut and liver (Wasserman & Cherrington, 1996). Gut proteolysis releases a number of amino acids that are delivered to the liver and are ultimately released into the systemic circulation as substrates for skeletal muscle to use (Young & Ajami, 2000). For example, alanine and glutamine display strong communications and interactions between the muscle and liver while aspartate and glutamate reflect an intimate link between liver, gut and skeletal muscle (Wasserman & Cherrington, 1996). Active skeletal muscle is also the principal consumer of BCAA, and data derived from canines suggest that amino acids, such as BCAA, released from gut proteolysis are used to attenuate net muscle catabolism during and following exercise (Williams et al., 1996). Thus, amino acids are not only key to our understanding of nitrogen balance and the metabolic control of energy provision during exercise, but also important in interorgan interactions. While the distinct purpose of these interactions is unclear, it is possible that these amino acids may act as substrates or signals in tissue 16

29 Figure 2.3 Whole body amino acid metabolism during dynamic exercise. 17

30 communication for regulating and attending to the metabolic demands of the body and maintaining energy homeostasis. These interactions are further exacerbated with increased metabolic demands such as exercise. In general, amino acids and proteins contribute minimally as substrate energy sources during exercise (Wagenmakers et al., 1989). Amino acid oxidation accounts for less than ~1% of energy expenditure during short-term, intense exercise (Graham et al., 1995a), whereas during prolonged exercise amino acid oxidation accounts for ~3-6% of total ATP production (Phillips et al., 1993; Tarnopolsky et al., 1995). However, when the initial supply of carbohydrate supply is low, the amino acids can account for more than 10% of total energy expenditure via gluconeogenesis (Lemon & Mullin, 1980). Despite their insignificant role in substrate oxidation, amino acids might have an active role in intermediary metabolism in addition to the regulation of energy metabolism during exercise as well as tissue communication. Different types of exercise challenges lead to varying metabolic responses. While resistance exercise results in numerous changes in amino acid and protein metabolism, this type of exercise challenge is very different from dynamic exercise and is beyond the scope of this thesis. Therefore, the focus of this section of the literature review will be on the effects of aerobic exercise and energy demands on amino acid metabolism. 18

31 2.2.1 The Effects of Short-Term Exercise on Amino Acid Metabolism During short-term exercise (less than 1 h), muscle amino acid concentrations typically exhibit little change (Henriksson, 1991; Katz et al., 1986); however, there are a few alterations observed in certain amino acids, in muscle and flux rates, that may have regulatory roles in oxidative metabolism (Figures 2.1 and 2.2). Work conducted in the 1970 s (Ahlborg et al., 1974; Felig & Wahren, 1971; Wahren et al., 1973), has shown that alanine release exceeded all other amino acid release rates. They reported that alanine accounted for ~35-40% of all amino acids released from the exercising leg (Ahlborg et al., 1974; Felig & Wahren, 1971; Wahren et al., 1973). Although glutamate, glutamine, aspartate, and ammonia are important in metabolism, they were not measured. Furthermore, while the work of Wahren and coworkers was innovative for that research era, an incomplete picture was portrayed without these amino acid and ammonia measurements and without muscle biopsies. However, they did demonstrate some of the finest early examples of interorgan communication during exercise. They suggested that while alanine was released by the exercising muscle, it was extracted by splanchnic metabolism, for gluconeogenesis, to a greater extent that any other amino acid measured. In more current research, muscle glutamate has been shown to decrease concomitantly with an increase in muscle alanine during short-term exercise (Bergström et al., 1985; Dawson et al., 2003; Gibala et al., 1997a; Henriksson, 1991; Katz et al., 1986; Rennie & Tipton, 2000; Sahlin et al., 1990). In addition to these alterations, there is an increase muscle glutamine (Henriksson, 1991; Katz et al., 1986), aspartate (Katz et al., 1986) and ammonia (Henriksson, 1991; Katz et al., 1986) with short-term exercise. These changes 19

32 occur to a greater extent than can be attributed to protein degradation (Bergström et al., 1985; Henriksson, 1991; Katz et al., 1986; Sahlin et al., 1990). Furthermore, the decrease in glutamate occurs, to the same degree, in all fiber types (Essén-Gustavsson & Blomstrand, 2002), suggesting that the role of glutamate may not be specific to aerobic or anaerobic activity. Intramuscular glutamate concentrations may be more sensitive than alanine to muscle contraction since low exercise intensity (25%VO 2max ) can result in a fall of about ~20% in glutamate without affecting alanine (Gibala, 2001; Sahlin et al., 1995). During exercise of more than 50% VO 2max, the decrease in muscle glutamate concentrations is reciprocal to the increase in alanine; however, the relative drop in glutamate is usually greater than the increment in muscle alanine concentrations (Henriksson, 1991; Sahlin et al., 1990). Furthermore, exercise intensity seems to parallel the magnitude of change; thus, the more intense the exercise bout, the greater the drop in muscle glutamate and increase in muscle alanine (Sahlin et al., 1995). Within the initial period of moderately intense, dynamic exercise, there is an increased uptake of glutamate (1-3 fold) and increased release of alanine and glutamine (Gibala et al., 1997a; MacLean et al., 1994; van Hall et al., 1995). What is particularly interesting, is that modifications in muscle glutamate and alanine concentrations can occur within the first few minutes of moderately, intense exercise (Gibala et al., 1997a, 2002b). These intramuscular changes occur concomitantly with increased glutamate uptake into muscle and augmented release rates of glutamine, alanine and ammonia from muscle within the 20

33 first minute of exercise (Gibala et al., 1997a) which ultimately result in elevated plasma ammonia, alanine and glutamine concentrations (Henriksson, 1991; Katz et al., 1986; Wagenmakers et al., 1990). Although it appears that several alterations occur in the free amino acid pool in early exercise, the functional significance of these fluctuations remains to be elucidated Prolonged Exercise Effects on Amino Acid Dynamics Amino acid dynamics has been studied for prolonged exercise durations that range from 1 to 4 hours. While there are limited studies of amino acids during prolonged exercise in humans, there are some interesting findings from these studies. It is also important to acknowledge that few studies have measured entire amino acid profiles in blood/plasma and especially in muscle. The proposed objectives and protocols of these studies are variable (summarized in Table 2.1) but they do share the common interest in understanding the potential role of certain amino acids in nitrogen balance with prolonged exercise as well as their putative effects on fatigue and interorgan interactions. In general, the decrease in muscle glutamate that is observed in early exercise is maintained throughout the duration of exercise bouts that last up to 3 hours (Gibala et al., 2002a; MacLean et al., 1991, 1996; Sahlin et al., 1990). Henriksson et al (1991) observed that muscle amino acids were stable throughout exercise with the exception of alanine. Alanine has been noted to gradually decline in muscle between 60 and 90 minutes of steady-state exercise whereby muscle glutamate is stable during this period (Henriksson, 1991; Sahlin et al., 1990). The carbon skeletons of glutamate, aspartate, BCAA and 21

34 Table 2.1 Summary of prolonged exercise (>60min) studies examining amino acid metabolism in humans. 22 Publication Ahlborg et al. (1974) J.Clin. Invest. 53:1080. Rennie et al. (1981) Clin. Sci. 61:627. Sahlin et al. (1990) Am.J.Physiol. Cell Physiol. 259:C834. Wagenmakers et al. (1990) Int.J.Sports Med. 11:S101. Exercise Type, Duration & Intensity Type: cycling Duration: 4 h Intensity: 30% VO 2max Type: treadmill Duration: Trial 1: 3h 45min, n=4; Trial 2: 4h, n=2 and 2h 15min, n=2 Intensity: 50% VO 2max -one of the earliest studies of stable isotopes in protein turnover in humans Type: cycling Duration: 1 h 15 min Intensity: 75% VO 2max Type: cycling Intensity: Incremental to max Key Points -arterial ala rose 40% above rest after 40 min of exercise & all other aa didn t change - in arterial ala from 40min to 4h & leu, ile, tyr, & phe slightly -net output of ala at 40 min & 3x rest levels at 4h -net BCAA uptake by leg at 4 h -ala splanchnic uptake rose by 20% at 40 min compared to rest & by 50% at 4 h -splanchnic BCAA output at 4h -amino acid catabolism -whole-body protein synthesis while whole-body protein breakdown -exercise fractional rate of myofibrillar protein breakdown in contrast with whole-body breakdown -catabolism of BCAA may regulate changes in whole-body protein turnover via changes in wholebody free BCAA pools -continuous muscle gln -muscle glu rapidly in 1 st 5 min & remained low until fatigue; -muscle ala & was partially reverted at fatigue -NH 3, ala, & gln exchange almost equal -McArdle s patients have larger uptake of BCAA & a more rapid activation of BCOADH than healthy - NH 3 & gln release greater in McArdle s vs healthy Limitations -no muscle biopsies -glu, gln, & NH 3 not measured -4 time points examined -n=4 -subjects studied 2 occasions separated by several months -wide fitness range of subjects -assumptions made without concrete validations -only 3 TCAIs measured -few time points examined -only 2 McArdle s patients -healthy vs patient

35 23 Wagenmakers et al. (1991) Am.J.Physiol. (Endocrinol. Metab.) 260: E883. Henriksson et al. (1991) J.Exp.Biol. 160:149. MacLean et al. (1991) JAP 70:2095. Type: cycling Duration: 2h Intensity: 50-75% W max Duration: 3.5 h Type: cycling Duration: to exhaustion = 1h 42min (Mixed diet) & 2h 25min (high CHO diet) -CHO loading eliminated BCAA oxidation -NH 3 production from deamination of aa -BCAA aminotransferase may drain TCA cycle -plasma glu & ala greater with CHO loading -BCAA at rest with CHO loading -depletion of plasma aa pool -except ala, muscle free aa pool was stable -activation of BCAA metabolism in trained vs untrained - plasma glutamine -no diet effect - muscle total & EAA at exhaustion -no change in muscle or plasma BCAA -BCAA for energy & NH 3 production exercise not very comparable -intensities were varied between control & high CHO tests -no muscle aa -no TCAIs -unpublished results -intensity & type of exercise not provided -no flux measurements -mixed diet not controlled Spencer et al. (1991) Am.J.Physiol.(Cell Physiol.) 30:C71. MacLean et al. (1994) Am.J.Physiol. (Endocrinol. Metab.) 30:E1010. Intensity: 75% VO 2max Type: cycling Duration: to EXH Intensity: 70% VO 2max Type: knee-extensor Duration: 1h Intensity: 71% W max -muscle ala more with CHO -muscle glu to same extent as control -muscle asp to same extent as control -BCAA in blood & muscle with BCAA supplementation -release of EAA during BCAA trial independent of BCAA exchange - NH 3 production greater with BCAA -only 3 aa measured -muscle biopsies only at rest & exercise of CON vs CHO -no plasma aa nor flux -few muscle biopsies taken 23

36 Blomstrand et al. (1995) Acta Physiol. Scand.153(2):87. Graham et al. (1995) JAP 78:725. Type: cycling Duration: to EXH Intensity: 75% VO 2max n=5 3 trials: water/cho only/bcaa+cho supplementation Type: knee-extensor Duration: 3 h Intensity: 60% W max supplementation & suppressed endogenous muscle protein breakdown - plasma gln with CHO+BCAA than CHO or water -no change in muscle gln - muscle glu in all 3 conditions -no changes in aromatic aa thus, BCAA + CHO supplementation doesn t influence net protein degradation -examined trained & untrained subjects -muscle glu & asp -muscle total aa pool remained despite efflux -large glu uptake + efflux of gln, ala, EAA & NH 3 -muscle glu in 10 min & quantitatively equal to NH 3 production but NH 3 continued to be released -muscle gln, ala, & BCAA did not change much -no flux data -only n=5 therefore the lack of significance may be due to small sample size -few sample points -no NH 3 measured -measurements limited to aa s & NH 3 24 van Hall et al. (1995) J.Physiol. 489(Pt1):251. Blomstrand et al. (1996) Nutrition 12(7-8):553. MacLean et al. (1996) J.Physiol. 493:909. Type: knee-extensor Duration: 1.5 h Intensity: 60-65% W max -exhaustive submax exercise -BCAA vs placebo Type: knee-extensor Duration: 1.5 h Intensity: 64% W max -smaller in muscle glu with BCAA supplementation -no change in other aa -possible glycogen sparing effect with BCAA supplementation - plasma & muscle alan with BCAA ingestion -plasma arg in plasma with BCAA ingestion -constant uptake & arterial levels of glu while in muscle glu -BCAA supplementation resulted in NH 3 & BCAA release from muscle + muscle NH 3, ala, and gln & -few time points measured -only other metabolites measured were adenine nucleotides & glycogen -no flux data -few data points during exercise - no NH 3 measurements -pulmonary VO 2 not measured for comparison -no TCAIs 24

37 25 Jackman, M.L. et al (1997) Am.J.Physiol. (Endocrinol. Metab.) 35:E233. Blomstrand & Saltin (1999) J.Physiol. 514: 293. Blomstrand & Saltin (2001) Am.J.Physiol. (Endocrinol. Metab.)281:E365. (same control used in both) van Hall et al. (1999) Clin.Sci. 97:557. Type: cycling Duration: to fatigue Intensity: 75% VO 2max -low muscle glycogen(low)/low + BCAA/high muscle glycogen (CON) -biopsies: rest, 15 min, EXH for LOW trial 49min Type: knee-extensor Duration: 1 h Intensity: 75% VO 2max -compared low & normal glycogen content Type: knee-extensor Duration: 1.5 h Intensity: 60-65% W max no effect on glu uptake -BCOAD activity was not different at rest -higher with LOW & LOW+BCAA than CON -BCOAD activity not dependent on glycogen -muscle glu & ala not affected by glycogen or BCAA -plasma NH 3 more with LOW & LOW+BCAA than CON -less NH 3 produced with low glycogen leg yet total N lost over exercise was less with normal glycogen leg -more ala & gln lost with low glycogen leg -glu uptake constant between low & normal glycogen content -no difference in muscle glu, ala, BCAA during ex or recovery in either low & normal glycogen leg -muscle gln lower in low glycogen leg & NH 3 produced may have been incorporated into glu & released as gln -in a second study, BCAA supplementation showed a protein-sparing effect -total aa production was 1.5-fold greater with lowglycogen vs normal-glycogen - net muscle protein degradation -glu, BCAA, asp, & asn from protein + glu & BCAA from uptake used for gln synthesis and TCAIs measured -few muscle aa measured -no plasma aa -only one exercise measurement; focus on recovery -no TCAIs measured -tracer not used to decipher which carbons are transferred to gln McKenzie et al. Type: cycling -leucine turnover from rest in untrained subjects -no flux data 25

38 26 (2000) Am.J.Physiol. Endo.Metabol. 278: E580. Essén-Gustavsson & Blomstrand (2002) Acta Physiol. Scand. 174:275. Gibala et al. (2002) J.Physiol. 545: 705. Duration: 1.5 h Intensity: 60% VO 2max -n=5-60 min submax + 20 min near max -fibre typing Type: knee-extensor Duration: 1.5 h Intensity: 70% W max - leu flux with exercise & in nonoxidative leu disposal with exercise -muscle aa were same between type I & II fibres except glu, asp, & arg which were 10% greater in the type II fibres -muscle glu in both type I & II fibres -no effect on tau, ala, BCAA or gln between fibres - ala release & muscle ala until 30 min exercise & subsequently -muscle glu as early as 5 min & remained at same concentration - muscle pyruvate similar to ala but no change in 2- oxoglutarate -limited muscle biopsies during exercise -only leu turnover measured -max workload not controlled -no other amino acids measured -NH 3 not measured Abbreviations: glu = glutamate; gln = glutamine; ala = alanine; pro = proline; asn = asparagine; arg = arginine; asp = aspartate; leu = leucine; tau = taurine; aa = amino acids; EAA = essential amino acids; BCAA = branched-chain amino acids; BCOA = branched-chain oxo-acids; BCOADH = branched-chain oxo-acid dehydrogenase; TCA cycle = tricarboxylic acid cycle; TCAI = tricarboxylic acid cycle intermediates; ex = exercise. 26

39 other amino acids that are partially metabolized in skeletal muscle are probably not used for the synthesis of muscle alanine but are primarily converted into TCA cycle intermediates (van Hall et al., 1999b). In contrast to Wahren s suggestion that alanine is the most important gluconeogenic precursor (Ahlborg et al., 1974; Wahren et al., 1973), it is now thought that the transport of the carbon skeleton of glutamine from muscle may be more important than alanine for gluconeogenesis in the liver and kidney (Sahlin et al., 1990; van Hall et al., 1999b). While BCAA generally are maintained at constant levels in the muscle (Graham et al., 1995b; Henriksson, 1991; MacLean et al., 1991), there appears to be a decrease in muscle aspartate past 1 hour of exercise (MacLean et al., 1991). The biochemical interrelationship and homologous structure of aspartate and glutamate (Young & Ajami, 2000), may suggest that aspartate is important in maintaining glutamate concentrations in muscle during the later phase of exercise. Furthermore, there appears to be an increase by about 20% in non-essential amino acids in muscle during prolonged exercise which may be attributed to an increase in net protein degradation (MacLean et al., 1991). Circulating amino acids and their fluxes into and out of skeletal muscle are particularly interesting and provide a more dynamic schema of amino acid metabolism during exercise (Figures 2.1 and 2.2). Some of the classic work conducted by Wahren and his colleagues in the early 1970 s (Ahlborg et al., 1974; Wahren et al., 1973), focused on the interactions of amino acids between the splanchnic region and skeletal muscle. They proposed that hepatic glycogenolysis can only be maintained for a limited time; 27

40 subsequently, glucose homeostasis will more heavily rely on gluconeogenesis and/or diminish glucose uptake by the muscle. Their subjects exercised for 4 hours at 30% VO 2max whereby hepatic and femoral arterio-venous catheters were used to examine the flux of substrates between the exercising muscle and the splanchnic region (Ahlborg et al., 1974;Wahren et al., 1973). They found that up to 40 minutes of exercise, there was a 40% increase in arterial alanine from resting concentrations while there were no significant changes in other amino acids (Ahlborg et al., 1974;Felig & Wahren, 1971;Wahren et al., 1973). However, between 40 minutes and 4 hours, they observed a decrease in alanine and a slight increase in leucine, isoleucine, tyrosine and phenylalanine concentrations (Ahlborg et al., 1974;Wahren et al., 1973). Interestingly, net flux of alanine from the exercising muscle rose slightly from rest to 40 minutes but efflux was 3 times the resting flux rate by 4 hours of exercise. Furthermore, splanchnic uptake of alanine increased by 50% from rest to 4 hours. Enhanced net uptake of BCAA into muscle was also measured at 4 hours which was attributed to an elevated BCAA release from splanchnic tissues by the end of exercise. Wahren s investigations generated further questions about the gluconeogenic role of amino acids in exercise physiology. While there were few time points (~4 points) measured, there were also certain important measurements missing from Wahren s studies: glutamate, aspartate, glutamine and ammonia measurements as well as muscle biopsies. The drop in arterial alanine and increased release of alanine from muscle was confirmed by several other studies that followed (Gibala et al., 2002a; Graham et al., 1995b; Rennie et al., 1981; van Hall et al., 1999b). It has been suggested that alanine is 28

41 produced in amounts that are equal to its relative occurrence in proteins (van Hall et al., 1999b), while glutamine is released in much larger amounts which further supported the idea that glutamine is a more important precursor for gluconeogenesis (Graham et al., 1995b; van Hall et al., 1999b). There are some researchers that have found steady plasma concentrations of amino acid up to 90 minutes and subsequently BCAA concentrations tend to decrease following 90 minutes (MacLean et al., 1996; Rennie et al., 1981; Wagenmakers et al., 1990, 1991), which is in agreement with Wahren s work. A net depletion of the plasma amino acid pool has also been attributed to increased consumption of amino acids by different organs for protein synthesis (Blomstrand & Saltin, 1999; Henriksson, 1991; Rennie et al., 1981). Rennie et al (1981) found that with the use of isotopes, protein degradation resulted from whole-body protein degradation and not from skeletal muscle and these changes were attributed to enhanced BCAA metabolism. However, they only studied 4 subjects and there were various limitations to this study as outlined in Table 2.1. In contrast, a net efflux of EAA has been reported during 3 hours of knee-extensor exercise suggesting net protein degradation within muscle (Graham et al., 1995b). BCAA and glutamate are both readily taken up by the muscle despite their relatively high concentrations in muscle and net increased availability from intramuscular protein degradation (Graham et al., 1995b; MacLean et al., 1996; van Hall et al., 1999b). Since glutamate and BCAA are the only amino acids that are taken up in significant quantities, they may contribute to nitrogen balance (Figure 2.1). However, glutamate, glutamine, alanine and BCAA are also 29

42 intricately associated through various transamination reactions with the TCA cycle and may be key to the regulation of oxidative metabolism. Wahren and his colleagues emphasized the significance of amino acid metabolism in both the muscle and splanchnic tissues during exercise and demonstrated important findings in interorgan communication. New techniques have allowed us to attain more information about amino acids and their dynamic metabolism. It is remarkable that skeletal muscle can respond to such prolonged exercise challenges and still display minimal changes in the muscle free amino acid pool despite continual protein turnover as well constant uptake and release of particular amino acids. The small changes in muscle amino acids indicate the tight regulatory response of amino acid metabolism which suggests that these compounds are important to modulatory mechanisms in metabolism. While it is clear that there is a distinct and intimate relationship between glutamate, glutamine, alanine and BCAA in the muscle (Figure 2.2) and in other organs (Figure 2.3), their roles and specific functions are ambiguous Training Effects on Amino Acid Metabolism Endurance training protocols are used to assess metabolic responses to enhanced oxidative capacity. Thus, metabolic processes interlinked with oxidative metabolism exhibit adaptive responses to training which would ultimately provide an approach to better understanding the relationship between a process of interest and oxidative metabolism. Studies examining the effects of training on amino acid metabolism in humans are incredibly limited (studies summarized in Table 2.2). Hence, the review of 30

43 Table 2.2 Summary of studies examining the effects of training on amino acid metabolism in humans. 31 Publication Henriksson et al. (1991) J.Exp.Biol. 160:149. Graham et al. (1995) JAP 78:725. McKenzie et al. (2000) Am.J.Physiol. Endo.Metabol. 278: E580. Dawson et al. (2003) JAP 95:999. Training Type & Duration -trained vs untrained male subjects -compared at rest -trained vs untrained male subjects -3 h knee-extensor exercise (60% W max ) -Test: 1.5 h cycling at 60%VO 2max -Training: 38d 60 min/d for 5 60% VO 2peak with intervals 1d/wk -5 days aerobic training in females -70% VO 2peak for 45 min per bout of cycling Key Points - BCAA metabolism with prolonged exercise in trained subjects -higher basal amino acid concentrations in trained subjects but differences were small between untrained and trained subjects -muscle amino acids were in 8 amino acids; significant in glu & tau -lower muscle glu, tau, & ala at rest & exercise in untrained -plasma glu, ala, gly, & pro in trained -training leu oxidation & BCOADH activation - in muscle glu attenuated by 40% - in ala attenuated by 25% Limitations -cross-sectional study -unpublished observations -only 11 amino acids measured -cross-sectional study -limited muscle biopsies during exercise -only leu turnover measured -females studied & be difficult to compare results to males -short training program Abbreviations: glu = glutamate; ala = alanine; gly = glycine; pro = proline; leu = leucine; tau = taurine; aa = amino acids; EAA = essential amino acids; BCAA = branched-chain amino acids; BCOADH = branched-chain oxo-acid dehydrogenase 31

44 training effects on amino acid metabolism will also extend to work that has been carried out with animal models as well. In general, aerobic training is anticipated to result in enhanced oxidative capacity resulting from improved metabolic and cardiovascular parameters such as increased mitochondrial density, enhanced capillarization, and efficient use of carbohydrates along with increased fat use. If amino acids have a role in oxidative metabolism to regulate energy supply and attain homeostasis, training should also induce an adaptive response to amino acid metabolism. Overall, Henriksson et al (1991) reported a significant activation of BCAA metabolism with prolonged exercise that was more pronounced in trained subjects. In a different cross-sectional study by Graham et al (1995b), a complete amino acid profile demonstrated the differences in amino acids in trained and untrained subjects during 3 hours of knee-extensor exercise. They found that the untrained muscle was lower in intramuscular glutamate, taurine and alanine concentrations at rest and during exercise as compared to the trained muscle. While they did not find any differences in muscle glutamine concentrations nor in amino acid flux between trained and untrained subjects, the trained subjects did exhibit decreases in arterial glutamate, alanine, glycine and proline. Both untrained and trained subjects demonstrated efflux of EAA and had constant intramuscular concentrations of all amino acids measured. Furthermore, they all experienced large releases of ammonia, glutamine and alanine. More recently, Dawson et al (2003) also reported an attenuated drop in muscle glutamate concentration following 5 consecutive days of endurance training in female subjects. 32

45 They found no changes in muscle alanine between pre- and post-training during endurance exercise. In contrast, data from Hood and Terjung (1994) demonstrated an increase in alanine production in trained rats. These results are supported by the increased alanine aminotransferase protein following training in rats (Mole et al., 1973) and horses (Guy & Snow, 1977). Other enzymes important to amino acid metabolism that have been examined following training include glutamate dehydrogenase (Wibom & Hultman, 1990) and branched-chain oxo-acid dehydrogenase (McKenzie et al., 2000) enzymes which were both elevated with training. Although these enzymes are enhanced following training, they may not be maximally active during exercise since the data on BCAA and glutamate metabolism following training suggests that glutamate and BCAA are used to a lesser extent. Although there are very few training studies of humans that examine amino acid metabolism, it is evident that there are various discrepancies. Some of these discrepancies may stem from the different exercise protocols and the training duration. For example, Graham et al (1995b) examined subjects who had been training for years and the protocol employed the knee-extensor ergometer which elicits few systemic effects. In contrast, Dawson et al (2003) examined female subjects that underwent a 5- day, aerobic training protocol on a cycling ergometer. As the metabolism of amino acids such as glutamate, alanine, glutamine, and BCAA was altered with training, future investigations are necessary to further evaluate their potential role oxidative metabolism. 33

46 2.2.4 Ammonia Production in Relation to Amino Acid Metabolism Nitrogen balance rarely undergoes alterations without changes in amino acid metabolism. At rest, there is a small amount of ammonia taken up by muscle (Graham, 1994), whereas during exercise there is a sharp increase in ammonia produced in the muscle and released into the circulation (Graham et al., 1990, 1994; MacLean et al., 1994; Rennie, 1996; Wagenmakers et al., 1990). With training, at the same absolute intensity there is more ammonia generated in the untrained state versus the trained state; however, at the same relative intensity there are no differences in ammonia production with or without training (Graham et al., 1995b; Lo & Dudley, 1987). The source of ammonia generated during exercise has been frequently debated. Ammonia can be produced from a myriad of reactions including glutamate dehydrogenase, the purine nucleotide cycle, protein breakdown, and/or other amino acid deamination reactions (Figure 2.2). Amino groups from amino acids can also be removed via deamination before they are oxidized or be transferred to another metabolite by transamination. Glutamate and 2-oxoglutarate are the central amino acid and oxoacid, respectively, that are involved in all transamination reactions. During moderate intensity and steady-state exercise, it is more likely that ammonia is derived from BCAA deamination (MacLean et al., 1996; Rennie, 1996). This is supported by work from MacLean et al (1994, 1996) that showed BCAA ingestion followed by prolonged, steady-state exercise resulted in increased ammonia release and elevated arterial ammonia concentrations compared to control while adenine nucleotides 34

47 were constant during exercise. In addition, Wagenmakers et al (1990) have shown that although patients with McArdle s disease exhibit large increases in plasma ammonia concentrations, these are further exacerbated with BCAA ingestion. Furthermore, alanine and glutamine release have also been elevated following BCAA ingestion (MacLean et al., 1994, 1996). Since glutamate interacts with several amino acids; the decrease in muscle glutamate concentrations may in part account for the ammonia production rate (Figure 2.2). However, this decrease in glutamate as well as its continual uptake from circulation cannot account for all amino groups released (ammonia, glutamine and alanine) as shown in Figure 2.1. Thus, it has been suggested that BCAA may account for the release of some of these amino groups (MacLean et al., 1996; Rennie, 1996). While the complex relationships between these amino acids have emerged in the last decade, their role in metabolism largely remains unclear The Relationship of Amino Acids with the TCA cycle During Exercise There are a number of transamination and transdeamination reactions that couple several amino acids with the TCA cycle intermediates (Figure 2.2). As such, it is possible that particular amino acids, such as glutamate, may play an integral role in oxidative metabolism through the TCA cycle. The TCA cycle is a series of reactions that oxidize acetyl-coa, predominantly produced from fat and carbohydrate oxidation, to generate reducing equivalents (ie. NADH and FADH 2 ) that can be used to produce ATP through oxidative phosphorylation via the electron transport chain. Thus, the TCA cycle provides 35

48 an important platform for this large production of energy generated by a blending of various fuels. Several studies have now shown that with the onset of muscle contraction, there is a rapid anaplerotic phenomenon that occurs whereby the TCA cycle intermediates (TCAI) increase several fold within the first few minutes of exercise (Aragón & Lowenstein, 1980; Bruce et al., 2001; Dawson et al., 2003; Gibala et al., 1997b, 1997a, 1999, 2002a; Sahlin et al., 1990, 1995; Spencer et al., 1991). Malate has consistently shown the largest absolute changes, accounting for ~50% of the net increase of the TCAI pool (Gibala et al., 1997b, 1997a, 1998, 1999; Sahlin et al., 1990). The first of the intermediates to begin to decrease, in early exercise, is 2-oxoglutarate (Gibala et al., 1997a) while the rest of the intermediates gradually decrease during prolonged exercise (Gibala et al., 1997b, 2002a; Spencer et al., 1991). The sum of the TCAI pool has been shown to begin declining by 10 minutes into moderately intense exercise (Gibala et al., 2002a). When exhaustion is reached in prolonged exercise, oxaloacetate and 2- oxoglutarate decline to concentrations that are similar to resting levels (Gibala et al., 1997b). Furthermore, with endurance training, there is an attenuated increase in TCAI pool (Dawson et al., 2003). In spite of the extensive work conducted to identify the changes that occur in the TCA cycle during exercise, little is known about the functional significance of these fluctuations in the TCAI pool. It was originally thought that the expansion of the TCAI pool stemmed from the purine nucleotide cycle since one of its products is fumarate (Aragón & Lowenstein, 1980). 36

49 However, the cycling of the purine nucleotide reactions during exercise is questionable (Meyer & Terjung, 1980; Rennie, 1996; Sahlin & Broberg, 1990). Williamson and Cooper (1980) suggested that since different NAD + -dependent dehydrogenases are regulated in accordance to their sensitivity by fluctuations in the NAD + /NADH ratio, then the redox potential of NAD + for a particular reaction depends on the concentration and availability of substrate in the mitochondria. Unfortunately, it has not been possible to make the vital determinations of mitochondrial redox state in vivo. In particular, there are 3 non-equilibrium reactions (excluding pyruvate dehydrogenase) in the TCA cycle: citrate synthase, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase. Isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase are NAD + - dependent, and thus, would limit flux through the TCA cycle. Therefore, the more substrate and product available for these reactions, the greater the flux through the TCA cycle. Several other labs also support this idea that the generation of energy equivalents is dependent on anaplerosis of the TCA cycle (Sahlin et al., 1990, 1995; Wagenmakers, 1998a). However, if anaplerosis is required to drive the TCA cycle flux, then oxygen consumption could also alter in parallel to changes in the TCAI pool. Yet, when the sum of the TCAIs decreased with prolonged exercise or when the TCAI pool was manipulated, mitochondrial respiration was not compromised (Bruce et al., 2001; Constantin-Teodosiu et al., 1999; Dawson et al., 2003; Gibala et al., 2002a, 2002b; Gibala & Saltin, 2000). 37

50 Another theory has been put forth that associates the decrease in the TCAI pool during prolonged exercise with fatigue. Sahlin et al (1990) observed that when the total TCAI that they measured (malate, citrate, and fumarate) decreased, they proposed that this decrease in the TCAI pool was associated with fatigue. They further examined this idea in another study where carbohydrate supplementation further increased the TCAI pool and was linked to a delay in fatigue during prolonged exercise (Spencer et al., 1991). However, Gibala et al (1997b, 2002a) have shown that, during prolonged exercise, phosphocreatine was relatively stable and oxygen uptake remained elevated despite the decrease in TCAIs. Furthermore, the TCAI pool has been shown to begin decreasing after 10 minutes of exercise at which point the subjects were not fatigued (Gibala et al., 2002a). Wagenmakers (1990,1998a) proposed that the decrease in 2-oxoglutarate may be associated with fatigue particularly because it decreases to almost resting levels during prolonged exercise. He suggests that McArdle s patients had difficulty regenerating 2- oxoglutarate which could result in a lower carbon flux through the TCA cycle and thereby impede energy production (Wagenmakers et al., 1990). These patients also produce a greater amount of ammonia in muscle which has been shown to inhibit the activity of 2-oxoglutarate dehydrogenase in muscle (Wagenmakers et al., 1990) and other tissues (Lai & Cooper, 1986). Yet, in a study by Gibala and his colleagues there was no significant change in 2-oxoglutarate concentrations during prolonged exercise (Gibala et al., 2002a). Wagenmakers (1991, 1998a) has also discussed the possibility that the increased activity of the branched-chain oxo-acid dehydrogenase enzyme may cause a 38

51 potential drain on the TCA cycle carbon availability. However, Gibala et al (1999) have shown that BCAA ingestion does not affect TCA cycle anaplerosis. An alternative hypothesis has been put forth proposing that the expansion of the TCAI pool occurs as a result of a mismatch between pyruvate production and oxidation, whereby it is the result of a change in metabolism and not the regulator of it. Extensive work has been done, particularly in the last decade, that attributes the initial rapid expansion of the TCAI pool to the rightward shift of the near-equilibrium reaction that is catalyzed by alanine aminotransferase (pyruvate + glutamate alanine + 2- oxoglutarate) (Gibala et al., 1997a,1997b; Sahlin et al., 1990, 1995). With the onset of exercise, pyruvate production is elevated with increased glycolytic flux which exceeds the oxidation rate of pyruvate through the TCA cycle (Gibala, 2001). It is important to note that while these changes are dramatic, this mismatch is relatively small when considering that from the pyruvate generated ~1-2% results in lactate and less than that is utilized in the alanine aminotransferase reaction. This would result in a rapid influx of carbon which would be swiftly converted to succinate, fumarate and malate resulting in anaplerosis. Other reactions have enzyme capacities that are too low to account for such a rapid increase in TCAI (Gibala et al., 1997a; Newsholme & Leech, 1983). However, near-equilibrium reactions respond to change in substrate and product concentrations and may be important in providing the initial flux of carbon and then fine-tuning the levels of the TCAI pool. 39

52 The rapid expansion of the TCAI pool as well as oxidative energy provision was not impeded by low, pre-exercise muscle glycogen concentrations (Gibala et al., 1999, 2002b). It was anticipated that low glycogen concentration would impair anaplerosis; rather, the TCAI pool was expanded to a similar extent as with normal glycogen content suggesting that pyruvate flux was adequate to provide carbon through the alanine aminotransferase reaction for the TCA cycle (Gibala et al., 1999). Furthermore, Sahlin et al (1995) observed only a 2-fold increase in TCAI in patients with McArdle s disease in addition to a relatively small increase in alanine as compared to the healthy subjects. Sahlin and his colleagues (1995) suggest that anaplerosis McArdle s patients was unlikely to occur through the alanine aminotransferase reaction because of the decreased pyruvate production. Instead, anaplerosis was likely generated from glutamate dehydrogenase or AMP deaminase since a greater release in ammonia was observed (Sahlin et al., 1995). However, in healthy subjects the alanine aminotransferase reaction appeared to be important to increasing the TCAI pool. Several other studies have attempted to manipulate TCAI concentrations by altering pyruvate availability via pyruvate infusion, dichloroacetate administration, as well as epinephrine infusion, and have resulted in manipulating anaplerosis without affecting aerobic energy provision (Constantin-Teodosiu et al., 1999; Gibala & Saltin, 2000). Overall, TCA cycle anaplerosis and its functional significance remains to be elucidated. To better understand TCA cycle anaplerosis through the alanine aminotransferase reaction, the role of particular amino acids should be more critically examined. There are a number of amino acids that participate in the ancillary reactions that are coupled with 40

53 the TCA cycle that may be important to anaplerosis. Glutamate is central to the transamination and deamination reactions that link alanine, aspartate, BCAA and glutamine into the TCA cycle (Figure 2.1). Interestingly, glutamate, alanine, glutamine and BCAA demonstrated consistent changes in concentrations and fluxes in different tissues during exercise. A few studies have examined these interrelationships between amino acids and the TCA cycle via dietary manipulation. BCAA ingestion was one form of dietary manipulation used to examine BCAA interactions with the TCA cycle and it was found that BCAA ingestion did not affect the total TCAI pool (Gibala et al., 1999). Despite that the BCAA indirectly interact with TCAIs at various points of the TCA cycle (Figure 2.2), BCAA ingestion did not affect the TCA cycle anaplerosis. Glutamine supplementation has also been used to manipulate the TCAI pool (Bruce et al., 2001). While glutamine supplementation did increase intramuscular glutamine and tended to increase succinate, fumarate and malate, there was no effect on oxidative metabolism (Bruce et al., 2001). Under control conditions, it has consistently been shown that intramuscular glutamate decreases and alanine increases in early exercise (Gibala et al., 1997a, 1998, 2002a). Glutamate and pyruvate transaminate via the alanine aminotransferase reaction to form alanine, which is released by the muscle, and 2-oxoglutarate which enters the TCA cycle. Furthermore, glutamate was shown to decrease early in exercise along with 2- oxoglutarate (Gibala et al., 1997a). Therefore, the key focus may be in critically examining the changes in amino acids and how they affect the TCA cycle pool through 41

54 their corresponding, near-equilibrium aminotransferse reactions. Since glutamate is central to all transamination reactions, glutamate may be key to energy metabolism and homeostasis. 2.3 Glutamate Metabolism Glutamate is one of the most abundant free amino acids in liver, kidney, skeletal muscle and brain (Brosnan, 2000). The molecular structure of glutamate has several reactive characteristics that make glutamate favourable for diverse roles in facilitating metabolic processes (Young & Ajami, 2000). Glutamate is capable of releasing its amino group to a greater extent than other amino acids and it is one of the few amino acids with which other amino acids will exchange amino groups (Young & Ajami, 2000). The ã-glutamyl transamination properties of glutamine and glutamate allow them to traffick nitrogen within tissues as well as transfer nitrogen between tissues, predominantly from muscle (Young & Ajami, 2000). These characteristics of glutamate may explain its abundance in nature and may potentially explain its essential, central role in transamination and transdeamination reactions. There has been extensive research that has been conducted on glutamate metabolism in several different tissues. While bearing in mind that all tissues serve different purposes and even different compartments within tissues may have metabolites with diverse roles, there is a lot to be gained from this research towards unraveling glutamate s role in skeletal muscle and in interorgan communications. 42

55 2.3.1 Glutamate and Insulin To attain a better understanding of the potential roles of glutamate, its regulation for movement across different tissues should be discussed. It appears that glutamate is associated with insulin (Aoki et al., 1972; Graham et al., 2000; Maechler et al., 2002; Molnár et al., 1995; Mourtzakis & Graham, 2002; Thomassen et al., 1991). While it has been shown that elevated insulin levels can increase glutamate uptake into the muscle (Aoki et al., 1972), glutamate also appears to affect circulating insulin levels (Graham et al., 2000; Maechler et al., 2002; Maechler & Wollheim, 1999; Molnár et al., 1995; Mourtzakis & Graham, 2002; Rorsman & Renström, 1999; Thomassen et al., 1991; Weaver et al., 1998). Following glutamate ingestion in humans, an insulin rise has been demonstrated (Graham et al., 2000;Mourtzakis & Graham, 2002;Thomassen et al., 1991), which likely resulted from increased insulin secretion as there was a similar rise in C- peptide levels (Mourtzakis & Graham, 2002). Furthermore, Bertrand et al (1995) showed that glutamate stimulated insulin secretion in rats in vivo via an excitatory receptor which improved glucose tolerance. Other studies have also shown that there is a potential role for glutamate in insulin regulation through the glutamate dehydrogenase reaction whereby mutations in this enzyme have resulted in hyperinsulinemia and hyperammonemia in infants (Fang et al., 2002). Glutamate receptors have been identified in pancreatic â-cells suggesting that glutamate may have a putative role in stimulating insulin secretion (Maechler et al., 2002; Molnár et al., 1995; Weaver et al., 1998). Moreover, glutamate appears to act as an intracellular messenger that couples glucose metabolism to insulin secretion through 43

56 calcium activation (Maechler & Wollheim, 1999; Rorsman & Renström, 1999). Glutamate does not actually initiate the secretion of insulin; rather, a rise in mitochondrial calcium is necessary to initiate the response and glutamate is proposed to act as a second messenger in insulin exocytosis (Maechler & Wollheim, 1999; Rorsman & Renström, 1999). Therefore, it is also possible that in skeletal muscle, glutamate may act as a second messenger for metabolic processes, such as those involved in exercise metabolism, that are associated with the entry of calcium into the sarcolemma. The relationship between glutamate and calcium is being investigated to a greater extent in the last few years (Frank et al., 2002; Lerea, 1997; Yamada et al., 2001). Activation of glutamate receptors has been shown to result in an initial increase in intracellular Ca 2+ (Lerea, 1997). Moreover, using a C2C12 cell-line of myotubes, glutamate-induced increase in Ca 2+ concentrations seems to allow Ca 2+ entry by activating the gateway of the endoplasmic Ca 2+ channels (Frank et al., 2002). In neuronal cells, it has been suggested that the activation of anion conductance during glutamate uptake secures the membrane potential at a relatively negative potential which could dampen the neuronal excitability (Tanaka, 2000). However, it is difficult to anticipate whether these events take place in the skeletal muscle, particularly during exercise, since nerve tissues have disparate purposes in metabolism than skeletal muscle. Although studies are necessary to further examine glutamate uptake in muscle and its association with Ca 2+ channel activation, this potential relationship may reveal a very important role for glutamate in exercise physiology. 44

57 2.3.2 Glutamate Transport in Skeletal Muscle Glutamate is a pivotal intermediary metabolite in many tissues including skeletal muscle. However, the tissue-to-blood exchange of this amino acid usually occurs at very low rates in most tissues probably as a result of the low activity of its membrane-bound transporter (Rennie, 1996). What is particularly intriguing is that glutamate is taken up by the skeletal muscle at rest and during exercise but the rate of glutamate uptake increases by approximately 10-fold when going through the transition of rest to exercise (Gibala et al., 1997a; van Hall et al., 1999b), since the decrease in muscle glutamate creates a more favourable concentration gradient. Potentially, glutamate transporters have a greater influence on regulating skeletal muscle metabolism than was originally thought. Many amino acid transporters have a low affinity but are adequately active so that they do not limit metabolic processes (Rennie & Tipton, 2000). However, the glutamate transporter found in muscle is saturated at low concentrations and has a low maximal capacity which could have implications in the role of glutamate in skeletal muscle (Rennie et al., 1996; Rennie & Tipton, 2000). Glutamate is transported, along with aspartate, by System-X - AG which was thought to be insulin- and sodium-independent (Rennie, 1996; Wagenmakers, 1999b) but H + -dependent (Frank et al., 2002; Rennie, 1996; Wagenmakers, 1999b). Most of this work has been carried out in the rat and has assumed to be similar in human skeletal muscle. However, it has been shown that insulin can increase glutamate uptake in human skeletal muscle (Aoki et al., 1972). Furthermore, in more recent work, glutamate uptake may be sodium-dependent in skeletal muscle and is proposed to be coupled to the Na + /K + -pump (Frank et al., 2002). 45

58 Various aspects of glutamate transport that may be important to skeletal muscle glutamate transport have been examined in renal and neural tissues (Hediger, 1999; Munir et al., 2000; Welbourne & Matthews, 1999). For example, glutamate transport in renal cells can regulate ATP utilization and cellular acidification processes as well as oxidative deamination (Hediger, 1999; Welbourne & Matthews, 1999). The role of glutamate transport may be specific to different types of cells within the kidney (Hediger, 1999; Welbourne & Matthews, 1999). Furthermore, in one type of glutamate transporter, increased ammonia concentrations inhibit the uptake of glutamate in cortical astrocytes of rat cell culture (Chan et al., 2000). While these effects of ammonia have not been examined in skeletal muscle, the implications of ammonia potentially inhibiting glutamate uptake in skeletal muscle during exercise would be of particular interest and may explain the rapid clearance of ammonia by the liver. Thus, these findings in other tissues should be explored in skeletal muscle to better understand glutamate metabolism. One of the most interesting aspects of glutamate transport is that a >50-fold concentration gradient is maintained between larger concentrations in skeletal muscle and smaller concentrations in blood during rest and exercise conditions (Rennie, 1996; Wagenmakers, 1999b). Despite this remarkably large gradient, the muscle extracts substantial amounts of glutamate from the circulation especially during exercise when the gradient is decreased. Under conditions where glutamine concentrations drop, the capacity of the skeletal muscle glutamate transporter is elevated and this upregulation appears to result in further adaptations via gene transcription and translation (Low et al., 1994). Thus, to 46

59 further understand how glutamate transport affects the metabolic regulation of diverse processes in skeletal muscle, progressive transporter research specific to human skeletal muscle is essential Glutamate Metabolism: Interactions with Nitrogen-based Metabolites and the TCA cycle Several amino acids enter the TCA cycle by forming TCA cycle intermediates. For an intermediate to be formed, the amino group from amino acids needs to be removed via deamination or transferred to another metabolite through transamination before the amino acid can enter the TCA cycle. Glutamate and 2-oxoglutarate, its corresponding oxo-acid, are central to all transamination reactions in skeletal muscle (Brosnan, 2000). Thus, glutamate is pivotal to the formation of ammonia, aspartate, alanine, BCAA and glutamine ( Gibala et al., 1999; Häussinger & Gerok, 1984; Graham et al., 1997; Graham & MacLean, 1998; Rennie, 1996; Wagenmakers, 1998a). During exercise, there is an elevated release of ammonia, glutamine, and alanine from the working muscle suggesting that glutamate is important role in the transfer of amino groups and in the TCA cycle in skeletal muscle (Blomstrand & Saltin, 1999; Gibala et al., 1997a; Gibala & Saltin, 1999; Graham et al., 1995b; van Hall et al., 1999b). Glutamate uptake has been suggested to promote ammonia production (Rennie, 1996; van Hall et al., 1999b), alanine production (Gibala et al., 1997a, 2001; Rennie, 1996) and glutamine production (Rennie, 1996) as well as for the synthesis of the TCA cycle intermediates in skeletal muscle (Gibala et al., 1997a, 2002a, 2002b) and cardiac muscle 47

60 (Gibala et al., 2000; Pietersen et al., 1998; Thomassen et al., 1991). Collectively, the literature suggests that glutamate is most likely involved in alanine and TCAI production and to a lesser extent glutamate may generate ammonia and glutamine (Gibala et al., 1997a, 2001, 2002a). Since glutamate may be involved in TCA cycle anaplerosis, it is important to focus on the predominant reactions that involve glutamate and TCA cycle intermediates. These reactions are catalyzed by glutamate dehydrogenase, aspartate aminotransferase, BCAA aminotransferase, and alanine aminotransferase (Figure 2.2). It has been proposed that the drop in glutamate that is exhibited in muscle early in exercise may partially account for ammonia and 2-oxoglutarate production via the glutamate dehydrogenase reaction (glutamate + NAD + 2-oxoglutarate + ammonia + NADH) (Rennie, 1996). However, this enzyme has a low activity and is regulated by GTP and ADP (Fang et al., 2002). In the rabbit tibialis anterior muscle, the alanine aminotransferase enzyme has an activity level that is 2 times greater than glutamate dehydrogenase, while it is 3 times greater in the soleus (Passoneau & Lowry, 1993). The aspartate aminotransferase reaction (aspartate + 2-oxoglutarate glutamate + oxaloacetate) as well as the BCAA aminotransferase reaction (leucine/isoleucine/valine + 2-oxoglutarate glutamate + á-kmv/á-kmv/á-kiv) are near-equilibrium reactions that are directly affected by any change in glutamate concentration. Due to the nature of near-equilibrium reactions, muscle glutamate is also influenced by alterations in substrate/products involved in the aminotransferase reaction. However, the alanine 48

61 aminotransferase reaction has been proposed as being essential to TCA cycle anaplerosis (Gibala et al., 1997a, 1997b; Sahlin et al., 1990, 1995). Glutamate and pyruvate are transaminated by alanine aminotransferase to form 2- oxoglutarate and alanine. Because pyruvate flux is altered with the onset of exercise and anaplerosis is a rapid phenomenon that occurs as exercise begins, the alanine aminotransferase reaction is more likely to have an important role early in exercise. With this in mind, it is also interesting that the Km for pyruvate in the alanine aminotransferase reaction is ~0.3 mm which is similar to the concentrations of intramuscular pyruvate in humans (Bergmeyer, 1974). However, the Km for muscle glutamate in this reaction is 25 mm which is about 2 times greater than the intramuscular concentration of glutamate (Bergmeyer, 1974). Thus, the kinetics of this enzyme suggests that increased glutamate concentration can optimize the activity of this enzyme and may have certain implications on TCA cycle anaplerosis and potentially oxidative metabolism. As a vital energy process, the TCA cycle provides a central platform for not only fat and carbohydrate pathways to merge but also for amino acids to integrate and play a putative role in regulating oxidative metabolism. Since muscle glutamate is a central amino acid to several transamination reactions, it may be integral to substrate metabolism and oxidative capacity through the TCA cycle. With the onset of exercise, there is an increased production of pyruvate via glycolysis from muscle glycogen and blood glucose uptake. Concomitantly, there is a 40-80% drop in muscle glutamate concentrations, increase in alanine production (Blomstrand & Saltin, 1999; Gibala et al., 1997a; Gibala & 49

62 Saltin, 1999; Graham et al., 1995b; van Hall et al., 1999b) as well as a rapid rise in 2- oxoglutarate that declines after a few minutes of exercise (Gibala et al., 1997a, 2002b). Furthermore, there is the expansion of the TCAI pool that occurs within the first few minutes of exercise (Aragón & Lowenstein, 1980; Bruce et al., 2001; Dawson et al., 2003; Gibala et al., 1997b, 1997a, 1999, 2002a; Sahlin et al., 1990, 1995; Spencer et al., 1991). With prolonged exercise, as glycogen stores deplete there is an attenuated production of pyruvate which may shift substrate utilization from carbohydrate to fat (Watt et al., 2002), which may affect glutamate metabolism through the alanine aminotransferase reaction. In a study by Gibala et al (2002b), subjects exercised on 2 separate occasions whereby there were normal and low glycogen stores in their legs. At rest, muscle glutamate concentrations were greater with low glycogen than normal glycogen stores and the absolute drop in muscle glutamate by 1 minute of exercise was similar in both conditions (Gibala et al., 2002b). However, muscle glutamate reached lower concentrations during the first minute of the control exercise and remained at this level for the duration of exercise whereas these same levels were attained by 10 minutes under low glycogen conditions (Gibala et al., 2002b). In contrast, Blomstrand and Saltin (1999) conducted a study on fewer subjects where initial low glycogen concentration did not affect muscle glutamate concentrations nor glutamate uptake. While there are a few discrepancies and timing of muscle biopsies may be important, it is possible that a potential association between glutamate and pyruvate availability exists. 50

63 Investigating glutamate metabolism may be important to understanding the functional significance of anaplerosis of the TCA cycle. Because the TCA cycle is a significant source of reducing equivalents during exercise, its regulation is vital to understanding metabolism. The ultimate purpose of all metabolic processes is maintaining a steadystate concentration of ATP and ATP/ADP ratio in contracting muscle (Rennie, 1996). It would be anticipated that amino acids and their metabolites, particularly those that exhibit the most notable changes in concentrations, are intimately linked with pathways of fuel metabolism that demonstrate the most rapid changes during contractile activity (Rennie, 1996). Therefore, a decrease in muscle glutamate early in exercise may be associated with energy metabolism, and hence, ATP turnover. The proposed association between glutamate and oxidative metabolism can be further explored with endurance training whereby oxidative capacity is enhanced. There have been few studies that have examined amino acids, but clearly glutamate is one of the few amino acids that is affected by training (Dawson et al., 2003; Graham et al., 1995b). In other tissues, such as the brain, glutamate plays a very significant role in energy metabolism (Daikhin & Yudkoff, 2000; Hertz et al., 2000). In renal cells, glutamate has several roles including acid-base balance, energy metabolism, and urea metabolism (Hediger, 1999; Welbourne & Matthews, 1999). There are several aspects that can be learned from studies in various tissues that may reveal the putative role(s) that glutamate may have in skeletal muscle. 51

64 2.4 Approaches to Studying Glutamate Metabolism There are several approaches that can be taken to studying glutamate metabolism. While studies and comparisons have been carried out on various animal models, the focus of this thesis is glutamate metabolism in humans (Graham & MacLean, 1998; Stegink et al., 1979b). To study glutamate metabolism during exercise in humans, two predominant exercise models will be used: whole body and knee-extensor exercise. While they are commonly used, it is important to understand the limitations that these models present for a more accurate interpretation of results. The whole body exercise model involves two-legged exercise usually of either cycling or running. While this model is a very practical method of examining the effects of exercise, this model can be very complicated to work with for several reasons. Pulmonary oxygen consumption is usually used in this model to measure oxygen uptake. However, this measurement may not accurately represent the exercising muscle since not all muscles are active. To alleviate this factor, researchers normally refer to an approximate 8 kg of mass of exercising muscle to relate the flux of metabolites to the active muscle (Graham & MacLean, 1998; van Hall et al, 1999). However, the mass of the active muscle varies for different individuals and of course varies with muscle intensity which can result in some inaccuracies. Catheter placement can also create confounding results since a slight change in catheter placement can generate imprecision (Graham & MacLean, 1998). Furthermore, it is difficult to isolate the effects solely to metabolic changes in this model because whole body exercise induces vast alterations in hormonal and cardiovascular parameters. 52

65 Another model that has been used to study amino acid metabolism in humans is the knee extensor model. In this model one or two legs exercise the quadriceps alone. In this scenario, exercise is focused on the quadriceps muscle and therefore femoral arterial and venous catheters provide more accurate measurement of flux of metabolites entering and leaving the exercising muscle. However, catheter placement can also present errors in this model (van Hall et al., 1999a) and blood flow per unit of muscle during exercise is much greater than in whole body exercise (Graham & MacLean, 1998). In turn, this suggests that the dynamics of delivery and clearance are very different from whole body exercise rendering a different set of results. This model can be beneficial though in studying the isolated exercising muscle because there is minimal disturbance of the endocrine and cardiovascular system; however, whole body exercise is also important to understanding the integrated systems Methodological Tools in Studying Glutamate Metabolism There are various tools that have been used to examine amino acid metabolism and there are limited studies that have focused on glutamate metabolism. The use of the stable isotope methodology has exponentially increased over the past two decades. For the most part, tracers have been used to study the incorporation of amino acids into proteins in both healthy and unhealthy conditions (ie. burns, sepsis) (Wagenmakers, 1999c). Few studies have used tracers to examine the regulatory aspects of certain amino acids, such as glutamate. The centrality and dynamics of such amino acids makes it difficult to examine and quantify their changes during exercise. Since glutamate is central to 53

66 numerous amino acid reactions and other metabolic processes, it is challenging to accurately utilize stable isotope methodology in studying glutamate metabolism. However, there are a few studies that have utilized glutamate tracers. Labelled nitrogen has been used in a renal cell-line to assess the fate of cytosolic glutamate (Welbourne & Nissim, 2001). Welbourne and Nissim (2001) found that glutamate is highly compartmentalized in the cell line that they examined and that there was a significant cycling of glutamate and glutamine across the plasma membrane. There also one study that has examined the use of [1-13 C]glutamate in the heart of human subjects and found that there was uptake of glutamate and release of 13 CO 2 and increased glutamine by the heart following the infusion (Pietersen et al., 1998). Furthermore, it appeared that 85% of the labelled carbon that was infused as glutamate was oxidized to CO 2 suggesting that the infused glutamate was incorporated into the TCA cycle for the production of CO 2 (Pietersen et al., 1998). Therefore, labelled glutamate may be a method that should be further developed and considered to a greater degree for future investigations for studying glutamate in skeletal muscle. There are other studies that have combined tracer methodology and nuclear magnetic resonance (NMR) spectroscopy to noninvasively measure tissue oxygen consumption. Jeffrey et al (1999) examined the measurement of oxygen consumption using NMR and the flux of glutamate in the TCA cycle. A similar approach in using NMR can also provide information about glutamate and its effects on regulating gluconeogenesis in liver (Jones et al., 2001; Yang & Brunengraber, 2000), insulin release in the pancreas 54

67 (Brennan et al., 2002) and glucose metabolism in the brain (Chhina et al., 2001). While this method of examining glutamate and its involvement in various aspects of metabolism could be advantageous, there is an abundance of developmental work that is required before this approach can be used extensively. Glutamate administration has also been used to study the effects of glutamate on metabolism (Ghezzi et al., 1985; Stegink et al., 1982b, 1983a, 1985; Zanda et al., 1973). The majority of these studies focused on the neurotoxic, endocrine and absorptive effects of glutamate (Redding et al., 1971; Stegink et al., 1973, 1979a,1982b, 1983a; Zanda et al., 1973). Originally, early studies of oral glutamate ingestion used monosodium glutamate (MSG) to understand the effects of MSG as it related to Chinese Restaurant Syndrome (Ghadimi & Kumar, 1972; Zanda et al., 1973). Chinese Restaurant Syndrome involves headache, flushing, facial pressure, chest pressure, faint feeling, gastric distress, nausea, etc. following the ingestion of MSG. These studies led to investigating MSG as a neurotoxin (Ghadimi & Kumar, 1972; Merritt & Williams, 1990). Further interest in the effects of glutamate emerged by using MSG as a tool to examine the effects of glutamate on different thyroid hormones (Fernstrom et al., 1996; Redding et al., 1971), insulin (Graham et al., 2000; Mourtzakis & Graham, 2002; Thomassen et al., 1991), ammonia (Graham et al., 2000; Mourtzakis & Graham, 2002; Systrom et al., 1991), gut absorption of glutamate (Battezzati et al., 1995; Ghezzi et al., 1985; Matthews et al., 1993; Reeds et al., 1997; Stegink et al., 1973, 1979a, 1982b, 1983a, 1985), heart function (Langenberg et al., 2001; Thomassen et al., 1990, 1991) and skeletal muscle (Graham et al., 2000; Mourtzakis & Graham, 2002; Thomassen et al., 1991). From the studies that have 55

68 focused on the examination of skeletal muscle, MSG administration appears to be a useful tool in examining the effects of glutamate in oxidative metabolism as well as its interactions with other amino acids. 2.5 Summary Although amino acid metabolism is convoluted and intricate, it is integral and important to the regulation of metabolism. While the roles of different amino acids may vary between tissues and within different compartments of tissues and individual cells, there are certain amino acids that stand out as potentially having significant modulatory roles in exercise metabolism. Glutamate is a central amino acid to numerous metabolic processes within skeletal muscle and may be intricately involved in the regulation of oxidative and substrate metabolism. While it appears to have an important role in skeletal muscle, there are data that strongly suggest that it may be key to interorgan communications during elevated metabolic demands. While the distinct purpose of these interactions is unclear, glutamate may act as a substrate or signal in regulating and meeting metabolic demands. In addition to being the predominant amino acid to be taken up in significant amounts by skeletal muscle, glutamate is integral to several nearequilibrium reactions in different tissues which suggests that it may be involved in regulatory mechanisms of the TCA cycle and energy metabolism. Thus, it is possible that the unique biochemical properties of glutamate may provide this amino acid with the capacity to contribute in diverse ways to the regulation of energy homeostasis within skeletal muscle and across other tissues. 56

69 CHAPTER 3 STUDY I GLUTAMATE METABOLISM AND PROLONGED EXERCISE 3.0 Introduction The regulation of substrate utilization in skeletal muscle during exercise is an aspect of energy metabolism that is frequently debated. The working muscle usually derives the majority of its energy primarily from carbohydrates, and as exercise continues, energy derived from fat oxidation gradually increases as carbohydrate utilization declines (Ahlborg et al., 1974; Coyle, 1999; Romijn et al., 1993; Wahren et al., 1971, 1973). Although the contributions from amino acids towards substrate oxidation are minimal, they may be important regulatory factors in carbohydrate-lipid metabolism through their interactions in the TCA cycle. The role of amino acids in prolonged (>3h) exercise, during which there is a shift in substrate utilization from carbohydrate to fat oxidation, is rarely studied in humans (Ahlborg et al., 1974; Rennie et al., 1981; Wahren et al., 1973). Through the interactions that amino acids have with the TCA cycle, it is possible that some amino acids may have a modulatory impact on carbohydrate flux. Amino acids have been proposed to play key roles in TCA cycle anaplerosis (Gibala et al., 1997a, 2001, 2002a; Rennie, 1996; Wagenmakers, 1998a). For example, glutamate along with the BCAA may affect the decrease in 2-oxoglutarate that occurs within the first few minutes of exercise (Aragón & Lowenstein, 1980; Gibala et al., 1997a, 2002a; Wagenmakers, 1998a). Despite that amino acids are potentially important for oxidative metabolism and substrate utilization through the TCA cycle, there is little known about the roles of amino acids during prolonged exercise. 57

70 Fundamental to oxidative metabolism is the pyruvate dehydrogenase (PDH) reaction as well as various amino acid transamination reactions such as the AAT, that interlink with the TCA cycle and may be key to the regulation of substrate utilization. The PDH and the AAT reactions are of particular interest because they both share pyruvate as a substrate for their respective reactions; however, these enzymes enter the TCA cycle at various points and have different fluxes in addition to being regulated in diverse manners. The PDH enzyme is a non-equilibrium complex located on the mitochondrial membrane and is covalently and allosterically modulated by several factors including Ca 2+ and the energy status of the cell. On the other hand, the AAT reaction transaminates pyruvate and glutamate to form 2-oxoglutarate, a TCA cycle intermediate, as well as alanine. As a near-equilibrium enzyme, it is regulated by fluctuations in the concentrations of its substrates and products, which correspondingly shift this reaction to the right with increased substrate concentrations or the left with increased product concentrations. Thus, the nature and location of these enzymes may shape their role in oxidative metabolism. Glutamate is not only a substrate of the AAT reaction, it is also central to all other amino acid transamination reactions that interact with the TCA cycle. It is particularly interesting that this amino acid exhibits a drop in muscle concentration near the onset of exercise (Gibala et al., 1997a, 2002a) and has not been shown to recover during exercise nor in a 2 hour recovery period (Blomstrand & Saltin, 1999). As such, glutamate may be influenced by pyruvate flux through the AAT reaction and potentially have a regulatory role in oxidative metabolism. Initially, the increased flux through AAT could result in 58

71 decreased muscle glutamate and prolonged exercise would gradually attenuate pyruvate flux. Ultimately, the dynamics of glutamate metabolism would be affected via AAT by potentially allowing some accumulation of muscle glutamate. Since glutamate is central to various other aminotransferase reactions, any potential alterations in glutamate metabolism will also affect other interrelated amino acids such as alanine, glutamine and BCAA that may impose changes on TCA cycle anaplerosis. The PDH reaction would also be affected by the drop in pyruvate flux during prolonged exercise; however, the product of PDH, acetyl-coa, would not be affected by a reduction of pyruvate flux since it can also be produced through fat oxidation. There are no studies that have combined the potential changes in substrate utilization during prolonged exercise with possible regulatory effects of amino acids, especially glutamate, on oxidative metabolism. Furthermore, since glutamine, alanine, BCAA and glutamate are metabolized in various tissues (Kunishima et al., 2000; Molnár et al., 1995; Newsholme & Leech, 1983; Rennie, 1996; Wagenmakers, 1998a; Wasserman & Cherrington, 1996), it is likely that each of these amino acids has a specific metabolic function in different tissues. Splanchnic tissues are particularly dynamic and release a number of amino acids as well as clear various amino acids during exercise (Newsholme & Leech, 1983; Wasserman & Cherrington, 1996). Those amino acids released by the splanchnic region are usually made available for other tissues such as muscle to use. Since these amino acids have minor contributions towards energy expenditure and are part of a small plasma pool, they are dynamic and may have roles in interorgan communication towards regulating a steady energy supply. 59

72 Therefore, the present study was designed to: 1) investigate the potential effects of alterations in pyruvate flux on glutamate and alanine metabolism via the AAT reaction and their putative regulatory roles in oxidative metabolism, 2) examine the role of PDH in substrate utilization when pyruvate production becomes compromised in prolonged exercise, 3) examine the potential alterations in glutamate in relation to other amino acids as well as nitrogen balance during prolonged exercise, and 4) study the regulation and dynamics of nonmuscle tissue communication. 3.1 Methods Six healthy, recreationally active males were recruited to participate in this study. The mean + SE age, body mass and VO 2max of the subjects was years, kg, ml/kg/min ( L/min) respectively. Subjects were informed verbally and in writing of the purpose of the study as well as the procedures and risks involved with the experiment. The experimental protocol was approved by the Copenhagen and Frederiksberg Ethics Committee in Denmark Experimental Protocol After an overnight fast, subjects arrived at the laboratory and rested in the supine position. Prior to catheterization, pulmonary and blood flow resting measurements were taken. Arterial blood flow was measured at rest and recovery using Ultrasound Doppler as described by Rådegran et al (1997) and was estimated during exercise using measured femoral arteriovenous differences and rate of pulmonary oxygen consumption at a given workload according to Fick s principle (Wade & Bishop, 1972). Cannulation of the 60

73 femoral artery and vein was performed under local anesthesia. The femoral arterial catheter was inserted ~2 cm below the inguinal ligament and set proximally ~10 cm, while the femoral venous catheter was placed ~2-5 cm below the inguinal ligament and forwarded ~10 cm in the distal direction. After the insertion of the catheters, subjects rested for 20 minutes before resting blood samples were drawn. Subsequently, a biopsy was obtained from the vastus lateralis muscle under local anesthesia using the needle biopsy technique as described by Bergström (1975). Following the preparatory procedures and the resting measurements, subjects cycled at % VO 2max (mean + SE) with a cadence of 70 rpm until exhaustion. Pulmonary measurements were taken 30 minutes into the exercise protocol. Thereafter, pulmonary measurements, blood and muscle samples were taken at hourly intervals. When subjects could not maintain 70 rpm for longer than 30 seconds, this was denoted as exhaustion (EXH) at which point they stopped exercising and a muscle biopsy was taken immediately. The subjects remained in the supine position and refrained from eating during the following 3 h of recovery where pulmonary measurements, blood flow readings, and blood samples were taken hourly. Muscle biopsies were obtained at 1 and 3 h post-exercise (Figure 3.1) Blood Analysis Heparinized syringes were used to collect blood samples for measuring blood PCO 2 and PO 2 (ABL5, Radiometer, Denmark), hemoglobin, oxygen saturation (OSM3 hemoximeter, Radiometer, Denmark) and hematocrit. Arterial and venous plasma 61

74 Figure 3.1 The experimental protocol used. Blank box depicts the exercise period and the grey box depicts the recovery period. 62

75 samples from the heparinized syringes were used to analyze amino acids using UV detection on HPLC (Henrikson & Meredith, 1984) and ammonia using a fluorometric method (Bergmeyer, 1974). Arterial blood samples were collected in tubes containing EDTA and glutathione which were centrifuged and stored at -40ºC for the analysis of insulin (DAKO Ltd., UK), glucagon (Linco Research Inc., Montana, USA) and catecholamines. Catecholamines were analyzed using HPLC with electrochemical detection as described by Hallman et al (1978). Arterial and venous samples were collected for the analysis of glucose, lactate, and free fatty acids. Whole blood glucose and lactate were measured on an automatic analyser (EML105, Radiometer, Denmark) while plasma lactate and glucose were analyzed with an enzymatic method on an automatic analyser (Cobas Fara, Roche, Switzerland). Plasma free fatty acids were measured using a WAKO NEFA-C kit (Wako Chemical, Germany) Muscle Analysis Biopsy samples were immediately frozen (<10 s) and stored in liquid nitrogen. A 10 to 20 mg piece of muscle sample was used to measure PDHa (Cederblad et al., 1990; Constantin-Teodosiu et al., 1991) as modified by Putman et al (1993). These samples were corrected for differences in blood and connective tissue by adjusting to the highest total creatine concentration within a subject from the neutralized perchloric acid extracts of wet muscle homogenates used for the PDHa analyses. Another part of the biopsy sample was freeze-dried, powdered and stored at 80 C. The freeze-dried muscle was then extracted using 0.5 M perchloric acid (with 1mM EDTA) and neutralized with 2.2 M KHCO 3. The extract was then used to measure muscle amino acids (Henrikson & 63

76 Meredith, 1984), glycogen (Passoneau & Lowry, 1993), pyruvate (Passoneau & Lowry, 1993) and acetyl-coa (Cederblad et al., 1990). Adenine nucleotides, phosphocreatine and creatine were also measured and are presented in Appendix A (Wojtaszewski et al., 2002) Statistics and Calculations Values are expressed as means + SE. All data were analyzed using one-way repeated measures ANOVA. Statistical significance was accepted at P < 0.05 and analysis was conducted using Tukey s post-hoc test. Furthermore, all flux calculations were made using the Fick principle using blood flow measurements. The term flux is used interchangeable with net flux and they maintain the same meanings. Where plasma flows were required, hematocrit data were used to make the appropriate calculations. Data that were presented as flux per hour was calculated by multiplying the flux per minute by 60. For the first hour of exercise, resting concentrations were not considered and the flux at 1 h was assumed to be equivalent to the onset of exercise. For the period from 3 h to exhaustion, flux rate per minute was multiplied by the number of minutes between 3h and exhaustion for each subject and then were averaged. Moreover, data presented as exercise versus recovery is the average of each individual subject s total exercise time and total recovery time. Although glutamine consists of 2 amino groups, it was not multiplied by 2 for the nitrogen distribution graphs as the source of the amino groups was uncertain. Depending on the source of glutamine could represent a net exchange of 1 or 2 amino groups but will be assumed to be 1 amino group for these calculations. 64

77 Energy use (in kcal) from fat and carbohydrate sources during prolonged exercise was calculated while assuming that only the exercising legs expended energy (ie. RER = RQ) (Frayn, 1983). The energy sources depicted include whole body carbohydrate utilization, blood glucose uptake, estimated muscle glycogen, whole body fat utilization, blood free fatty acid uptake as well as estimated intramuscular triglycerides. Rate of whole body carbohydrate and fat utilization were calculated using pulmonary VO 2 while muscle carbohydrate and fat utilization were calculated using muscle VO 2. Blood glucose uptake (mmol/min/leg) was multiplied by the appropriate time (as per subject) and then converted to g/leg by multiplying the molecular weight of glucose (180 g) and further multiplied by calories per gram of carbohydrate (~4.2 kcal/g). Finally, these results were multiplied by 2 to represent both legs. The same calculations were used for free fatty acid uptake only molecular weight of palmytoyl-stearoyl-oleoyl-glycerol was used (861g) which accounts for a combination of fatty acids as suggested by Frayn (1983). Subsequently, this calculation was multiplied by 9.5 kcal/g. Although muscle biopsies were taken, glycogen was estimated because at low intensity exercise, the homogeneity of fibre recruitment is uncertain. Thus, muscle biopsies may not represent a clear representation of glycogenolysis in the entire leg. The use of intramuscular triacylglycerol (IMTG) and glycogen was derived by calculating total calories of carbohydrates used and fat used in muscle and subtracting the blood glucose and free fatty acid calculations from the total use, respectively. 65

78 Total amino acids (TAA) were calculated as the sum of all amino acids measured. The sum of threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine was referred to as total EAA. Tryptophan was not included because it could not be measured precisely. BCAA were calculated as the sum of valine, leucine and isoleucine. Since muscle exchange of amino acids and ammonia were measured, non-muscle exchanges (presumably splanchnic tissue) of glutamate, glutamine, alanine, BCAA and ammonia could also estimated during exercise and recovery. To determine non-muscle clearance, the muscle release or uptake of a given amino acid or ammonia was calculated for each hour except from 3h to exhaustion whereby individual exhaustion times were considered. Change in plasma pool was estimated by the difference in arterial concentrations during each hour (eg. 2h 1h = difference 2-1h ) which was added to the hourly flux and multiplied by 3.5 (estimated volume of plasma) to provide non-muscle clearance. Non-muscle release was calculated in a similar manner whereby the uptake/release of the metabolite by muscle over each hour was calculated and exhaustion times were also considered. The difference in arterial concentrations was then added to the hourly flux. These calculations were carried out under the assumption that inactive muscles had negligible contributions to the ammonia and amino acid dynamics. Also, since the release of alanine from muscle seems to occur within the first few minutes of exercise (Gibala et al., 1997a), it was assumed that alanine reached its peak release rate with the onset of exercise. Since other amino acids changed minimally in 1 hour of exercise, all amino acids were calculated similarly. FFA and glucose non-muscle production was calculated in similar manner to compare the capacity of regulation. 66

79 3.2 Results Pulmonary VO 2, Respiratory Exchange Ratio (RER), blood flow, and muscle VO 2 Subjects exercised at 44% VO 2max for 3 h 23min + 11min (3 h 32 min for n=5). With the onset of exercise, pulmonary and muscle VO 2 as well as blood flow increased and remained elevated (p<0.05) for the entire exercise duration suggesting that the subjects were exercising at a metabolic steady-state (Table 3.1). RER was elevated during exercise and tended to decline towards the end of exercise (Table 3.1). The calculated percent oxidation of fat versus carbohydrate during exercise presents a very slight increase in fat oxidation while carbohydrate flux was generally steady with a modest, reciprocal decline (~8%) from 1h to 3h of exercise (Figures 3.2a & 3.2b). More importantly, there was an increase in calories derived from FFA uptake while there was a decreased use of intramuscular triacylglycerol (IMTG) use in muscle as exercise progressed (Figure 3.2c & 3.2d). What is particularly interesting is that glycogen and blood glucose contribute almost equally (~10-14% of total caloric expenditure each) to the production of pyruvate in the muscle during exercise except at exhaustion where exogenous glucose contribution is reduced ~3% of the total calories expended (Figure 3.2d). However, ~40-50% of combined fat and carbohydrate sources are derived from non-muscle tissues such as kidney, adipose, and splanchnic tissues during any given time 67

80 Table 3.1 Pulmonary VO 2, blood flow, muscle VO 2 and RER during prolonged exercise and recovery. Rest 30 min 1 h 2 h 3 h EXH Post 1 h Post 2 h Post 3 h Pulmonary VO a a a a bcde bcde bcde (mlo 2 /min) Blood flow a a a ab bcde bcde bcde (ml/min/leg) Muscle VO a a a a bcde bcde bcde (mlo 2 /min/leg) RER b abcd abcd Values presented as mean (+ SE). EXH = exhaustion. Significance indicated by a = p<0.05 from rest; b = p<0.05 from 1 h; c = p<0.05 from 2 h; d = p<0.05 from 3 h; e = p<0.05 from EXH 68

81 Figure 3.2 Whole body and muscle energy expenditure. The whole body (a) and muscle (b) energy expenditure derived from carbohydrates and fat during prolonged exercise and recovery in calories. Muscle energy expenditure derived from blood glucose uptake, estimated muscle glycogen, FFA uptake and estimated intramuscular triacylglycerol (IMTG) is presented (c). Various sources of energy during prolonged exercise and recovery in calories in whole body metabolism (d). Data are presented as mean values. 69