Regulation of Muscle Glycogen Metabolism during Exercise: Implications for Endurance Performance and Training Adaptations

Since the introduction of the muscle biopsy technique in the late 1960s, our understanding of the regulation of muscle glycogen storage and metabolism has advanced considerably. Muscle glycogenolysis and rates of carbohydrate (CHO) oxidation are affected by factors such as exercise intensity, duration, training status and substrate availability. Such changes to the global exercise stimulus exert regulatory effects on key enzymes and transport proteins via both hormonal control and local allosteric regulation. Given the well-documented effects of high CHO availability on promoting exercise performance, elite endurance athletes are typically advised to ensure high CHO availability before, during and after high-intensity training sessions or competition. Nonetheless, in recognition that the glycogen granule is more than a simple fuel store, it is now also accepted that glycogen is a potent regulator of the molecular cell signaling pathways that regulate the oxidative phenotype. Accordingly, the concept of deliberately training with low CHO availability has now gained increased popularity amongst athletic circles. In this review, we present an overview of the regulatory control of CHO metabolism during exercise (with a speci c emphasis on muscle glycogen utilization) in order to discuss the effects of both high and low CHO availability on modulating exercise performance and training adaptations, respectively. Keywords:muscle glycogen; glucose; athletes; train-low 1. Introduction The study of carbohydrate (CHO) metabolism in relation to the eld of sport and exercise is an area of investigation that is now over 100 years old. Almost a century ago, Krogh and Lindhard [1] reported the ef ciency of CHO as a fuel source during exercise and demonstrated that fatigue occurs earlier when subjects consume a low CHO diet (as compared with a high CHO diet) in the days preceding

of sport and exercise is an area of investigation that is now over 100 years old. Almost a century ago, Krogh and Lindhard [1] reported the ef ciency of CHO as a fuel source during exercise and demonstrated that fatigue occurs earlier when subjects consume a low CHO diet (as compared with a high CHO diet) in the days preceding an exercise bout undertaken at a xed workload. Levine et al. [2] also observed that runners who completed the 1923 Boston marathon exhibited hypoglycemia (<4 mmol L 1 ) immediately post-exercise, thus suggesting that low CHO availability may be linked to fatigue. These early studies provided the initial evidence that CHO was an important fuel source for sustaining exercise performance. Nonetheless, much of the foundation of our understanding of CHO metabolism was developed by Scandinavian researchers in the late 1960s with the introduction of the muscle biopsy technique [3–6]. These researchers provided the platform for modern day sports nutrition practice in a series of studies that collectively demonstrated that (1) muscle glycogen is depleted during exercise in an intensity dependent manner; (2) high CHO diets increase muscle glycogen storage and subsequently improve exercise capacity and (3) muscle glycogen storage is acutely enhanced following prior glycogen depletion (i.e., the super-compensation effect), the magnitude of which is dependent on high CHO Nutrients2018,10, 298; doi:10.3390/nu10030298

Nutrients2018,10, 298 2 of 21 availability [4]. This body of work remains some of the most highly cited papers in the eld and is referenced accordingly in the most recent sport nutrition guidelines [7]. The eld continued to develop throughout the 1980s and 1990s with the consistent nding that CHO feeding during exercise also improved exercise performance and capacity [8–12]. Such studies relied on the use of stable isotope methodology (to quantify exogenous CHO oxidation) as well as magnetic resonance imaging to quantify liver glycogen depletion during exercise [13]. As such, it is now generally accepted that liver glycogen depletion is also a major contributing cause of fatigue during endurance exercise. It is noteworthy, however, that CHO feeding can also improve physical performance via non-metabolic effects through modulating regions of the brain associated with reward and motor control [14,15]. In addition to a simple “fuel store”, our understanding of CHO metabolism has advanced considerably in recent years with the use of more sophisticated molecular biology techniques. In this regard, it is now accepted that glycogen is more than a store [16], acting as a regulator of many key cell signaling pathways related to promoting the oxidative phenotype, insulin sensitivity, contractile processes, protein degradation and autophagic processes [16,17]. When considered this way, it is remarkable that whole body storage of only 500 g of substrate can exert such profound effects on multiple tissues, organs and systems, the results of which have considerable effects on human health and performance. The aim of this review is to, therefore, present a contemporary discussion of our understanding of CHO metabolism (focusing on muscle glycogen metabolism) with speci c reference to sport and exercise. We begin by presenting an overview of CHO storage followed by outlining regulatory steps in the control of both muscle glycogen metabolism and muscle glucose uptake. We then proceed to discuss how manipulating substrate availability (i.e., CHO availability itself) and alterations to speci cs of the exercise protocol (e.g., intensity, duration) and training status of the athlete can all affect the magnitude of CHO utilized during exercise. The previous section, therefore, provides

regulatory steps in the control of both muscle glycogen metabolism and muscle glucose uptake. We then proceed to discuss how manipulating substrate availability (i.e., CHO availability itself) and alterations to speci cs of the exercise protocol (e.g., intensity, duration) and training status of the athlete can all affect the magnitude of CHO utilized during exercise. The previous section, therefore, provides the platform to discuss the well-documented effects of both endogenous (i.e., liver and muscle glycogen) and exogenous (i.e., CHO feeding during exercise) CHO availability on exercise performance. Finally, we close by discussing the role of CHO availability on modulating aspects of training adaptation, a eld of research that has grown rapidly in the last decade. 2. Overview of CHO Storage Carbohydrate is predominantly stored as glycogen in both the liver (approximately 100 g) and muscle (approximately 400 g) with 5 g also circulating in the blood stream as glucose. In skeletal muscle, glycogen is typically expressed as mmol kg 1 of dry muscle (d.w.) where concentrations in whole muscle homogenate can vary from 50 to 800 mmol kg 1 d.w., depending on training status, fatigue status and dietary CHO intake (see Figure). Muscle glycogen can also be expressed as mmol kg 1 of wet muscle (ww), as commonly reported in the early classical literature, where values range between 10 to 180 mmol kg 1 ww [3–6]. The glycogen granule itself is essentially a tiered assembly of glucose units (i.e., polymers) that is formed in a branch-like structure via 1:4 and 1:6 -glycosidic bonds. Glycogen granules are formed on the protein, glycogenin, and can be as large as 42 nm in diameter as well as potentially having 12 tiers. At its maximal size, the granule can consist of as much as 55,000 glucosyl units [18]. Nonetheless, the majority of glycogen granules in human skeletal muscle are reported to be 25 nm in diameter with approximately eight tiers [19]. Although muscle glycogen has traditionally been quanti ed through acid hydrolysis in whole muscle homogenate, it is, of course, apparent that glycogen is expressed and utilized in bre type speci c patterns as

55,000 glucosyl units [18]. Nonetheless, the majority of glycogen granules in human skeletal muscle are reported to be 25 nm in diameter with approximately eight tiers [19]. Although muscle glycogen has traditionally been quanti ed through acid hydrolysis in whole muscle homogenate, it is, of course, apparent that glycogen is expressed and utilized in bre type speci c patterns as well as being located in speci c intracellular locations within muscle cells themselves. Using histochemical techniques, it has typically been reported that resting glycogen content is not apparently different between type I and type II bres [20–22]. Nonetheless, using biochemical quanti cation (a more quantitative measure), it has been reported that type II bres may contain 50–100 mmol kg 1 d.w. more glycogen than type I

Nutrients2018,10, 298 3 of 21 bres [9,23]. Regardless of method of quanti cation, glycogen depletion during exercise is dependent on bre type recruitment patterns, according to the speci cs of the exercise protocol. For example, during prolonged steady state type protocols, type I bres show preferential depletion, whereas during near maximal or supra-maximal type activity, type II bres become recruited and show considerable glycogen depletion [24]. In activities involving high-intensity intermittent exercise (e.g., a soccer match), considerable glycogen depletion is observed in both muscle bre types (54% and 46% of type I and II bres classed as completely or almost empty, respectively) thus re ecting recruitment patterns to support both moderate and high-intensity running speeds [25]. The use of transmission electron microscopy (TEM) has also revealed that glycogen is stored in three distinct sub-cellular pools contained in the myo brils (intra-myo brillar glycogen, 5–15% of total glycogen pool), between myo brils (inter-myo brallar glycogen, 75% of total glycogen pool) and beneath the sarcolemmal region (sub-sarcolemmal glycogen, 5–15% of total glycogen pool). In endurance trained athletes, it appears that both intra-myo brillar and sub-sarcolemmal glycogen stores are greater in type I bres compared with type II bres, whereas inter-myo brillar glycogen storage is greater in type II bres [26]. In relation to acute exercise itself, it is also apparent that intra-myo brillar glycogen stores show preferential depletion [27], and the failure to restore this speci c pool in the immediate hours after exercise is associated with impaired Ca 2+ release from the sarcoplasmic reticulum [28,29]. Clearly, our understanding of muscle glycogen storage has advanced considerably in recent years, and there remains a de nitive need to further quantify intracellular glycogen utilization in a variety of exercise settings, according to training status, age and gender.Nutrients 2018, 10, x FOR PEER REVIEW 3 of 21 Regardless of method of quantification, glycogen depletion during exercise is dependent on fibre type recruitment patterns, according to the specifics of the exercise protocol. For example, during prolonged steady state type protocols, type I fibres show preferential depletion, whereas during near maximal or supra-maximal type activity, type II

age and gender.Nutrients 2018, 10, x FOR PEER REVIEW 3 of 21 Regardless of method of quantification, glycogen depletion during exercise is dependent on fibre type recruitment patterns, according to the specifics of the exercise protocol. For example, during prolonged steady state type protocols, type I fibres show preferential depletion, whereas during near maximal or supra-maximal type activity, type II fibres become recruited and show considerable glycogen depletion [24]. In activities involving high-intensity intermittent exercise (e.g., a soccer match), considerable glycogen depletion is observed in both muscle fibre types (54% and 46% of type I and II fibres classed as completely or almost empty, respectively) thus reflecting recruitment patterns to support both moderate and high-intensity running speeds [25]. The use of transmission electron microscopy (TEM) has also revealed that glycogen is stored in three distinct sub-cellular pools contained in the myofibrils (intra-myofibrillar glycogen, 5–15% of total glycogen pool), between myofibrils (inter-myofibrallar glycogen, 75% of total glycogen pool) and beneath the sarcolemmal region (sub-sarcolemmal glycogen, 5–15% of total glycogen pool). In endurance trained athletes, it appears that both intra-myofibrillar and sub-sarcolemmal glycogen stores are greater in type I fibres compared with type II fibres, whereas inter-myofibrillar glycogen storage is greater in type II fibres [26]. In relation to acute exercise itself, it is also apparent that intra- myofibrillar glycogen stores show preferential depletion [27], and the failure to restore this specific pool in the immediate hours after exercise is associated with impaired Ca 2+ release from the sarcoplasmic reticulum [28,29]. Clearly, our understanding of muscle glycogen storage has advanced considerably in recent years, and there remains a definitive need to further quantify intracellular glycogen utilization in a variety of exercise settings, according to training status, age and gender. Figure 1. Variations in muscle glycogen storage according to fatigue status, training status and dietary carbohydrate (CHO) intake (data are compiled from males only and from several studies including Taylor et al. [30]; Bartlett et al. [31]; Arkinstall et al. [32]; Gollnick et al. [24]; Coyle et al. [8]). 3. Regulation of CHO Metabolism There are a number of potential sites of control

in muscle glycogen storage according to fatigue status, training status and dietary carbohydrate (CHO) intake (data are compiled from males only and from several studies including Taylor et al. [30]; Bartlett et al. [31]; Arkinstall et al. [32]; Gollnick et al. [24]; Coyle et al. [8]). 3. Regulation of CHO Metabolism There are a number of potential sites of control that can regulate the interaction of CHO and lipid metabolism during endurance exercise (see Figure 2). These include the availability of intra- muscular and extra-muscular substrate (controlled by diet and the action of key hormones such as the catecholamines and insulin), the abundance of transport proteins involved in transporting substrates across both the plasma and mitochondrial membranes and, of course, the activity of the key regulatory enzymes involved in the metabolic pathways. The activity of regulatory enzymes can be modified acutely through covalent modification (i.e., phosphorylation and dephosphorylation, largely under hormonal control) and/or allosteric regulation via important signalling molecules that Figure 1. Variations in muscle glycogen storage according to fatigue status, training status and dietary carbohydrate (CHO) intake (data are compiled from males only and from several studies including Taylor et al. [30]; Bartlett et al. [31]; Arkinstall et al. [32]; Gollnick et al. [24]; Coyle et al. [8]). 3. Regulation of CHO Metabolism There are a number of potential sites of control that can regulate the interaction of CHO and lipid metabolism during endurance exercise (see Figure). These include the availability of intra-muscular and extra-muscular substrate (controlled by diet and the action of key hormones such as the catecholamines and insulin), the abundance of transport proteins involved in transporting substrates across both the plasma and mitochondrial membranes and, of course, the activity of the key regulatory enzymes involved in the metabolic pathways. The activity of regulatory enzymes can be modi ed acutely through covalent modi cation (i.e., phosphorylation and dephosphorylation, largely under hormonal control) and/or allosteric regulation via important signalling molecules that are produced

the metabolic pathways. The activity of regulatory enzymes can be modi ed acutely through covalent modi cation (i.e., phosphorylation and dephosphorylation, largely under hormonal control) and/or allosteric regulation via important signalling molecules that are produced

Nutrients2018,10, 298 4 of 21 in the muscle as a result of contraction (e.g., adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inorganic phosphate (Pi), calcium (Ca 2+ ), hydrogen ion (H + ). Enzyme activity can also be modi ed through substrate activation or product inhibition such that increasing the substrate concentration increases catalysis, whereas increased product concentration may inhibit the reaction. Finally, enzyme activity can be regulated chronically through increasing the muscle cell's content of the actual enzyme protein (i.e., more of the enzyme is actually present) as would occur with endurance training. Clearly, muscle cells possess a highly coordinated and regulatory network of signalling and feedback pathways which function to ensure ATP demand is matched by ATP synthesis. From a physiological perspective, key factors, such as exercise intensity, duration, nutritional status, training status etc., can all regulate substrate utilization during exercise, largely through in uencing the potential regulatory control points discussed above. This section will outline the regulation of CHO utilization during endurance exercise, where we pay particular attention to what are currently considered the predominant sites of regulation that are relevant to the speci c situation.Nutrients 2018, 10, x FOR PEER REVIEW 4 of 21 are produced in the muscle as a result of contraction (e.g., adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inorganic phosphate (Pi), calcium (Ca 2+ ), hydrogen ion (H + ). Enzyme activity can also be modified through substrate activation or product inhibition such that increasing the substrate concentration increases catalysis, whereas increased product concentration may inhibit the reaction. Finally, enzyme activity can be regulated chronically through increasing the muscle cell’s content of the actual enzyme protein (i.e., more of the enzyme is actually present) as would occur with endurance training. Clearly, muscle cells possess a highly coordinated and regulatory network of signalling and feedback pathways which function to ensure ATP demand is matched by ATP synthesis. From a physiological perspective, key factors, such as exercise intensity, duration, nutritional status, training status etc., can all regulate substrate utilization during exercise, largely through influencing the potential regulatory control points discussed

endurance training. Clearly, muscle cells possess a highly coordinated and regulatory network of signalling and feedback pathways which function to ensure ATP demand is matched by ATP synthesis. From a physiological perspective, key factors, such as exercise intensity, duration, nutritional status, training status etc., can all regulate substrate utilization during exercise, largely through influencing the potential regulatory control points discussed above. This section will outline the regulation of CHO utilization during endurance exercise, where we pay particular attention to what are currently considered the predominant sites of regulation that are relevant to the specific situation. Figure 2. Overview of CHO metabolism and main control points. Key regulatory enzymes are well recognized as phosphorylase (PHOS), hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH). Additionally, the rate of muscle glucose uptake can also determine the flux through glycolysis. Abbreviations: ADP, Adenosine diphosphate; AK, Adenylate kinase; AKT, Protein kinase B; AMP, Adenosine monophosphate; ATP, Adenosine triphosphate; Ca 2+ , Calcium; CHO, Carbohydrate; CK, Creatine kinase; Cr, Creatine; CS, Citrate synthase; ETC, Electron transport chain; G-1-P, Glucose-1-phosphate; G-6-P, Glucose-6-phosphate; Glu, Gluose; GLUT4, Glucose transporter 4; H + , Hydrogen ion; H2O, water; IRS-1, Insulin receptor substrate 1; HK, Hexokinase; LDH, Lactate dehydrogenase; O 2, Oxygen; NAD, Nicotinamide adenine dinucleotide; TCA cycle, Tricarboxylic acid cycle; P i, phosphate; PCr, Phosphocreatine; PFK, Phosphofructokinase; PhK, Phosphorylase kinase; PHOS, Glycogen phosphorylase; PI3-K, Phosphoinositide 3-kinase. 3.1. Effects of Exercise Intensity and Duration As exercise intensity progresses from moderate (i.e., 65% VO 2max) to high-intensity (85% VO2max), muscle glycogenolysis, liver glycogenolysis and glucose uptake increase such that CHO metabolism predominates [33,34]. In contrast, there is a reduction in whole body lipid oxidation due to a reduction in both plasma free fatty acids (FFA) and intramuscular triglyceride oxidation. Maximal rates of lipid oxidation are considered to occur around 65% VO 2max, though this is dependent on a number of other factors, such as training status, gender and diet [35]. Figure 2. Overview of CHO metabolism and main control points. Key regulatory enzymes are well recognized as phosphorylase (PHOS), hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and pyruvate dehydrogenase

Maximal rates of lipid oxidation are considered to occur around 65% VO 2max, though this is dependent on a number of other factors, such as training status, gender and diet [35]. Figure 2. Overview of CHO metabolism and main control points. Key regulatory enzymes are well recognized as phosphorylase (PHOS), hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH). Additionally, the rate of muscle glucose uptake can also determine the ux through glycolysis. Abbreviations: ADP, Adenosine diphosphate; AK, Adenylate kinase; AKT, Protein kinase B; AMP, Adenosine monophosphate; ATP, Adenosine triphosphate; Ca 2+ , Calcium; CHO, Carbohydrate; CK, Creatine kinase; Cr, Creatine; CS, Citrate synthase; ETC, Electron transport chain; G-1-P, Glucose-1-phosphate; G-6-P, Glucose-6-phosphate; Glu, Gluose; GLUT4, Glucose transporter 4; H + , Hydrogen ion; H 2O, water; IRS-1, Insulin receptor substrate 1; HK, Hexokinase; LDH, Lactate dehydrogenase; O 2, Oxygen; NAD, Nicotinamide adenine dinucleotide; TCA cycle, Tricarboxylic acid cycle; P i, phosphate; PCr, Phosphocreatine; PFK, Phosphofructokinase; PhK, Phosphorylase kinase; PHOS, Glycogen phosphorylase; PI3-K, Phosphoinositide 3-kinase. 3.1. Effects of Exercise Intensity and Duration As exercise intensity progresses from moderate (i.e., 65% VO2max) to high-intensity (85% VO2max), muscle glycogenolysis, liver glycogenolysis and glucose uptake increase such that CHO metabolism predominates [33,34]. In contrast, there is a reduction in whole body lipid oxidation due to a reduction in both plasma free fatty acids (FFA) and intramuscular triglyceride oxidation. Maximal rates of lipid oxidation are considered to occur around 65% VO2max, though this is dependent on a number of other factors, such as training status, gender and diet [35].

This review discusses the regulation of carbohydrate metabolism during exercise, focusing on muscle glycogen utilization.