REVIEW ARTICLE
Spons Medicine II (4): 232-243. 1991 0112-1642/91/0004-0232/$06.00/0 © Mis Inlernalional Limilcd. All righls reserved. SPOIOO6a
Regulation of Glycogen Resynthesis Following Exercise Dietary Considerations Jacob E. Friedman, P. Darrell Neufer and G. Lynis Dohm Department of Biochemistry. School of Medicine, East Carolina University, Greenville, North Carolina, USA
Contents 232 233 234 235 235 236 236 236 237 238 238 239 239 240
Summary
Summary I. Glucose Metabolism 2. Muscle Glycogen Resynthesis in Humans 2.1 Time Course for Muscle Glycogen Resynthesis 2.2 Type of Carbohydrate Ingested 2.3 Timing of Carbohydrate Ingestion 2.4 Role of Gastric Emptying in Glucose Availability 2.5 Impaired Glycogen Resynthesis After Eccentric Exercise 3. Glycogen Synthase Activation 4. Role of Glucose Transport in Regulation of Glycogen Synthesis 4.1 The Glucose Transporter 4.2 Glucose Disposal is Limited by Glucose Transport 4.3 Examples of Glucose Transport Regulating Glycogenesis 5. Conclusion
With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Understanding the rate-limiting steps that regulate glycogen synthesis will provide us with strategies to increase the resynthesis of glycogen during recovery from exercise, and thus improve performance. Given the importance of muscle glycogen to endurance performance, various factors which may optimise glycogen resynthesis rate and insure complete restoration have been of interest to both the scientist and athlete. The time required for complete muscle glycogen resynthesis after prolonged moderate intensity exercise is generally considered to be 24 hours provided "'500 to 700g of carbohydrate is ingested. Muscle glycogen synthesis rate is highest during the first 2 hours after exercise. Ingestion of O. 70g glucose/kg bodyweight every 2 hours appears 10 maximise glycogen resynthesis rate at approximately 5 to 6 jLmol/g wet weightjh during the first 4 to 6 hours after exhaustive exercise. Further enhancement of glycogen resynthesis rate with ingestion of greater than O.70g glucose/kg bodyweight appears to be limited by the constraints imposed by gastric emptying. Ingestion of glucose or sucrose results in similar muscle glycogen resynthesis rates while glycogen synthesis in liver is better served with the ingestion of fructose. Also, increases in muscle glycogen content during the first 4 to 6 hours after exercise are greater with ingestion of simple as compared with complex carbohydrate. Glycogen synthase activity is a key component in the regulation of glycogen resynthesis. Glycogen synthase enzyme exists in 2 states: the less active, more phosphorylated (D) form which
Glycogen Resynthesis After Exercise
233
is under allosteric control of glucose-6-phosphate, and the more active, less phosphorylated (I) form which is independent of glucose-6-phosphate. There is generally an inverse relationship between glycogen content in muscle and the percentage synthase in the activated (I) form. Exercise and insulin by themselves activate glycogen synthase by conversion to glycogen synthase I. Although small changes in the activity ratio (% I form) can lead to large changes in the rate of glycogen synthesis, glycogen synthase I appears to increase very little ("'25%) in response to glycogen depletion and returns to pre-exercise levels as glycogen levels return to normal. Thus glycogen resynthesis, which may increase 3- to 5-fold, may also be influenced by glucose-6-phosphate. which can activate glycogen synthase in the D form. There is considerable evidence that glucose transport across the cell membrane is the rate limiting step in the synthesis of muscle glycogen .. Thus, regulation of glucose transport may 'set the pace' for glycogen resynthesis after exercise. Contractile activity increases the permeability of muscle to glucose even in the absence of insulin and this increase in glucose transport persists for several hours after cessation of exercise. Increased glucose transport into muscle may persist for 16 to 20 hours following exercise in rats if carbohydrate intake is restricted. This suggests that glucose transport may be regulated by glycogen concentration. However, the rate of glucose transport is not increased when muscle glycogen concentration is reduced by an overnight fast, suggesting that contractile activity provides an important stimulus linking glucose transport and the resynthesis of muscle glycogen. The changes in glucose transport suggest that ingestion of carbohydrate immediately after cessation of exercise should result in the most rapid resynthesis of muscle glycogen. Muscle possesses a unique tissue specific glucose transporter protein, termed GLUT4, which regulates glucose transport across the muscle cell membrane. Exercise training increases the synthesis of GLUT4 glucose transporters in skeletal muscle. This adaptation, along with a similar increase in glycogen synthase enzyme with training, suggests the ability to increase glycogen stores in the trained state may in part be due to increased numbers of glucose transporters.
1. Glucose Metabolism The importance of muscle glycogen as an energy source for prolonged exercise has long been appreciated by athletes who are especially interested in how they might improve their performance by enhancing muscle glycogen stores and preventing glycogen depletion. Considerable attention has been given in prior reviews to dietary regimens that result in glycogen supercompensation and 'g1ycogensparing' and therefore we have not covered these topics. Instead we have concentrated on the mechanisms which may regulate the resynthesis of muscle glycogen during recovery from endurance exercise. Understanding the metabolic factors that govern glycogen resynthesis may be considered especially important for athletes training every day or for individuals competing in multiple events on consecutive days. The first section of this review considers the practical aspects of glycogen resynthesis by review-
ing dietary studies in human subjects in which glycogen resynthesis after exercise was investigated. The second part is devoted to a discussion of the biochemical mechanisms that regulate glycogen synthesis in muscle. Although much has been written about the metabolic pathway for glycogen synthesis, until recently very little was known about which step(s) were important in regulating glycogen resynthesis in muscle following exercise. The integration of information gleaned from dietary studies together with biochemical studies may provide effective strategies aimed at increasing muscle glycogen resynthesis during recovery from exercise, and thereby improving athletic performance. The metabolic pathway for the synthesis of glycogen is shown in figure I. The greatest portion of glucose for glycogen synthesis comes from the blood, and thus the first step in the pathway is the transport of glucose across the cell membrane. Once inside the cell glucose is rapidly phosphorylated to glucose-6-phosphate which is in equilibrium with
234
Sports Medicine II (4) 1991
Muscle glycogen Glucose
PhOSPhOrYlase
!
Glucose·l·phosphate
Blood glucose
t
Hexokinase
Glucose,S' phosphate ATP .......-... ADP
Lactate
~
~
Pyruvate
~l
OXIdation
Fig. 1. Pathways of glucose metabolism.
glucose-I-phosphate. The interconversion of glucose-I-phosphate and glycogen occurs by 2 separate pathways with UDP-glucose pyrophosphorylase and glycogen synthase catalysing the synthesis of glycogen and phosphorylase catalysing the breakdown to glucose-I-phosphate. Nonequilibrium reactions in a pathway present possible points of metabolic regulation (Newsholme & Start 1973) and therefore we will focus attention on glucose transport, the hexokinase reaction and glycogen synthase as points of importance for control of glycogen synthesis. With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Glucose uptake (the combination of glucose transport and disposal) is high during the initial 2 to 4 hours after exercise (Bogardus et at. 1983) followed by a progressive decline over the next 18 hours (Cartee et at. 1989). The major pathway of glucose disposal in muscle is glycogen synthesis. After considering the evi-
dence, we believe it is likely that the overall rate of glycogen resynthesis is limited by glucose transport into the cell Thus, glucose transport after exercise may act as a 'pace-setter' for glycogen resynthesis.
2. Muscle Glycogen Resynthesis in Humans With the introduction of the muscle biopsy procedure in the early 1960s, the importance of muscle glycogen became apparent with respect to exercise endurance and performance. Several investigators, using the muscle biopsy procedure, demonstrated that muscle glycogen content decreased during exercise and that exhaustion coincided with nearly complete glycogen depletion (Ahlborg et at. 1967; Bergstrom & Hultman 1967; Bergstrom et at. 1967). Of particular interest was the finding that endurance capacity was enhanced when muscle glycogen concentration was elevated prior to exercise by dietary manipulation (Bergstrom et at. 1967; Karl-
235
Glycogen Resynthesis After Exercise
son & Saltin 1971). Thus, it became apparent that the complete restoration of glycogen content in previously active muscle was a key component to recovery and subsequent performance. 2.1 Time Course for Muscle Glycogen Resynthesis Findings from early feeding studies of Bergstrom et al. (1967) and Piehl (1974) suggested that following exhaustive exercise, glycogen level~. do not return to normal for 2 to 3 days. Costill et al. (1971) extended these observations, demonstrating a progressive decline in pre-exercise glycogen levels in subjects performing repeated days of endurance running. Despite consuming a mixed diet (250 to 350g carbohydrate), the subjects were able to only partially restore glycogen levels between each daily run. To determine if a certain time is required for complete resynthesis of glycogen after exercise independent of carbohydrate intake, Maehlum and Hermansen (1978) fasted subjects for 12 hours after a prolonged exercise bout. Although glycogen levels increased slightly during the first 4 hours, muscle glycogen concentration remained only 35% of normal during the subsequent 8 hours. These studies raised the question of whether glycogen resynthesis is dependent upon the amount of carbohydrate ingested during the recovery period and whether a critical quantity of carbohydrate is necessary to attain complete restoration within 24 hours. Using a high intensity, intermittent exercise protocol to lower glycogen levels, MacDougal et al. (1977) found that consuming a mixed diet (380g carbohydrate) completely restored muscle glycogen concentration within 24 hours. Interestingly consuming an additional 600g of carbohydrate did not accelerate the rate of resynthesis higher than the normal diet. Costill et al. (1981) further examined the effects of consuming different quantities of carbohydrate in individuals in which glycogen levels were lowered by performing a 16.1 km run at 80% ~02max. Unlike the subjects of MacDougal et al. (1977), both skeletal muscle and liver carbohydrate stores were likely depressed in subjects participating in the study by Costill et al. (1981). Increasing
carbohydrate consumption from between 188 and 648 g/24h resulted in progressively greater amounts of glycogen resynthesis. The authors concluded that muscle glycogen values were normalised within 24 hours with a carbohydrate intake of 525 to 648g. More recent studies have attempted to determine the upper limit of glycogen synthesis rate in individuals when fed varying amounts of carbohydrate after exercise. 810m et al. (1987) found a mean 6-hour postexercise glycogen synthesis rate of 2.1 "mol/g wet weight/h when subjects were fed 0.35g glucose/kg bodyweight every 2 hours after exercise. Increasing the glucose load to 0.70g glucose/kg bodyweight more than doubled the rate of glycogen synthesis to 5.8 "mol/g wet weight/h. However, no further increase in glycogen synthesis rate was observed when the glucose load was again doubled to l.4g glucose/kg bodyweight. Ivy et al. ( 1988) recently confirmed these observations, demonstrating similar glycogen storage rates (4.5 to 5.1 "mol glucose/g wet weight/h) over a 4-hour period when subjects consumed either 1.5 or 3.0g glucose/kg bodyweight. Taken together, the available research suggest that providing approximately 0.70g glucose/kg bodyweight every 2 hours is sufficient to maximise glycogen resynthesis rate at approximately 5 to 6 l'Mol/g wet weight/h during the first 4 to 6 hours after exhaustive exercise (810m et al. 1986; Ivy et al. 1988; Keizer et al. 1986). 2.2 Type of Carbohydrate Ingested In an early study by Bergstrom and Hultman (1967), a 50% greater muscle glycogen resynthesis rate was observed during infusion of 4 g.lkg bodyweight of glucose than during corresponding fructose infusion. In comparing glucose with fructose ingestion, more recent research in both humans and rats suggests that fructose is indeed less effective in promoting a rapid postexercise muscle glycogen synthesis (810m et al. 1987; Conlee et al. 1982). However, Nilsson and Hultman (1974) reported a 3-fold greater increase in liver glycogen after 3 hours of fructose compared with glucose infusion. Higher fructokinase activity in liver than muscle likely accounts for the relatively low fructose metabolism
236
in muscle (Newsholme & Start 1973). Interestingly, however, ingestion of equal amounts of glucose or sucrose (fructose + glucose disaccharide) after exercise elicits similar muscle glycogen resynthesis rates (Blom et al. 1987). Thus, it appears that muscle glycogen synthesis after exercise is maximised with glucose or sucrose ingestion immediately following exercise, while glycogen synthesis in liver is better served with the ingestion of fructose. The possible influence of simple versus complex carbohydrate ingestion on muscle glycogen resynthesis has also been investigated. Costill and coworkers (1981) reported that muscle glycogen levels were similar 24 hours after exhaustive exercise when subjects were fed isocaloric amounts of simple sugars or starch. More recently, however, Ki~ns et al. (1990) observed a significantly greater Increase in muscle glycogen content during the first 6 hours after exercise with ingestion of simple than with complex carbohydrates. Higher plasma insulin responses with simple carbohydrate ingestion likely accounted for the initial greater resynthesis rate (Kiens et al. 1990). These findings are particularly relevant to endurance athletes competing in events requiring rapid recovery, suggesting that resynthesis immediately after exercise is maximised with the ingestion of simple carbohydrates. 2.3 Timing of Carbohydrate Ingestion In rats fed carbohydrate, it has also been demonstrated that glycogen resynthesis is most rapid during the first hour after exercise (Conlee et al. 1978; Terjung et al. 1974). To determine whether glycogen synthesis after exercise is influenced by the timing of carbohydrate ingestion in humans, Ivy and Katz (1988) fed subjects 2g carbohydrate/ kg bodyweight either immediately after or 2 hours after exercise. Their data demonstrated that delaying the ingestion of carbohydrate by 2 hours after exercise resulted in a significantly lower glycogen synthesis rate (2 to 4 hours) than ingestion immediately after exercise (0 to 2 hours). Glycogen synthase activity was not different between treatments and could not account for the differences in resynthesis rate. Thus, it is likely that glucose
Sports Medicine 11 (4) 1991
transport was accelerated during initial 2 hours after exercise, accounting for the resynthesis rate. 2.4 Role of Gastric Emptying in Glucose Availability If glucose transport is rate limiting for postexercise glycogen synthesis, the question arises as to why increasing quantities of carbohydrate ingested after exercise do not elicit increasing resynthesis rates (Blom et al. 1987; Ivy et al. 1988). As suggested by Ivy et al. (1988), variations in carbohydrate load may be overriden by limits in gastric emptying and intestinal absorption. Indeed, gastric emptying is controlled such that the concentration of glucose presented to the intestines for absorption remains constant irrespective of the concentration ingested (Brener et al. 1983). Thus, glycogen resynthesis rate after exercise may be limited to a large extent by gastric emptying. A number of investigators, in attempting to bypass the constraints imposed by gastric emptying, have examined the effects of glucose infusion on postexercise glycogen synthesis (Bergstrom & Hultman 1967; Reed et al. 1989; Roch-Norlund et al. 1972). Reed et al. (1989) compared the effects of ingesting 3g carbohydrate/kg bodyweight with constant infusion of an equivalent amount of glucose over 240 minutes of recovery from exercise. Despite a near doubling of the steady-state blood glucose level with infusion, glycogen synthesis rate did not differ between experiments (6.5 /Lmolfg wet weight). These results were likely due to the insulin responses observed with each treatment. Oral ingestion of glucose, although limiting the rate of increase in blood glucose, elicited a similar plasma insulin response to that of glucose infusion. Thus, it is likely that insulin-mediated activation of glucose transport and disposal was similar in these experiments. 2.5 Impaired Glycogen Resynthesis After Eccentric Exercise A special case in which the resynthesis of glycogen in muscle is impaired following exercise involves eccentric exercise. Eccentric exercise, in
Glycogen Resynthesis After Exercise
which a muscle lengthens as it develops tension, is a component of normal exercise but has been used experimentally to mimic the delayed postexercise muscle soreness and damage typically found following any unaccustomed exercise. In a study of marathon runners, Sherman et al. (1983) found that 5 days following a 42.2km race muscle glycogen levels were still only 67% of pre-race levels, although subjects consumed a carbohydrate-rich diet, which suggested that muscle glycogen repletion was impaired in these subjects. O'Reilly et al. (1987) found that following just 45 minutes of eccentric exercise in untrained subjects, muscle glycogen levels in vastus lateralis muscle remained significantly depleted up to 10 days. In contrast to the increase in whole body insulin sensitivity usually found following exercise, Lash et al. (1987) found that oral glucose tolerance was impaired in untrained subjects 48 hours following a 50-minute downhill run suggesting that whole body glucose disposal is disrupted by eccentric exercise. Although the mechanism responsible for impaired glycogen resynthesis is unknown, ultrastructural evidence suggests that eccentric exercise results in damage to the sarcolemmal membrane (Friden et al. 1981; O'Reilly et a1. 1987), which probably interferes with skeletal muscle glucose transport and glycogen resynthesis. This explanation seems most likely since muscle damage does not interfere with glycogen synthase activity (Sherman et al. 1983).
3. Glycogen Synthtue Acti,ation Numerous studies have focused on the activation of glycogen synthase in the regulation of glycogen resynthesis following exercise (Bergstrom et al. 1972; Conlee et al. 1978; Danforth 1965). Although a thorough description of the regulation of glycogen synthase is beyond the scope of this paper, a brief review of the mechanism of glycogen synthase activation is presented. The activation of glycogen synthase is thought to be an important regulatory mechanism in glycogen synthesis (Soderling & Park 1974). Glycogen synthase catalyses the transfer of the glycosyl unit in UDP-glucose onto the glycogen skeleton (fig. I). Glycogen synthase is present in the cell in less active, more
237
phosphorylated (D) form and more active, less phosphorylated (I) form. Exercise and insulin by themselves induce an increase in glycogen synthase activity by increasing the percent of the enzyme in the I form. Glycogen synthase can be allosterically activated in the D form by glucose-6-phosphate, while the dephosphorylated I-form of glycogen synthase is spontaneously active and virtually independent from the allosteric activation by glucose-6-phosphate. There is generally an inverse relationship between glycogen content in muscle and percentage synthase I activity (Bergstrom et al. 1972; Danforth 1965). The explanation for this appears to be that glycogen binds glycogen synthase and protects it against the action of phosphoprotein phosphatase, which is also bound to glycogen and is responsible for dephosphorylating and thereby activating glycogen synthase. When glycogen concentration decreases, both enzymes are released, enabling phosphoprotein phosphatase to catalyse conversion of synthase D to the I form (Villar-Palasi 1969). Although small changes in the activity ratio (% I form) of glycogen synthase can lead to large changes in the rate of glycogen synthesis, glycogen synthase I appears to increase very little in response to glycogen depletion, and returns to preexercise levels as glycogen levels return to normal (Bergstrom 1972; Conlee 1978; Kochan et al. 1979). For example, Richter et al. (1982) found that insulin stimulates glycogen synthesis up to 8-fold in muscle following intensive exercise, yet synthase I increased only by 25% (Richter et al. 1982). Thus, attempts to explain glycogen resynthesis based on changes in either total glycogen synthase activity or increase in the active I form of glycogen synthase have been unsuccessful. Evidence from human muscle biopsies obtained following exercise suggest, however, that glycogen synthase activity may increase 24 to 48 hours after exercise through a mechanism involving increased sensitivity of glycogen synthase D to activation by glucose-6-phosphate (Bogardus et al. 1983; Kochan et al. 1979). These studies suggest that glycogen resynthesis may be the result of a small but significant activation of glycogen synthase D by lower levels of glucose-
238
6-phosphate. Thus, although glycogen levels play an important role in regulating its own synthesis, it appears that the mechanism of regulation of glycogen synthase activation, at least during glycogen resynthesis, depends on the rate of glucose-6-phosphate formation. These findings imply, but do not prove, that glycogen reformation may be limited by glucose transport since glucose availability is governed by the rate of glucose transport in muscle.
4. Role of Glucose Transport in Regulation of Glycogen Synthesis Following exercise, insulin binding and insulin receptor activation (tyrosine kinase activity) in muscle are unchanged (Treadway et al. 1989), suggesting that the mechanism for increased insulinstimulated glucose transport and increased glycogen deposition following exercise is mediated by a mechanism beyond the level of the insulin receptor. Whereas classic physiological and biochemical studies have focused on the importance of muscle glycogen synthase in regulation of glycogen resynthesis following exercise, recent knowledge of the molecular basis of glucose transport indicates the glucose transport system plays a fundamental role in regulating glucose utilisation in skeletal muscle. 4.1 The Glucose Transporter Our understanding of the mechanism of glucose transport in muscle has begun to expand recently with the discovery of a family of glucose transporter proteins (for review see Mueckler 1990). Glucose is transported across the membrane down a concentration gradient by a membrane-spanning glucose transporter protein. Several different tissue specific glucose transporter proteins have been identified and cloned and at least 2 of these transporters are expressed in muscle. The GLUT-l transporter isoform is expressed in many tissues and is present in very low abundance in skeletal muscle. The GLUT-4 transporter is the major transporter species in muscle and is responsible for the increase in glucose transport in response to insulin. Several studies have found that the mech-
Sports Medicine 11 (4) 1991
anism of increased glucose transport by skeletal muscle following exercise is through translocation of an intracellular pool of transporters to the cell surface (Douen et al. 1989; Fushiki et al. 1989; Goodyear et al. 1990; Hirshman et al. 1988; King et al. 1989; Sternlicht et al. 1989). Insulin also stimulates glucose transport in muscle by increasing the number of glucose transporters in the cell sarcolemmal membrane and by increasing the 'intrinsic activity' of the glucose transporters (for review see Klip & Paquet 1990). Glucose transporters can be detected in partially purified skeletal muscle membranes using cytochalasin-B, a fungal metabolite which binds both species of glucose transporters. Using cytochalasinB binding combined with subcellular fractionation that separated the plasma membrane from the transverse tubules (T-tubules), Burdett et al. (1987) found the transporters were enriched several-fold higher in T-tubules compared with surface plasma membrane. More recently, using antibodies specific to the GLUT-l and GLUT-4 transporters, our laboratory has provided direct morphological evidence at the electron microscopic level that in human vastus lateralis muscle the GLUT-1 transporter is located along the plasma membrane and capillary endothelial cell, while GLUT-4 transporters are exclusively found between the myofibrils within the triad (T-tubules and terminal cisternae) [Friedman et al. 1991]. This distribution suggests that GLUT-I, located primarily on the plasma membrane, may regulate glucose transport into muscle under basal conditions, while GLUT4 is responsible for the increased rate of glucose transport following exercise or insulin stimulation. The close proximity of GLUT-4 to the transverse tubule lumen and the highly complex membrane system in muscle suggests that GLUT-4 may be involved in transporting glucose from the T-tubule lumen to the interior of the cell without translocation to the surface plasma membrane. This notion is supported by the observation that hexokinase is found in close proximity between myofibrils (Lawrence & Trayer 1985) and the finding that Ttubule membranes contain an abundance of insulin receptors (Burdett et al. 1987).
239
Glycogen Resynthesis After Exercise
4.2 Glucose Disposal is Limited by Glucose Transport Given the high activity of hexokinase in muscle, an increase in glucose transport can stimulate glycogen formation. As soon as glucose enters the cell, it is phosphorylated via hexokinase to form glucose-6-phosphate. Since reversal of the hexokinase reaction is thermodynamically unfavourable and glucose-6-phosphatase activity is neglible in muscle, phosphorylation traps glucose inside the cell where it must then be utilised through glycolysis and oxidation or stored as glycogen. Hexokinase is allosterically inhibited by glucose-6-phosphate (Newsholme & Start 1913) so that if glucose-6phosphate accumulates as a result of a block in glycolysis and/or glycogenesis, then hexokinase would become rate limiting and muscle intracellular free glucose would accumulate. However, over a wide range of glucose uptake rates, free glucose does not accumulate (Katz et al. 1988; Ziel et al. 1988) demonstrating that glucose transport is rate limiting for glucose utilisation in muscle. Contractile activity increases the permeability of muscle to glucose even in the absence of insulin (Holloszy & Narahara 1965; Holloszy et at. 1986; Ivy 1987). Ivy and Holloszy (1981) first reported that 1 hour after exercise at a moderate intensity, glucose uptake was 100fold higher in the basal (noninsulin-stimulated) state. Likewise, Richter et at. (1985) and Plough et at. (1984) also reported that hindlimb glucose transport in diabetic rats perfused without insulin increased substantially following contractile activity. It is worth noting that increased glucose transport following contractions occurred only in muscles which had been significantly depleted of their glycogen stores, and that the return of glucose transport to normal levels was associated with the restoration of glycogen levels (Fell et al' 1982). These studies indicate that glucose transport increases even in the absence of insulin and suggests that increased glucose transport may be related to muscle glycogen concentration. Increased glucose transport into muscle may persist for 16 to 20 hours following exercise in rats if carbohydrate intake is restricted (Wallberg-Hen-
riksson et al. 1988; Young et al. 1983). However, Young et al. (1983) found the rate of glucose transport was not increased when muscle glycogen concentration was reduced by an overnight fast, suggesting that contractile activity provides an important stimulus linking glucose transport and resynthesis of muscle glycogen. The insulin sensitivity of glucose transport also increases 5- to 100foid in skeletal muscle following acute endurance exercise (Richter et al. 1982; Zorzano et al. 1985). Insulin sensitivity refers to the concentration of insulin necessary to elicit a biological response to insulin. Thus an increase in insulin sensitivity indicates that less insulin is required to elicit a biological response. Cartee et al. (1989) has recently shown that in rats fed carb0hydrate after a vigorous bout of exercise insulin sensitivity is reversed within 18 hours after exercise. However, in rats fed a carbohydrate-deficient diet the increase in insulin sensitivity is prolonged for at least 48 hours following exercise. Richter et al. (1988) examined the extent to which muscle glycogen concentrations can be increased .by prolonged insulin stimulation and whether the limiting step in glycogen synthesis was glucose transport, the intracellular disposal of glucose or both. Rats perfused for 7 hours in the presence of pharmacological levels of insulin and 11 to 13 mmol/L glucose increased muscle glycogen levels to maximal values 2, 3, and 3.5 times greater than normal in fast twitch white, slow twitch red and fast twitch red fibres, respectively. During the perfusion muscle glycogen synthase activity decreased and free intracellular glucose and glucose6-phosphate increased indicating that glucose disposal was impaired. However, glucose transport was also markedly decreased after 5 and 1 hours perfusion compared with initial values, suggesting both glucose transport and glucose disposal limited glycogen storage under maximal insulin stimulation. 4.3 Examples of Glucose Transport Regulating Glycogenesis Recent findings suggest that expression of GLUT-4 protein may regulate maximal glucose transport rates in muscle, and may therefore be
240
partially responsible for variations in glucose disposal, i.e. glycogen synthesis. Friedman et al. (1990), demonstrated that endurance exercise training induced a significant 2.4-fold increase in GLUT-4 protein in gastrocnemius muscle of trained rats. This adaptation, along with a similar increase in percentage glycogen synthase I observed previously by others (James & Kraegen 1984), suggests that the ability to increase glycogen stores in the trained state is associated with a higher GLUT-4 protein content. James et a!. (1985) used the in vivo euglycaemic clamp with the administration of glucose tracers to study glucose transport and glycogen synthesis in several tissues of rats in response to insulin. They found that red muscle fibres transported glucose faster than white muscle. We have confirmed this observation and find that the higher transport rate in red muscles is most probably a result ofa greater number of glucose transporters in red muscle (Kern et a!. 1990). James et a!. (1985) also observed that glycogen synthesis was more rapid in red than white muscle fibres. Using a perfused rat muscle preparation, Richter et a!. (1988) confirmed the close correlation between glucose transport and glycogen synthesis in red and white muscle fibres. In addition, Richter et al. (1988) found that glycogen synthesis rate decreased dramatically after 3 hours of hyperglycaemic perfusion and this change was paralleled by a decrease in the rate of glucose transport. There was also a decrease in the fractional aetivity of glycogen synthase and the intracellular concentrations of glucose and glucose-6-phosphate. These studies thus confirm the close relationship betweeil glucose transport, glucose transporters and glycogen formation. Further evidence for the importance of glucose transport in the regulation of glucose utilisation by muscle comes from studies of patients with noninsulin-
Spons Medicine II (4): 232-243. 1991 0112-1642/91/0004-0232/$06.00/0 © Mis Inlernalional Limilcd. All righls reserved. SPOIOO6a
Regulation of Glycogen Resynthesis Following Exercise Dietary Considerations Jacob E. Friedman, P. Darrell Neufer and G. Lynis Dohm Department of Biochemistry. School of Medicine, East Carolina University, Greenville, North Carolina, USA
Contents 232 233 234 235 235 236 236 236 237 238 238 239 239 240
Summary
Summary I. Glucose Metabolism 2. Muscle Glycogen Resynthesis in Humans 2.1 Time Course for Muscle Glycogen Resynthesis 2.2 Type of Carbohydrate Ingested 2.3 Timing of Carbohydrate Ingestion 2.4 Role of Gastric Emptying in Glucose Availability 2.5 Impaired Glycogen Resynthesis After Eccentric Exercise 3. Glycogen Synthase Activation 4. Role of Glucose Transport in Regulation of Glycogen Synthesis 4.1 The Glucose Transporter 4.2 Glucose Disposal is Limited by Glucose Transport 4.3 Examples of Glucose Transport Regulating Glycogenesis 5. Conclusion
With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Understanding the rate-limiting steps that regulate glycogen synthesis will provide us with strategies to increase the resynthesis of glycogen during recovery from exercise, and thus improve performance. Given the importance of muscle glycogen to endurance performance, various factors which may optimise glycogen resynthesis rate and insure complete restoration have been of interest to both the scientist and athlete. The time required for complete muscle glycogen resynthesis after prolonged moderate intensity exercise is generally considered to be 24 hours provided "'500 to 700g of carbohydrate is ingested. Muscle glycogen synthesis rate is highest during the first 2 hours after exercise. Ingestion of O. 70g glucose/kg bodyweight every 2 hours appears 10 maximise glycogen resynthesis rate at approximately 5 to 6 jLmol/g wet weightjh during the first 4 to 6 hours after exhaustive exercise. Further enhancement of glycogen resynthesis rate with ingestion of greater than O.70g glucose/kg bodyweight appears to be limited by the constraints imposed by gastric emptying. Ingestion of glucose or sucrose results in similar muscle glycogen resynthesis rates while glycogen synthesis in liver is better served with the ingestion of fructose. Also, increases in muscle glycogen content during the first 4 to 6 hours after exercise are greater with ingestion of simple as compared with complex carbohydrate. Glycogen synthase activity is a key component in the regulation of glycogen resynthesis. Glycogen synthase enzyme exists in 2 states: the less active, more phosphorylated (D) form which
Glycogen Resynthesis After Exercise
233
is under allosteric control of glucose-6-phosphate, and the more active, less phosphorylated (I) form which is independent of glucose-6-phosphate. There is generally an inverse relationship between glycogen content in muscle and the percentage synthase in the activated (I) form. Exercise and insulin by themselves activate glycogen synthase by conversion to glycogen synthase I. Although small changes in the activity ratio (% I form) can lead to large changes in the rate of glycogen synthesis, glycogen synthase I appears to increase very little ("'25%) in response to glycogen depletion and returns to pre-exercise levels as glycogen levels return to normal. Thus glycogen resynthesis, which may increase 3- to 5-fold, may also be influenced by glucose-6-phosphate. which can activate glycogen synthase in the D form. There is considerable evidence that glucose transport across the cell membrane is the rate limiting step in the synthesis of muscle glycogen .. Thus, regulation of glucose transport may 'set the pace' for glycogen resynthesis after exercise. Contractile activity increases the permeability of muscle to glucose even in the absence of insulin and this increase in glucose transport persists for several hours after cessation of exercise. Increased glucose transport into muscle may persist for 16 to 20 hours following exercise in rats if carbohydrate intake is restricted. This suggests that glucose transport may be regulated by glycogen concentration. However, the rate of glucose transport is not increased when muscle glycogen concentration is reduced by an overnight fast, suggesting that contractile activity provides an important stimulus linking glucose transport and the resynthesis of muscle glycogen. The changes in glucose transport suggest that ingestion of carbohydrate immediately after cessation of exercise should result in the most rapid resynthesis of muscle glycogen. Muscle possesses a unique tissue specific glucose transporter protein, termed GLUT4, which regulates glucose transport across the muscle cell membrane. Exercise training increases the synthesis of GLUT4 glucose transporters in skeletal muscle. This adaptation, along with a similar increase in glycogen synthase enzyme with training, suggests the ability to increase glycogen stores in the trained state may in part be due to increased numbers of glucose transporters.
1. Glucose Metabolism The importance of muscle glycogen as an energy source for prolonged exercise has long been appreciated by athletes who are especially interested in how they might improve their performance by enhancing muscle glycogen stores and preventing glycogen depletion. Considerable attention has been given in prior reviews to dietary regimens that result in glycogen supercompensation and 'g1ycogensparing' and therefore we have not covered these topics. Instead we have concentrated on the mechanisms which may regulate the resynthesis of muscle glycogen during recovery from endurance exercise. Understanding the metabolic factors that govern glycogen resynthesis may be considered especially important for athletes training every day or for individuals competing in multiple events on consecutive days. The first section of this review considers the practical aspects of glycogen resynthesis by review-
ing dietary studies in human subjects in which glycogen resynthesis after exercise was investigated. The second part is devoted to a discussion of the biochemical mechanisms that regulate glycogen synthesis in muscle. Although much has been written about the metabolic pathway for glycogen synthesis, until recently very little was known about which step(s) were important in regulating glycogen resynthesis in muscle following exercise. The integration of information gleaned from dietary studies together with biochemical studies may provide effective strategies aimed at increasing muscle glycogen resynthesis during recovery from exercise, and thereby improving athletic performance. The metabolic pathway for the synthesis of glycogen is shown in figure I. The greatest portion of glucose for glycogen synthesis comes from the blood, and thus the first step in the pathway is the transport of glucose across the cell membrane. Once inside the cell glucose is rapidly phosphorylated to glucose-6-phosphate which is in equilibrium with
234
Sports Medicine II (4) 1991
Muscle glycogen Glucose
PhOSPhOrYlase
!
Glucose·l·phosphate
Blood glucose
t
Hexokinase
Glucose,S' phosphate ATP .......-... ADP
Lactate
~
~
Pyruvate
~l
OXIdation
Fig. 1. Pathways of glucose metabolism.
glucose-I-phosphate. The interconversion of glucose-I-phosphate and glycogen occurs by 2 separate pathways with UDP-glucose pyrophosphorylase and glycogen synthase catalysing the synthesis of glycogen and phosphorylase catalysing the breakdown to glucose-I-phosphate. Nonequilibrium reactions in a pathway present possible points of metabolic regulation (Newsholme & Start 1973) and therefore we will focus attention on glucose transport, the hexokinase reaction and glycogen synthase as points of importance for control of glycogen synthesis. With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Glucose uptake (the combination of glucose transport and disposal) is high during the initial 2 to 4 hours after exercise (Bogardus et at. 1983) followed by a progressive decline over the next 18 hours (Cartee et at. 1989). The major pathway of glucose disposal in muscle is glycogen synthesis. After considering the evi-
dence, we believe it is likely that the overall rate of glycogen resynthesis is limited by glucose transport into the cell Thus, glucose transport after exercise may act as a 'pace-setter' for glycogen resynthesis.
2. Muscle Glycogen Resynthesis in Humans With the introduction of the muscle biopsy procedure in the early 1960s, the importance of muscle glycogen became apparent with respect to exercise endurance and performance. Several investigators, using the muscle biopsy procedure, demonstrated that muscle glycogen content decreased during exercise and that exhaustion coincided with nearly complete glycogen depletion (Ahlborg et at. 1967; Bergstrom & Hultman 1967; Bergstrom et at. 1967). Of particular interest was the finding that endurance capacity was enhanced when muscle glycogen concentration was elevated prior to exercise by dietary manipulation (Bergstrom et at. 1967; Karl-
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Glycogen Resynthesis After Exercise
son & Saltin 1971). Thus, it became apparent that the complete restoration of glycogen content in previously active muscle was a key component to recovery and subsequent performance. 2.1 Time Course for Muscle Glycogen Resynthesis Findings from early feeding studies of Bergstrom et al. (1967) and Piehl (1974) suggested that following exhaustive exercise, glycogen level~. do not return to normal for 2 to 3 days. Costill et al. (1971) extended these observations, demonstrating a progressive decline in pre-exercise glycogen levels in subjects performing repeated days of endurance running. Despite consuming a mixed diet (250 to 350g carbohydrate), the subjects were able to only partially restore glycogen levels between each daily run. To determine if a certain time is required for complete resynthesis of glycogen after exercise independent of carbohydrate intake, Maehlum and Hermansen (1978) fasted subjects for 12 hours after a prolonged exercise bout. Although glycogen levels increased slightly during the first 4 hours, muscle glycogen concentration remained only 35% of normal during the subsequent 8 hours. These studies raised the question of whether glycogen resynthesis is dependent upon the amount of carbohydrate ingested during the recovery period and whether a critical quantity of carbohydrate is necessary to attain complete restoration within 24 hours. Using a high intensity, intermittent exercise protocol to lower glycogen levels, MacDougal et al. (1977) found that consuming a mixed diet (380g carbohydrate) completely restored muscle glycogen concentration within 24 hours. Interestingly consuming an additional 600g of carbohydrate did not accelerate the rate of resynthesis higher than the normal diet. Costill et al. (1981) further examined the effects of consuming different quantities of carbohydrate in individuals in which glycogen levels were lowered by performing a 16.1 km run at 80% ~02max. Unlike the subjects of MacDougal et al. (1977), both skeletal muscle and liver carbohydrate stores were likely depressed in subjects participating in the study by Costill et al. (1981). Increasing
carbohydrate consumption from between 188 and 648 g/24h resulted in progressively greater amounts of glycogen resynthesis. The authors concluded that muscle glycogen values were normalised within 24 hours with a carbohydrate intake of 525 to 648g. More recent studies have attempted to determine the upper limit of glycogen synthesis rate in individuals when fed varying amounts of carbohydrate after exercise. 810m et al. (1987) found a mean 6-hour postexercise glycogen synthesis rate of 2.1 "mol/g wet weight/h when subjects were fed 0.35g glucose/kg bodyweight every 2 hours after exercise. Increasing the glucose load to 0.70g glucose/kg bodyweight more than doubled the rate of glycogen synthesis to 5.8 "mol/g wet weight/h. However, no further increase in glycogen synthesis rate was observed when the glucose load was again doubled to l.4g glucose/kg bodyweight. Ivy et al. ( 1988) recently confirmed these observations, demonstrating similar glycogen storage rates (4.5 to 5.1 "mol glucose/g wet weight/h) over a 4-hour period when subjects consumed either 1.5 or 3.0g glucose/kg bodyweight. Taken together, the available research suggest that providing approximately 0.70g glucose/kg bodyweight every 2 hours is sufficient to maximise glycogen resynthesis rate at approximately 5 to 6 l'Mol/g wet weight/h during the first 4 to 6 hours after exhaustive exercise (810m et al. 1986; Ivy et al. 1988; Keizer et al. 1986). 2.2 Type of Carbohydrate Ingested In an early study by Bergstrom and Hultman (1967), a 50% greater muscle glycogen resynthesis rate was observed during infusion of 4 g.lkg bodyweight of glucose than during corresponding fructose infusion. In comparing glucose with fructose ingestion, more recent research in both humans and rats suggests that fructose is indeed less effective in promoting a rapid postexercise muscle glycogen synthesis (810m et al. 1987; Conlee et al. 1982). However, Nilsson and Hultman (1974) reported a 3-fold greater increase in liver glycogen after 3 hours of fructose compared with glucose infusion. Higher fructokinase activity in liver than muscle likely accounts for the relatively low fructose metabolism
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in muscle (Newsholme & Start 1973). Interestingly, however, ingestion of equal amounts of glucose or sucrose (fructose + glucose disaccharide) after exercise elicits similar muscle glycogen resynthesis rates (Blom et al. 1987). Thus, it appears that muscle glycogen synthesis after exercise is maximised with glucose or sucrose ingestion immediately following exercise, while glycogen synthesis in liver is better served with the ingestion of fructose. The possible influence of simple versus complex carbohydrate ingestion on muscle glycogen resynthesis has also been investigated. Costill and coworkers (1981) reported that muscle glycogen levels were similar 24 hours after exhaustive exercise when subjects were fed isocaloric amounts of simple sugars or starch. More recently, however, Ki~ns et al. (1990) observed a significantly greater Increase in muscle glycogen content during the first 6 hours after exercise with ingestion of simple than with complex carbohydrates. Higher plasma insulin responses with simple carbohydrate ingestion likely accounted for the initial greater resynthesis rate (Kiens et al. 1990). These findings are particularly relevant to endurance athletes competing in events requiring rapid recovery, suggesting that resynthesis immediately after exercise is maximised with the ingestion of simple carbohydrates. 2.3 Timing of Carbohydrate Ingestion In rats fed carbohydrate, it has also been demonstrated that glycogen resynthesis is most rapid during the first hour after exercise (Conlee et al. 1978; Terjung et al. 1974). To determine whether glycogen synthesis after exercise is influenced by the timing of carbohydrate ingestion in humans, Ivy and Katz (1988) fed subjects 2g carbohydrate/ kg bodyweight either immediately after or 2 hours after exercise. Their data demonstrated that delaying the ingestion of carbohydrate by 2 hours after exercise resulted in a significantly lower glycogen synthesis rate (2 to 4 hours) than ingestion immediately after exercise (0 to 2 hours). Glycogen synthase activity was not different between treatments and could not account for the differences in resynthesis rate. Thus, it is likely that glucose
Sports Medicine 11 (4) 1991
transport was accelerated during initial 2 hours after exercise, accounting for the resynthesis rate. 2.4 Role of Gastric Emptying in Glucose Availability If glucose transport is rate limiting for postexercise glycogen synthesis, the question arises as to why increasing quantities of carbohydrate ingested after exercise do not elicit increasing resynthesis rates (Blom et al. 1987; Ivy et al. 1988). As suggested by Ivy et al. (1988), variations in carbohydrate load may be overriden by limits in gastric emptying and intestinal absorption. Indeed, gastric emptying is controlled such that the concentration of glucose presented to the intestines for absorption remains constant irrespective of the concentration ingested (Brener et al. 1983). Thus, glycogen resynthesis rate after exercise may be limited to a large extent by gastric emptying. A number of investigators, in attempting to bypass the constraints imposed by gastric emptying, have examined the effects of glucose infusion on postexercise glycogen synthesis (Bergstrom & Hultman 1967; Reed et al. 1989; Roch-Norlund et al. 1972). Reed et al. (1989) compared the effects of ingesting 3g carbohydrate/kg bodyweight with constant infusion of an equivalent amount of glucose over 240 minutes of recovery from exercise. Despite a near doubling of the steady-state blood glucose level with infusion, glycogen synthesis rate did not differ between experiments (6.5 /Lmolfg wet weight). These results were likely due to the insulin responses observed with each treatment. Oral ingestion of glucose, although limiting the rate of increase in blood glucose, elicited a similar plasma insulin response to that of glucose infusion. Thus, it is likely that insulin-mediated activation of glucose transport and disposal was similar in these experiments. 2.5 Impaired Glycogen Resynthesis After Eccentric Exercise A special case in which the resynthesis of glycogen in muscle is impaired following exercise involves eccentric exercise. Eccentric exercise, in
Glycogen Resynthesis After Exercise
which a muscle lengthens as it develops tension, is a component of normal exercise but has been used experimentally to mimic the delayed postexercise muscle soreness and damage typically found following any unaccustomed exercise. In a study of marathon runners, Sherman et al. (1983) found that 5 days following a 42.2km race muscle glycogen levels were still only 67% of pre-race levels, although subjects consumed a carbohydrate-rich diet, which suggested that muscle glycogen repletion was impaired in these subjects. O'Reilly et al. (1987) found that following just 45 minutes of eccentric exercise in untrained subjects, muscle glycogen levels in vastus lateralis muscle remained significantly depleted up to 10 days. In contrast to the increase in whole body insulin sensitivity usually found following exercise, Lash et al. (1987) found that oral glucose tolerance was impaired in untrained subjects 48 hours following a 50-minute downhill run suggesting that whole body glucose disposal is disrupted by eccentric exercise. Although the mechanism responsible for impaired glycogen resynthesis is unknown, ultrastructural evidence suggests that eccentric exercise results in damage to the sarcolemmal membrane (Friden et al. 1981; O'Reilly et a1. 1987), which probably interferes with skeletal muscle glucose transport and glycogen resynthesis. This explanation seems most likely since muscle damage does not interfere with glycogen synthase activity (Sherman et al. 1983).
3. Glycogen Synthtue Acti,ation Numerous studies have focused on the activation of glycogen synthase in the regulation of glycogen resynthesis following exercise (Bergstrom et al. 1972; Conlee et al. 1978; Danforth 1965). Although a thorough description of the regulation of glycogen synthase is beyond the scope of this paper, a brief review of the mechanism of glycogen synthase activation is presented. The activation of glycogen synthase is thought to be an important regulatory mechanism in glycogen synthesis (Soderling & Park 1974). Glycogen synthase catalyses the transfer of the glycosyl unit in UDP-glucose onto the glycogen skeleton (fig. I). Glycogen synthase is present in the cell in less active, more
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phosphorylated (D) form and more active, less phosphorylated (I) form. Exercise and insulin by themselves induce an increase in glycogen synthase activity by increasing the percent of the enzyme in the I form. Glycogen synthase can be allosterically activated in the D form by glucose-6-phosphate, while the dephosphorylated I-form of glycogen synthase is spontaneously active and virtually independent from the allosteric activation by glucose-6-phosphate. There is generally an inverse relationship between glycogen content in muscle and percentage synthase I activity (Bergstrom et al. 1972; Danforth 1965). The explanation for this appears to be that glycogen binds glycogen synthase and protects it against the action of phosphoprotein phosphatase, which is also bound to glycogen and is responsible for dephosphorylating and thereby activating glycogen synthase. When glycogen concentration decreases, both enzymes are released, enabling phosphoprotein phosphatase to catalyse conversion of synthase D to the I form (Villar-Palasi 1969). Although small changes in the activity ratio (% I form) of glycogen synthase can lead to large changes in the rate of glycogen synthesis, glycogen synthase I appears to increase very little in response to glycogen depletion, and returns to preexercise levels as glycogen levels return to normal (Bergstrom 1972; Conlee 1978; Kochan et al. 1979). For example, Richter et al. (1982) found that insulin stimulates glycogen synthesis up to 8-fold in muscle following intensive exercise, yet synthase I increased only by 25% (Richter et al. 1982). Thus, attempts to explain glycogen resynthesis based on changes in either total glycogen synthase activity or increase in the active I form of glycogen synthase have been unsuccessful. Evidence from human muscle biopsies obtained following exercise suggest, however, that glycogen synthase activity may increase 24 to 48 hours after exercise through a mechanism involving increased sensitivity of glycogen synthase D to activation by glucose-6-phosphate (Bogardus et al. 1983; Kochan et al. 1979). These studies suggest that glycogen resynthesis may be the result of a small but significant activation of glycogen synthase D by lower levels of glucose-
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6-phosphate. Thus, although glycogen levels play an important role in regulating its own synthesis, it appears that the mechanism of regulation of glycogen synthase activation, at least during glycogen resynthesis, depends on the rate of glucose-6-phosphate formation. These findings imply, but do not prove, that glycogen reformation may be limited by glucose transport since glucose availability is governed by the rate of glucose transport in muscle.
4. Role of Glucose Transport in Regulation of Glycogen Synthesis Following exercise, insulin binding and insulin receptor activation (tyrosine kinase activity) in muscle are unchanged (Treadway et al. 1989), suggesting that the mechanism for increased insulinstimulated glucose transport and increased glycogen deposition following exercise is mediated by a mechanism beyond the level of the insulin receptor. Whereas classic physiological and biochemical studies have focused on the importance of muscle glycogen synthase in regulation of glycogen resynthesis following exercise, recent knowledge of the molecular basis of glucose transport indicates the glucose transport system plays a fundamental role in regulating glucose utilisation in skeletal muscle. 4.1 The Glucose Transporter Our understanding of the mechanism of glucose transport in muscle has begun to expand recently with the discovery of a family of glucose transporter proteins (for review see Mueckler 1990). Glucose is transported across the membrane down a concentration gradient by a membrane-spanning glucose transporter protein. Several different tissue specific glucose transporter proteins have been identified and cloned and at least 2 of these transporters are expressed in muscle. The GLUT-l transporter isoform is expressed in many tissues and is present in very low abundance in skeletal muscle. The GLUT-4 transporter is the major transporter species in muscle and is responsible for the increase in glucose transport in response to insulin. Several studies have found that the mech-
Sports Medicine 11 (4) 1991
anism of increased glucose transport by skeletal muscle following exercise is through translocation of an intracellular pool of transporters to the cell surface (Douen et al. 1989; Fushiki et al. 1989; Goodyear et al. 1990; Hirshman et al. 1988; King et al. 1989; Sternlicht et al. 1989). Insulin also stimulates glucose transport in muscle by increasing the number of glucose transporters in the cell sarcolemmal membrane and by increasing the 'intrinsic activity' of the glucose transporters (for review see Klip & Paquet 1990). Glucose transporters can be detected in partially purified skeletal muscle membranes using cytochalasin-B, a fungal metabolite which binds both species of glucose transporters. Using cytochalasinB binding combined with subcellular fractionation that separated the plasma membrane from the transverse tubules (T-tubules), Burdett et al. (1987) found the transporters were enriched several-fold higher in T-tubules compared with surface plasma membrane. More recently, using antibodies specific to the GLUT-l and GLUT-4 transporters, our laboratory has provided direct morphological evidence at the electron microscopic level that in human vastus lateralis muscle the GLUT-1 transporter is located along the plasma membrane and capillary endothelial cell, while GLUT-4 transporters are exclusively found between the myofibrils within the triad (T-tubules and terminal cisternae) [Friedman et al. 1991]. This distribution suggests that GLUT-I, located primarily on the plasma membrane, may regulate glucose transport into muscle under basal conditions, while GLUT4 is responsible for the increased rate of glucose transport following exercise or insulin stimulation. The close proximity of GLUT-4 to the transverse tubule lumen and the highly complex membrane system in muscle suggests that GLUT-4 may be involved in transporting glucose from the T-tubule lumen to the interior of the cell without translocation to the surface plasma membrane. This notion is supported by the observation that hexokinase is found in close proximity between myofibrils (Lawrence & Trayer 1985) and the finding that Ttubule membranes contain an abundance of insulin receptors (Burdett et al. 1987).
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Glycogen Resynthesis After Exercise
4.2 Glucose Disposal is Limited by Glucose Transport Given the high activity of hexokinase in muscle, an increase in glucose transport can stimulate glycogen formation. As soon as glucose enters the cell, it is phosphorylated via hexokinase to form glucose-6-phosphate. Since reversal of the hexokinase reaction is thermodynamically unfavourable and glucose-6-phosphatase activity is neglible in muscle, phosphorylation traps glucose inside the cell where it must then be utilised through glycolysis and oxidation or stored as glycogen. Hexokinase is allosterically inhibited by glucose-6-phosphate (Newsholme & Start 1913) so that if glucose-6phosphate accumulates as a result of a block in glycolysis and/or glycogenesis, then hexokinase would become rate limiting and muscle intracellular free glucose would accumulate. However, over a wide range of glucose uptake rates, free glucose does not accumulate (Katz et al. 1988; Ziel et al. 1988) demonstrating that glucose transport is rate limiting for glucose utilisation in muscle. Contractile activity increases the permeability of muscle to glucose even in the absence of insulin (Holloszy & Narahara 1965; Holloszy et at. 1986; Ivy 1987). Ivy and Holloszy (1981) first reported that 1 hour after exercise at a moderate intensity, glucose uptake was 100fold higher in the basal (noninsulin-stimulated) state. Likewise, Richter et at. (1985) and Plough et at. (1984) also reported that hindlimb glucose transport in diabetic rats perfused without insulin increased substantially following contractile activity. It is worth noting that increased glucose transport following contractions occurred only in muscles which had been significantly depleted of their glycogen stores, and that the return of glucose transport to normal levels was associated with the restoration of glycogen levels (Fell et al' 1982). These studies indicate that glucose transport increases even in the absence of insulin and suggests that increased glucose transport may be related to muscle glycogen concentration. Increased glucose transport into muscle may persist for 16 to 20 hours following exercise in rats if carbohydrate intake is restricted (Wallberg-Hen-
riksson et al. 1988; Young et al. 1983). However, Young et al. (1983) found the rate of glucose transport was not increased when muscle glycogen concentration was reduced by an overnight fast, suggesting that contractile activity provides an important stimulus linking glucose transport and resynthesis of muscle glycogen. The insulin sensitivity of glucose transport also increases 5- to 100foid in skeletal muscle following acute endurance exercise (Richter et al. 1982; Zorzano et al. 1985). Insulin sensitivity refers to the concentration of insulin necessary to elicit a biological response to insulin. Thus an increase in insulin sensitivity indicates that less insulin is required to elicit a biological response. Cartee et al. (1989) has recently shown that in rats fed carb0hydrate after a vigorous bout of exercise insulin sensitivity is reversed within 18 hours after exercise. However, in rats fed a carbohydrate-deficient diet the increase in insulin sensitivity is prolonged for at least 48 hours following exercise. Richter et al. (1988) examined the extent to which muscle glycogen concentrations can be increased .by prolonged insulin stimulation and whether the limiting step in glycogen synthesis was glucose transport, the intracellular disposal of glucose or both. Rats perfused for 7 hours in the presence of pharmacological levels of insulin and 11 to 13 mmol/L glucose increased muscle glycogen levels to maximal values 2, 3, and 3.5 times greater than normal in fast twitch white, slow twitch red and fast twitch red fibres, respectively. During the perfusion muscle glycogen synthase activity decreased and free intracellular glucose and glucose6-phosphate increased indicating that glucose disposal was impaired. However, glucose transport was also markedly decreased after 5 and 1 hours perfusion compared with initial values, suggesting both glucose transport and glucose disposal limited glycogen storage under maximal insulin stimulation. 4.3 Examples of Glucose Transport Regulating Glycogenesis Recent findings suggest that expression of GLUT-4 protein may regulate maximal glucose transport rates in muscle, and may therefore be
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partially responsible for variations in glucose disposal, i.e. glycogen synthesis. Friedman et al. (1990), demonstrated that endurance exercise training induced a significant 2.4-fold increase in GLUT-4 protein in gastrocnemius muscle of trained rats. This adaptation, along with a similar increase in percentage glycogen synthase I observed previously by others (James & Kraegen 1984), suggests that the ability to increase glycogen stores in the trained state is associated with a higher GLUT-4 protein content. James et a!. (1985) used the in vivo euglycaemic clamp with the administration of glucose tracers to study glucose transport and glycogen synthesis in several tissues of rats in response to insulin. They found that red muscle fibres transported glucose faster than white muscle. We have confirmed this observation and find that the higher transport rate in red muscles is most probably a result ofa greater number of glucose transporters in red muscle (Kern et a!. 1990). James et a!. (1985) also observed that glycogen synthesis was more rapid in red than white muscle fibres. Using a perfused rat muscle preparation, Richter et a!. (1988) confirmed the close correlation between glucose transport and glycogen synthesis in red and white muscle fibres. In addition, Richter et al. (1988) found that glycogen synthesis rate decreased dramatically after 3 hours of hyperglycaemic perfusion and this change was paralleled by a decrease in the rate of glucose transport. There was also a decrease in the fractional aetivity of glycogen synthase and the intracellular concentrations of glucose and glucose-6-phosphate. These studies thus confirm the close relationship betweeil glucose transport, glucose transporters and glycogen formation. Further evidence for the importance of glucose transport in the regulation of glucose utilisation by muscle comes from studies of patients with noninsulin-
