John O. Holloszy, Wendy M. Kohrt and Polly A. Hansen

Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA

Received 9/2/98 Accepted 9/8/98

7. REGULATION OF GLUCOSE AND GLYCOGEN UTILIZATION IN MUSCLE DURING EXERCISE AND THE EFFECT OF TRAINING

Transport of glucose through the sarcolemma is the primary rate-limiting step for glucose metabolism in striated muscle. This process has been reviewed in detail (3-5). Glucose transport into the muscle cell occurs by means of a passive transport mechanism that does not use ATP. It is a saturable process that is mediated by glucose transporter proteins, of which two isoforms are expressed in skeletal muscle (88-90). The less abundant GLUT1 isoform is thought to reside primarily in the sarcolemma and contributes to basal glucose transport. The GLUT4 isoform is the major glucose transporter in skeletal muscle. In the basal state, most of the GLUT4 are components of intracellular vesicles. Glucose transport into muscle is stimulated by at least two separate pathways, one of which is activated by insulin, the other by muscle contractions. The increases in glucose transport induced by contractions and by insulin are mediated by a translocation of the GLUT4 vesicles from intracellular sites to the sarcolemma

with fusion of the vesicles with, and incorporation of the GLUT4 protein into, the sarcolemma (88,91-95). Most of the GLUT4 containing vesicles are translocated to the transverse tubules (96,97). These invaginations of the sarcolemma extend deep into the muscle and are adjacent to the sarcoplasmic reticulum. This arrangement makes possible the transport of glucose directly into the interior of the muscle cells where much of the ATP utilized by the myofibrils during contractile activity is generated via aerobic glycolysis and oxidation of the pyruvate that is formed. The transverse tubules are also adjacent to the sarcoplasmic reticulum (SR), which is the region where much of the glycogen resynthesis after exercise occurs in the glycogen-protein complexes associated with the SR. The increases in GLUT4 at the cell surface and in glucose transport induced by maximally effective contraction and insulin stimuli are additive, providing evidence that two separate pathways are involved (93,98-102). The finding that it is possible to inhibit insulin-stimulated, but not contraction-stimulated, glucose transport with the phosphatidylinositol (PI) 3-kinase inhibitor wortmanin provides further evidence for two pathways (93,103,104).

Exercise has three separate, well documented effects on muscle glucose transport (3). The first of these is an insulin-independent stimulation of glucose transport that occurs during normal exercise such as running or swimming (105-108), as well as during electrical stimulation of muscle contractions (109,110). This effect persists sufficiently long to be measurable immediately after contractile activity, but usually wears off completely within 60 minutes or so, in muscles incubated in vitro (111,112). The mechanisms by which muscle contractile activity brings about a movement of GLUT4 into the sarcolemma have not yet been elucidated. However, considerable evidence suggests that the first step in the pathway by which exercise stimulates glucose transport is the increase in cytosolic Ca²⁺ that occurs as a result of release of Ca²⁺ from the sarcoplasmic reticulum during excitation-contraction coupling (113-116).

As the acute, insulin-independent effect of exercise wears off, it is replaced by a large increase in the sensitivity of the glucose transport process to insulin (112,117-121). Insulin sensitivity is defined in terms of the insulin concentration required to cause one-half of its maximal effect. The term insulin responsiveness refers to the increase in glucose transport caused by a maximally effective insulin stimulus. The increase in insulin sensitivity following exercise reverses as muscle glycogen supercompensation occurs (120). Feeding a high-carbohydrate diet speeds the reversal of the increase in muscle insulin sensitivity after exercise, while fasting or feeding a carbohydrate-free diet to prevent glycogen supercompensation results in persistence of the increase in insulin sensitivity for days (120). Glycogen supercompensation is the term used to describe the increase in muscle glycogen concentration above the usual, fed level in response to carbohydrate feeding following glycogen depleting exercise. The mechanism responsible for the increase in muscle insulin sensitivity after exercise has not yet been elucidated. However, it is known that a serum factor is required (122), that sensitivity of glucose transport to contractions and hypoxia is also increased (121), and that translocation of more GLUT4 into the sarcolemma in response to a given submaximal insulin stimulus is responsible for the increased glucose transport activity (123).

Another effect of exercise that influences muscle glucose transport is an adaptive increase in GLUT4 protein (figures 3,4). Endurance exercise-training induces a number of major adaptations in skeletal muscle. These include an increase in muscle mitochondria with an enhancement of the capacity to oxidize carbohydrate and fatty acids (124-126). The half-time of this adaptive increase in mitochondria is ~6 days (127-129). Increases in GLUT4 protein and hexokinase are components of this adaptive response (130-136). The GLUT4 protein has a very short half-life, and, as a result, the increase in GLUT4 occurs even more rapidly than the increases in most of the mitochondrial enzymes (137). In studies on rats, it was found that the adaptive increase in GLUT4 protein in response to a large exercise stimulus plateaus within ~48 hours (137). The adaptive increase in GLUT4 also reverses very rapidly in rats (within 40 hr), after exercise is stopped (figure 3) (138).

Figure 3. Effect of 5 days of exercise training and 40 hr of rest, posttraining on maximally insulin-stimulated glucose transport activity (open bars) and GLUT4 protein content (solid bars) in rat epitrochlearis muscles. Values are the means ± SE for 6-12 rats/group. *P<0.01, **P<0.001 versus sedentary control and 40-hr detrained. From reference (138).

Figure 4. Effect of 2 days of swim training on maximally insulin-stimulated glucose transport activity and GLUT4 protein concentration in rat epitrochlearis muscles. Following the last bout of exercise, animals were either fasted or fed standard rodent chow ad libitum for ~18 hr prior to assay of 2-deoxyglucose uptake and GLUT4 expression. Glycogen concentrations in muscles of the four groups at the time of the assay were as follows: control fasted, 13 mmol/g; trained fed, 81 mmol/g muscle wet weight; control fed, 28 mmol/g; trained fasted, 10 mmol/g; trained fed, 81 mmol/g. *P<0.001 versus sedentary groups. **P<0.001 versus all other groups. From reference (140).

In the absence of conditions, such as visceral obesity, that cause resistance of glucose transport to the actions of insulin and contractile activity, the GLUT4 content of a muscle determines its maximally stimulated glucose transport capacity (figure 3) (101,139). As a consequence, the adaptive increase in muscle GLUT4 induced by exercise is reflected in increases in maximally insulin-stimulated, maximally contraction-stimulated, and maximally contraction-plus insulin-stimulated glucose transport (131,137,138). However, if glycogen supercompensation is allowed to occur after exercise the effect of the increase in GLUT4 induced by training becomes masked so that, despite the increase in GLUT4, the effect of a maximal insulin stimulus on glucose transport is no greater than in control, untrained muscle (figure 4) (140).

The adaptations induced by endurance exercise training are associated with a marked sparing of carbohydrate during exercise, with a slower utilization of plasma glucose, liver glycogen and muscle glycogen during sustained exercise of the same intensity after, as compared to before, training (9,33,141-144). It seems surprising, in view of the increases in muscle insulin sensitivity and GLUT4 content in individuals who exercise regularly, that trained individuals have slower rates of glucose uptake and utilization during exercise of the same absolute exercise intensity than they did in the untrained state (9,84). This apparent discrepancy makes good sense from a teleological viewpoint, because the more rapidly glucose and glycogen are used during exercise, the sooner the individual is forced to stop exercising by either the development of hypoglycemia or depletion of muscle glycogen. Thus the slower utilization of blood glucose and muscle glycogen during exercise in the trained state are among the important mechanisms by which exercise training enhances endurance. However the biological mechanism responsible for the slower utilization of glucose in the face of increases in muscle insulin sensitivity and GLUT4 content has not yet been established. One possible mechanism that may partially explain the plasma glucose sparing effect of training could be that the GLUT4 vesicles in trained muscle are more resistant to translocation to the cell surface by contractile activity (145).

While it is well established that glucose utilization during exercise of the same absolute intensity is slower in the trained state, the effect of exercise training on the rate of plasma glucose utilization during exercise of the same relative intensity, i.e. at the same percentage of VO₂max, is less clear cut. In one study, in which the same individuals were tested before and after 10 weeks of endurance exercise training, it was found that the rate of blood glucose utilization during a standardized exercise bout of the same relative intensity was the same before and after training (84). Because the same relative exercise intensity following adaptation to endurance training requires an increased rate of energy expenditure and involves a larger muscle mass, the proportion of energy derived from carbohydrate was decreased while the proportion provided by fat oxidation was increased in this study. In another study in which untrained individuals were compared to trained athletes, a situation in which the difference in level of training was much more marked, it was found that the same relative exercise intensity resulted in a slower rate of blood glucose utilization in the trained individuals (86). In any case, it is well established that training results in a proportionally lower reliance on carbohydrate utilization and a greater reliance on fatty acid oxidation for generation of the energy required during prolonged steady state exercise.

7.1 Regulation of Glycogenolysis

It has been known since the late 1960's that muscle glycogen is required for strenuous exercise. When muscle glycogen stores are depleted muscle fatigue develops and vigorous exercise can no longer be continued (44,146-148). It is still not clear why muscle glycogen is essential for strenuous exercise when other substrates including fatty acids and blood glucose are still available. This is a question that requires further investigation. On the other hand, our understanding of how glycogenolysis is geared to work rate and the mechanism by which endurance exercise training spares muscle glycogen have been largely elucidated in recent years. During vigorous exercise, or electrical stimulation of muscles to contract in situ, at a rate at which a steady state can be maintained, there is a large initial burst of glycogenolysis followed by a marked slowing of the rate of glycogen breakdown (149-152). The increase in lactate that results from the initial burst of glycogenolysis is followed by a decrease in muscle and blood lactate levels, despite continued muscle contractile activity, as a result of the marked slowing of glycogenolysis (151-154).

The initial burst of glycogenolysis results from activation of phosphorylase by the increase in cytosolic calcium that occurs during excitation contraction coupling. Glycogen phosphorylase, which catalyzes glycogen breakdown, exists in two molecular forms. Phosphorylase b, which is inactive under the conditions usually found in resting muscle cells, is converted to the (or its’) active a form by the enzyme phosphorylase kinase which requires calcium for activity (155-158). Phosphorylase kinase also exists in a dephosphorylated, less active or b form that is converted to the more active a form by protein kinase A, which is activated by the increase in cyclic AMP induced by increased catecholamine levels (155-158).

Phosphorylase kinase a can activate phosphorylase at the low intracellular Ca²⁺ concentrations that are found in the cytoplasm of resting muscle cells; in contrast, phosphorylase kinase b is inactive in resting muscle, but becomes active at the cytosolic calcium levels that are attained when muscles are activated to contract (155-158). The classical, textbook picture of the regulation of glycogenolysis in skeletal muscle was based on the concept that glycogenolysis does not occur in resting muscle because phosphorylase is essentially completely in the inactive b form (156,157,159,160). However, it has become clear during the last 20-30 years that approximately 10% of phosphorylase is in the active a form under physiological conditions (161-163). The presence of phosphorylase a in resting muscle was initially attributed to a preparation artifact as a result of contraction of the muscle during freezing or homogenization. However, the methodology has improved to the point where muscles can be clamp frozen almost instantaneously, and it has become evident that there is always a considerable amount of phosphorylase in the active form in resting muscle (150,161-163).

According to the classical concept, it was though that glycogen breakdown is geared to muscle contraction by the transient increases in calcium concentration in the cytoplasm during each contraction. It was thought that the rate of glycogenolysis is determined by the frequency of muscle contraction, with release of Ca²⁺ from the SR during each excitation-contraction coupling resulting in conversion of inactive phosphorylase b to active phosphorylase a and a burst of glycogenolysis (155,157,159,164-166). The studies on which this original concept of glycogenolysis regulation was based were done either on purified enzymes, on glycogen-enzyme particles, or on muscles that were subjected to brief tetanic stimulation. More recently, during studies involving longer periods of stimulation, it was found that phosphorylase activation reverses within a few minutes during continued contractile activity (150,167-170), and that the reversal occurs despite sustained contractile activity in the absence of fatigue (150). If phosphorylase activation did not reverse after a short time, resulting in a slowing of glycogenolysis, prolonged vigorous exercise would be impossible. Activation of phosphorylase by the calcium mechanism results in a marked overshoot of glycogen breakdown relative to the need for energy, and it would result in rapid depletion of glycogen, massive accumulation of lactate and rapid development of fatigue if it persisted.

It has now become apparent that the initial Ca²⁺-mediated, massive burst of glycogenolysis shuts off within a few minutes after making a large supply of pyruvate available to the mitochondria. The proportion of phosphorylase in the a form then drops back to the level found in resting muscle, i.e. approximately 10% (150,168-170). Most of the phosphorylase in muscle is bound to a glycogen-enzyme-sarcoplasmic reticulum complex that also contains phosphorylase kinase (165,171). Although the mechanism by which phosphorylase activation reverses during continuous exercise is not fully understood, it seems likely that the reversal is at least in part due to release of phosphorylase from the glycogen particle as the glycogen breaks down, thus uncoupling phosphorylase kinase and the calcium activating mechanism from phosphorylase. This mechanism may explain why the activation of phosphorylase both by contractile activity and by epinephrine is severely inhibited in glycogen depleted muscles following exercise (172)

It is well documented that prolonged strenuous exercise to the point of exhaustion can result in almost complete glycogen depletion (44). This raises the question: if the calcium mechanism for activating phosphorylase becomes inactivated soon after the onset of exercise, how does muscle glycogen depletion continue to occur? One possible explanation that has been suggested is that there may be a reactivation of phosphorylase during continued exercise via the beta adrenergic stimulation-cAMP mechanism (169,173). Arguing against this possibility is the lack of evidence that a reactivation of phosphorylase occurs during continuous exercise and the finding that, after the initial burst of glycogenolysis, the rate of glycogen breakdown is rather closely geared to the energy requirement of the working muscles.

In this context, a new explanation for the continuous breakdown of glycogen during prolonged, strenuous exercise and the gearing of glycogenolysis to work rate has evolved. It is based on two concepts that have gradually won acceptance. One is that, contrary to the classical concept that essentially all of the phosphorylase in resting muscle is in the b form, a considerable proportion, in the range of 8-15%, of muscle phosphorylase is in the a form in resting muscle in vivo (161-163). The other is that it is the concentration of free inorganic phosphate (Pi), not phosphorylase activity, that limits glycogenolysis under most conditions, and this is why rapid glycogen breakdown does not occur in resting muscle despite the presence of considerable phosphorylase activity (161,163,170).

Direct support for this concept was provided by a study on rat epitrochlearis muscles incubated in vitro in which cytosolic Pi was raised by means of hypoxia, and phosphorylase was activated with epinephrine (163). Over an 80 minutes period, hypoxia resulted in a progressive ~70% decrease in epitrochlearis muscle glycogen concentration despite no increase in % phosphorylase a. Inorganic phosphate concentration increased rapidly in response to the hypoxia, roughly mirroring the decline in phosphocreatine concentration. Incubation of oxygenated muscles with a high concentration of epinephrine for 20 minutes resulted in a large increase % phosphorylase a. Despite the activation of phosphorylase, there was only a small decrease in muscle glycogen (2.5 mmol glucose/g) over a 20 minute period (figure 5). No decrease in glycogen occurred in well oxygenated control muscles. In contrast, the muscles incubated under hypoxic conditions, in which there was no activation of phosphorylase, showed a 12 mmol glucose/g decrease in muscle glycogen over the 20 minute period (figure 5).

Figure 5.Changes in phosphorylase a and glycogen concentration in rat epitrochlearis muscles elicited by a 20 min in vitro incubation in oxygenated Krebs Henseleit buffer (KHB) (control), KHB gassed with 95% N₂-5% CO₂ (hypoxia), or oxygenated KHB containing 0.1 mM epinephrine. From reference (163).

The very small glycogen breakdown caused by a more than six fold increase in phosphorylase activity in response to epinephrine is explained by the fact that Pi levels were unchanged and therefore limited the ability of

phosphorylase to catalyze the reaction: Glycogen + Pi ® Glucose 6-P. Inhibition of phosphorylase b with 2-deoxyglucose-6-phosphate had a negligible effect on the stimulation of glycogenolysis by hypoxia, providing evidence that phosphorylase b activation by AMP was not playing a significant role (163). These findings show that the basal level of phosphorylase a activity present in non-stimulated muscle can mediate a moderately rapid rate of glycogenolysis in response to an increase in inorganic phosphate. Since the magnitude of the increase in Pi during muscle contractile activity is a function of the work rate, this mechanism explains how glycogenolysis is geared to work rate after the % phosphorylase a returns to the basal level in contracting muscle.

One of the most important physiological effects of the rapid increase in muscle mitochondria in response to endurance exercise training is the sparing of muscle glycogen during submaximal exercise (143,144,152). This effect is evident both as a smaller initial burst of glycogenolysis and a slower subsequent rate of glycogen utilization during prolonged exercise. In studies on rat skeletal muscles stimulated to contract in situ, it was found that the effect of training on the initial, calcium-induced burst of glycogenolysis is markedly blunted in endurance exercise trained muscle (151,153) even though there is no difference in the extent of phosphorylase activation between trained and untrained muscles subjected to the same stimulation protocol (153). This glycogen sparing effect of the adaptive increase in mitochondria in trained muscle is mediated by a smaller decrease in creatine phosphate and, therefore, a smaller increase in Pi in response to the same work rate (151,153,174). Thus, the steady state concentration of inorganic phosphate attained in muscles during continued contractile activity is lower in the trained muscles (151,153,174). It, therefore, seems probable that both the smaller initial burst of glycogenolysis and the slower rate of glycogen breakdown during continuous contractile activity is explained by the lower level of inorganic phosphate attained in trained muscles.

7.2 The Muscle Glycogen Supercompensation Phenomenon

The term "glycogen supercompensation" refers to the large increase in muscle glycogen concentration, far above the levels found in the well-fed, sedentary state, that occurs in response to carbohydrate feeding following a glycogen-depleting exercise bout (44,175-177). Glycogen supercompensation is limited to the muscles in which glycogen was depleted by the exercise. This was clearly shown by Bergström and Hultman (176) who first discovered the supercompensation phenomenon in a study in which they used themselves as subjects.

Glycogen synthase D, the inactive form of the enzyme, is converted to the active form, glycogen synthase I, by the action of glycogen synthase phosphatase (178,179). Both of these enzymes are bound to glycogen, and when glycogen is broken down during exercise they are released into the cytosol. This makes the inactive glycogen synthase D accessible to glycogen synthase phosphatase, which converts it to the active form, glycogen synthase I (180,181).

There has been much interest in the concept that glycogen synthase activity limits and determines the rate of muscle glycogen synthesis (177,182-184). With regard to the synthesis of muscle glycogen after glycogen-depleting exercise, it is now well documented that the increase in the proportion of glycogen synthase in the active I form is only involved in the early, rapid phase of glycogen repletion. It does not play a role in glycogen supercompensation. The evidence for this is that the activation of glycogen synthase reverses, with a decline in the proportion of glycogen synthase in the I form to a low level, when muscle glycogen concentration attains the usual, fed sedentary level, i.e. before glycogen supercompensation begins (185,186). Thus, glycogen supercompensation occurs despite low glycogen synthase activity.

The factor that regulates the rate and extent of muscle glycogen accumulation appears to be the rate of glucose transport into muscle. The factors that determine the rate of glucose transport are the number of GLUT4 at the cell surface and the concentration of glucose in the interstitial space. One line of evidence that the rate glucose entry into muscle exerts the primary control on glycogen synthesis comes from studies on transgenic mice that overexpress the GLUT1 glucose transporter in their muscles (187). Basal glucose transport is increased approximately 7-fold in muscles of GLUT1 transgenic mice. This results in a massive accumulation of glycogen in their muscles, to values ten-fold higher than those found in muscles of fed normal wild-type mice despite a 50% reduction in glycogen synthase I activity (187).

Further evidence is provided by a more physiological model, rats that have adapted to exercise with an increase in the GLUT4 content of their muscles (137,186). This adaptation occurs very rapidly and also reverses quickly (137,138). Muscles of rats that have adapted to exercise with an increase in GLUT4 have increased rates of glucose uptake and glycogen synthesis than muscles of untrained animals when the muscles are incubated in vitro with glucose and insulin despite no difference in glycogen synthase I activity (137). Furthermore, exercise-trained rats and humans with increased numbers of GLUT4 in their muscles have markedly greater rates of muscle glycogen resynthesis and attain higher levels of muscle glycogen supercompensation than untrained controls (186,188) (Figure 6).

Figure 6. Time course of epitrochlearis muscle glycogen accumulation in trained (solid bars) and untrained (open bars) rats after a glycogen-depleting bout of exercise. After an overnight fast, animals performed a bout of swimming exercise, then were allowed to recover with free access to standard rodent chow and 5% sucrose in their drinking water. Muscles were harvested at the indicated times. *P<0.001 versus untrained. From reference (186).

What physiological advantage does this adaptation confer. There is extensive evidence that starting a prolonged bout of exercise requiring ~75% of VO₂max with a markedly supercompensated muscle glycogen store postpones fatigue and improves performance, because strenuous exercise can not be continued once muscle glycogen is depleted (9,44,148,189-192). However, this beneficial effect of marked glycogen supercompensation is limited to moderately strenuous exercise lasting in the range of ~90 to 180 minutes (see (190) for review). For shorter periods of moderately strenuous to strenuous exercise (i.e. ~60 to 100% of VO₂max), starting with glycogen supercompensated muscles provides no benefit (190), while more prolonged moderately strenuous exercise requires carbohydrate ingestion during the exercise. In this context, it seems likely that the major beneficial effect of the adaptive increase in GLUT4, that provides the survival benefit for which it was selected, is the increased ability to rapidly resynthesize glycogen to replenish depleted muscle glycogen stores. As reviewed earlier, vigorous exercise becomes impossible once muscle glycogen becomes depleted, making fight or flight impossible under life-threatening conditions. The rapid adaptive increase in muscle GLUT4 in response to exercise makes possible more rapid muscle glycogen repletion when carbohydrate is eaten during brief rest periods, or even on-the-run (193). This would provide a survival advantage in situations in which a sustained increase in physical activity becomes necessary as, for example, when an animal’s territory is invaded by predators.