Copyright 1976. All rights reserved

BIOCHEMICAL ADAPTATIONS

TO ENDURANCE EXERCISE

IN MUSCLE

John O. Holloszy and Frank W. Booth Department of Preventive Medicine, Washington University School of Medicine.

St. Louis, Missouri 63110

INTRODUCTION

+1151

Two quite distinct adaptive responses can be induced in skeletal muscle by regularly performed, strenuous exercise. The nature of the exercise stimulus determines the type of adaptation. One type of adaptation involves hypertrophy of the muscle cells with an increase in strength; it is exemplified in its most extreme form by the muscles of weight lifters and body builders. The second type of adaptation involves an increase in the capacity of muscle for aerobic metabolism with an increase in endurance and is found in its most highly developed form in the muscles of competi­tive middle- and long-distance runners, long-distance cross-country skiers, bicy­clists, and swimmers. Although many types of physical activity can bring about varying degrees of both types of adaptation in the same muscle, it does appear that these adaptations can occur quite independently of each other in their most extreme forms. For example, the hypertrophied muscles of weight lifters do not appear to have an increased respiratory capacity (45), whereas the muscles of rodents trained by prolonged daily running, which have a large increase in respiratory capacity, are not hypertrophied (56, 92) and show no increase in strength (12).

This review deals with the biochemical adaptations induced in skeletal muscle by the endurance type of exercise and with the physiological consequences of these adaptations.

BACKGROUND INFORMATION

Different Types of Skeletal Muscle Fiber

Most of the skeletal muscles in those mammalian species in which the chronic adaptive responses to exercise have been studied are a mixture of three different fiber types. In rodent muscles, these are the fast-twitch white fibers, which have a low respiratory capacity, a high glycogenolytic capacity, and high myosin ATPase

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274 HOLLOSZY & BOOTH

activity; the fast-twitch red fibers. which have a high respiratory capacity. a high glycogenolytic capacity, and high myosin ATPase activity; and the slow-twitch red fibers, which have a moderately high respiratory capacity, a low glycogenolytic capacity, low myosin ATPase activity, and are fatigue resistant (5, 7, 8, 33, 97). In many other mammalian species, including man, in contrast to rodents, the slow­twitch rather than the fast-twitch red fibers have the highest respiratory capacity (22, 30, 41).

The earlier studies of the biochemical adaptations of muscle to endurance exercise were performed on whole mixed muscles, such as the gastrocnemius, of rodents, and on biopsy specimens of mixed muscles in human subjects. In more recent studies, two approaches were used to evaluate the adaptive responses of various types of muscle fiber. One approach has been to evaluate the levels of various enzymes histochemically in rodent and human muscles by their staining intensity; this quali­tative approach has led to some problems in interpretation which are discussed later. In other studies on rodents, the soleus muscle, which consists predominantly of slow-twitch fibers, has been used for biochemical studies on slow-twitch red fibers. For biochemical studies on fast-twitch white fibers, the superficial portion of the whole quadriceps, or of the vast us lateralis of the rat has been used, as these consist essentially of white fibers. The deepest red portion, closest to the femur, of the whole quadriceps or of the vastus lateralis, which consist predominantly of fast-twitch red fibers in the rat were used for studies on fast-twitch red fibers, (5, 8, 9, 126, 130). It is much more difficult to study the different fiber types in human muscle biopsies because of the small amount of tissue and because the red and white fibers are closely intermingled in human muscle. However, biochemical studies on individual fibers are underway in a number of Scandinavian laboratories; portions of single fibers, dissected out of freeze-dried sections of biopsy specimens, are analyzed using mi­croanalytical methods (36).

In studies on rodents, running has been the most effective and commonly used form of training (II, 49, 56, 121). In one program used in numerous studies, young rats are trained to run for progressively longer periods on a motor-driven treadmill up 1m SO incline until at the end of 12 weeks they are running at 31 meters per minute for two hours per day, five days per week. They are maintained at this work level for a few more weeks. This program produces a high level of training, with a large increase in endurance but no muscle hypertrophy (40, 56, 92). In rats, swimming is less effective than running for inducing adaptive changes in the skeletal muscles, as these animals do only the minimum work necessary to keep afloat. Six hours of daily swimming for 14 weeks produced only 35% as great an increase in the respiratory capacity of leg muscles in rats as does the running program just de­scribed (4).

MITOCHONDRIAL ADAPTATIONS TO ENDURANCE EXERCISE

Studies on muscle homogenates and on the mitochondrial fraction from muscle have shown that endurance exercise-training increases the capacity of skeletal muscle to

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ADAPTATIONS TO EXERCISE 275

oxidize pyruvate. This increase in the capacity to oxidize carbohydrate has been demonstrated in rats (5, 56), humans (86), and guinea pigs (11). Subsequent studies on muscle mitochondria (84) and on muscle homogenates (5, 84) showed that the capacity to oxidize long chain fatty acids is also increased in exercise-trained rats. The increase in muscle respiratory capacity varies with the duration and intensity of the exercise. In rats subjected to the treadmill running program described earlier, the capacities to oxidize fat and carbohydrate both increase twofold (5, 56, 84). The rates at which homogenates of leg muscles oxidize ,8-hydroxybutyrate and acetoace­tate are also increased in trained rats (127). These adaptations involve all three types of muscle fiber (5, 126).

The mitochondria from the trained animals' muscles exhibit normal respiratory control and tightly coupled oxidative phosphorylation (11, 56, 84), providing evi­dence that the increases in the capacities to oxidize fat and carbohydrate are accom­panied by a parallel rise in the capacity to generate A TP via oxidative phos­phorylation.

Underlying the exercise-induced increase in the capacity of muscle to generate A TP from the oxidative metabolism of substrates are increases in the levels of the enzymes involved in the activation, transport, and ,8-oxidation of long chain fatty acids (58, 84), the enzymes involved in ketone oxidation (126, 127), the enzymes of

. the tricarboxylic acid (TCA) cycle (29, 47, 62), the components of the mitochondrial respiratory chain involved in the oxidation of DPNH and succinate (16, 56, 86, 124), and mitochondrial coupling factor 1 (88). These increases in the levels of activity of a wide range of mitochondrial enzymes appear to result from an increase in enzyme protein concentration. The increases in the concentrations of the cyto­chromes (14, 56, 62) and in the protein content of the mitochondrial fraction of skeletal muscle is in accord with this inference (56, 86). Electron-microscopic stud­ies on human (65, 86) and on rat (49) skeletal muscles have provided evidence that increases in both the size and number of mitochondria are responsible for the increase in total mitochondrial protein. In addition to the increase in size and number, there is also an alteration in the composition of skeletal muscle mito­chondria in rats that have adapted to endurance exercise (61, 62, 88, 126). For example, in rats adapted to the two-hour-Iong program of daily running described in the preceding section, a number ofTCA cycle enzymes, including citrate synthase (62, 126), aconitase (58), NAD-specific isocitrate dehydrogenase (62), and succinate dehydrogenase (62), increased twofold, and others, including a-ketoglutarate dehy­drogenase (62) and malate dehydrogenase (62, 83), increased 50-60%. The TCA cycle-related enzyme glutamate dehydrogenase increased only about 30% (62) and acetoacetyl-CoA thiolase increased approximately 50% (126, 127). A number of mitochondrial enzyme activities do not increase at all in muscle in response to training when expressed per gram of muscle and, because of the increase in mito­chondrial protein, are decreased when expressed per milligram of mitochondrial protein; these enzymes include mitochondrial creatine kinase (88), adenylate kinase (88), and a-glycerophosphate dehydrogenase (61). Creatine kinase and adenylate kinase activities in the cytoplasm are also unchanged in gastrocnemius muscle (88).

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276 HOLLOSZY & BOOTH

The exercise-induced adaptive responses of a number of mitochondrial enzymes have been studied in all three types of skeletal muscle fibers in the rat. Of these, citrate synthase (5, 126), carnitine palmityltransferase (5), cytochrome oxidase (5), cytochrome c (F. W. Booth and J. O. Holloszy, unpublished data), and acetoacetyl­CoA thiolase (126) increased to roughly the same extent, on a percentage basis, in the three fiber types. In contrast, !3-hydroxybutyrate dehydrogenase activity in­creased 2.6-fold in slow red muscle, and sixfold in fast red muscle, while changing from not detectable to just measurable in white muscle of rats subjected to the running program described earlier (126).

Mitochondria from normal mammalian tissues are impermeable to NADH; a number of mechanisms have been proposed to explain how NADH formed during glycolysis is oxidized (123). The best document�d of these are the malate-aspartate and the a-glycerophosphate shuttles (123). Muscles of trained individuals appear to produce less lactate than those of untrained individuals, even at comparable rates of glycogenolysis (111). This finding suggests that the capacity to transfer reducing equivalents to the respiratory chain from cytoplasmic NADH might be increased in response to exercise. In studies designed to test this possibility, mitochondrial a-glycerophosphate dehydrogenase, expressed per gram of muscle, was unaffected in rat gastrocnemius muscle (61). In contrast, the enzymes of the malate-aspartate shuttle were increased in the mitrochondria and the cytoplasm of leg muscles of trained rats (57).

A pathway for pyruvate removal in skeletal muscle is conversion to alanine via the alanine transaminase reaction. The quantitative importance of this pathway has been demonstrated by Felig & Wahren (38). Alanine transaminase activity increases in both the mitochondria and cytoplasm of gastrocnemius muscles of endurance exercise-trained rats (83). This adaptation could result in conversion of a greater proportion of the pyruvate formed in muscle to alanine and less to lactate, and thus help protect against the development of acidosis in muscle during strenuous exer­cise.

There is evidence that hyperthyroidism is associated with an increase in mito­chondria in many tissues (118), and there has been some interest in the possibility that thyroid hormones play a role in the increase in muscle mitochondria induced by exercise. Although thyroidectomized rats have lower levels of succinate dehy­drogenase activity in their skeletal muscles than euthyroid animals, a highly signifi­cant increase in the level of this enzyme occurred in thyroidectomized animals in response to a program of running (48). Hypophysectomized rats also show an increase in succinate dehydrogenase in skeletal muscle in response to training (48). Euthyroid exercised rats do not have any increase in triiodothyronine (T 3) and thyroxin (T4) concentrations in skeletal muscle (129). Prolonged, severe thyrotox­icosis induces only 40% as great an increase in mitochondria in rat gastrocnemius muscle as does a program of treadmill running (128). It seems clear from all these observations that the increase in muscle mitochondria induced by exercise is not mediated by thyroid hormones.

Skeletal muscles of protein-deficient and diabetic rats have lower levels of mito­chondrial enzymes than normal controls, but also undergo large increases in mito­chondrial content in response to endurance exercise (44, 66).

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ADAPTATIONS TO EXERCISE 271

Effects 0/ a Reduction in Contractile Activity on Muscle Mitochondria

Not surprisingly, some of the biochemical changes induced in skeletal muscle by a reduction in contractile activity run counter to those seen in response to a chronic increase in contractile activity. The magnitude of the changes brought about by immobilization depends on the extent to which contractile activity is reduced. Some techniques for producing immobilization are more effective in reducing contractile activity than others. For example, immobilization of both the knee and ankle joints reduces contractile activity more than does immobilization of the ankle joint alone (39). Also, immobilizing a muscle in a position shorter than its normal resting length produces greater atrophy than immobilizing it at at greater than normal resting length (116).

In one study, ten days of immobilization resulted in decreases in the capacities of rat plantaris and soleus muscles to oxidize J3-hydroxybutyrate, palmitate, and glucose (104). This decrease was significant when expressed either per muscle or per gram of muscle (104). The weights of the soleus and plantaris muscles decreased to 70% and 60%, respectively, of control values after 10 days of immobilization (104). In addition, significant decreases in the respiratory control index and ADP /0 ratio (81) and a significant increase in ATPase activity (82) of mitochondria from rat gastrocnemius muscles immobilized from one to nine days have been reported.

The decrease in the levels of mitochondrial enzymes during immobilization does not appear to be the same in the different types of skeletal muscle fiber. Cytochrome oxidase activity per gram of muscle was significantly reduced in fast-twitch red, but not in slow-twitch red or fast-twitch white, fibers in rats after four weeks of immobil­ization (19). The time course of the decrease in the levels of various mitochondrial enzymes in the same muscle varies considerably. It has been reported that in rat skeletal muscle, cytochrome oxidase and monoamine oxidase activities per milli­gram of mitochondrial protein are unchanged after nine days of immobilization, whereas mitochondrial malate dehydrogenase is significantly decreased after two days (103). It is not known whether the decrease in the capacity of immobilized muscle to generate A TP via aerobic metabolism plays a role in the development of muscle atrophy or just occurs concomitantly.

TRIGLYCERIDE METABOLISM

Endogenous triglyceride stores can contribute a considerable portion of the energy utilized by red skeletal muscle during exercise (6, 23, 43, 101). In a group of men trained by bicycling, the capacity of quadriceps muscle to incorporate fatty acids into triglycerides was increased (86). Intramuscular stores of triglycerides also appear to be increased in trained men (85). In rats trained by means of treadmill running, a significant increase occurred in the capacity of skeletal muscle to synthe­si�e triglycerides by esterification of glycerol-3-phosphate (2).

Exercise can result in a reduction in serum triglyceride levels (64). There is evidence that serum trigJycerides can be utilized by skeletal muscle (71). In this context, the finding in a recent study (21) that lipoprotein lipase activity is increased threefold in fast-red muscle, twofold in slow-red, and twofold in white muscle fibers

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278 HOLLOSZY & BOOTH

in rats subjected to a 12 week long program of running is of considerable interest, as it suggests that the capacity to hydrolyze triglycerides to free fatty acids (FFA) may be increased by exercise-training.

MYOGLOBIN

In the skeletal muscles of land mammals, myoglobin content generally closely parallels respiratory capacity; muscles that are dark red in color are rich in mito­chondria and myoglobin; white muscles have a low respiratory capacity and contain little myoglobin. Exercise-training can increase muscle myoglobin concentration (78, 92). In rats subjected to a program of running for 14 weeks, myoglobin in­creased approximately 80% in hindlimb muscles (92). Myoglobin increases the rate of O2 diffusion through a fluid layer (52). It seems likely that myoglobin may also facilitate O2 utilization in muscle by increasing the rate of its diffusion through the cytoplasm to the mitochondria.

PHYSIOLOGICAL CONSEQUENCES OF THE EXERCISE-INDUCED INCREASE IN THE CAPACITY OF SKELETAL MUSCLE FOR AEROBIC METABOLISM

Submaximal Exercise

Individuals who have adapted to endurance exercise derive more of their energy from fat and less from carbohydrate than untrained individuals during submaximal exercise (26, 54, 110). The term submaximal exercise is used here to mean work requiring less than the individual's maximum capacity to utilize O2 (V02 max). Serial biopsies of the quadriceps muscle during standardized submaximal exercise, showed that men deplete their muscle glycogen stores less rapidly when they are trained than when they are untrained (54, 110, 111). Exercise-trained rats deplete both muscle and liver glycogen less rapidly than untrained animals during standard­ized exercise tests (4, 40). In a recent study (40), the total amount of glycogen utilized during a standardized bout of exercise was inversely related to the respira­tory capacity of the rat's muscles; the magnitude of the increase in mitochondria in the animals' gastrocnemius muscles varied over a twofold range as a result of varying the duration of the different groups' daily exercise sessions between 10 and 120 minutes per day. There was a significant correlation between how long the animals could run before they became exhausted and the respiratory capacity of their muscles (40). It is also well documented that trained individuals have lower blood (35, 54, 105, 110, 111) and muscle (I 10, 111) lactate levels than untrained individuals during submaximal exercise.

For many years it was believed that the trained individual's lesser reliance on carbohydrate [as reflected in lower lactate levels, slower glycogen depletion, and a lower respiratory quotient (RQ) during submaximal exercise] and his greater endur­ance were a result of improved delivery of O2 to the working muscles. This concept that trained individuals' muscles are better supplied with blood and O2 and are

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ADAPTATIONS TO EXERCISE 279

therefore "less hypoxic" than muscles of untrained individuals during submaximal exercise now appears to have been invalidated by studies showing that blood flow per gram of working muscle is lower in trained than in untrained men at the same absolute work level (16, 27, 50, 124). The working muscles compensate for the lower blood flow in the trained state by extracting more O2; this is reflected in a greater arteriovenous O2 difference (35, 109). Muscle blood flow appears to be similar at the same relative work load, that is, at the same percentage of the individual's V02max, in the trained and untrained states (50). Further evidence against improved O2 delivery to hypoxic muscles is the finding that O2 uptake at the same absolute work load is the same in the trained and untrained states (27, 109, 110). If the working muscles were hypoxic, and if their O2 supply were increased by training, one would expect O2 uptake to be higher in the trained state at the same submaximal work level. In this context, it appears likely that the skeletal muscles' increased content of mitochondria and myoglobin, rather than improved O2 delivery, is responsible for the trained individual's lower lactate levels, slower glycogen depletion, and lower RQ during submaximal exercise.

The rate at which muscle cells consume oxygen during work is primarily deter­mined by the frequency of contraction when load is constant; the O2 uptake of muscle cells can therefore be varied over a wide range by varying the work rate (24, 42). The mechanism by which oxygen consumption is closely geared to work rate relates to the tight coupling of phosphorylation of ADP to electron transport. When O2 and substrate are not limiting, the rate of respiration appears to be an inverse function of the ratio ATP/(ADP + Pi) (79). When muscle contracts, ATP and creatine phosphate (CP) are split and the levels of ADP and Pi in the mitochondria rise, leading to an increase in the rate of respiration. The increase in mitochondrial ADP appears to follow a saturation curve and attains a steady-state level, which is determined by the frequency of contraction (68), and, in turn, is largely responsible for determining the rate of O2 consumption (79). Once steady-state levels of mito­chondrial ADP and O2 consumption are attained in a muscle cell contracting at a frequency that results in a submaximal rate of O2 uptake (i.e. aerobic work), the rate of A TP formation via oxidative phosphorylation during and between muscle con­tractions must be sufficiently great to balance the rate of ATP splitting during the contraction. In the period between the beginning of work and attainment of the steady state, before ATP hydrolysis is balanced .by oxidative phosphorylation, the concentrations of CP and ATP fall in muscle until steady state is attained. Simulta­neously, the concentrations of ADP and Pi must rise in the mitochondria until electron transport, O2 consumption, and oxidative phosphorylation increase suffi­ciently to balance ATP breakdown.

Oxygen consumption is the same in the trained and untrained states at the same submaximal work rate (27, 109, 110). As muscle adapted to endurance exercise has up to twice as many mitochondrial cristae per gram as untrained muscle, the steady-state levels of intramitochondrial ADP and Pi required to attain the same submaximal rate of O2 consumption at a given work rate must be lower in trained than in untrained muscle. This is so because, with more mitochondrial respiratory chains, the rate of electron transport and O2 consumption per respiratory chain must

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280 HOLLOSZY & BOOTH

be lower to attain the same total O2 consumption. In other words, the greater the number of mitochondrial respiratory chains per gram of muscle, the lower must be the O2 uptake per respiratory chain to maintain a given submaximal level of O2 uptake per gram of muscle. In this context, it seems reasonable that, in the process of attaining a given steady.state level of O2 consumption, CP and ATP levels must decrease less and ADP, Pi, and creatine levels must increase less in muscles of trained as compared to untrained individuals. Because muscle contains high levels of adenylate kinase, some of the ADP formed is converted to AMP, part of which is deaminated by the action of adenylate deaminase, resulting in formation of ammonia (80). With a smaller rise in ADP, it seems likely that AMP and ammonia levels may also be lower in trained muscles during submaximal exercise, although no information on this point is yet available.

The intracellular levels of ATP, CP, Pi' AMP, ADP, and ammonia play major roles in controlling the rate of glycolysis (76, 91, 122, 131). ATP and creatine phosphate inhibit phosphofructokinase, and this inhibition is counteracted by Pi' ADP, AMP, and ammonia (76, 91,122,131). Therefore, because of higher steady­state levels of ATP and CP and lower levels of Pi; ADP, and, possibly, of AMP and ammonia, glycolysis should occur at a slower rate in muscle adapted to endurance exercise than in untrained muscle at a given submaximal rate of work and O2 utilization. This could, in part, explain the slower rates of muscle glycogen depletion and lactate formation seen in the trained as compared to the untrained state during submaximal exercise. Experimental evidence supporting this line of reasoning has come from studies on muscle biopsies obtained from exercising men (1 I I). At the same submaximal leveIs of work and O2 consumption, the decreases in the steady­state concentrations of CP and ATP, the rate of glycogen depletion, and the increase in lactate in quadriceps muscle were all lower in the trained as compared to the untrained state in the same individual (I I I).

Another factor that helps to account for the slower glycogen depletion and lactate production is the shift in the carbon source of the TCA cycle. As discussed earlier, the trained individual derives a greater percentage of his energy from fat oxidation than does the untrained during submaximal exercise. It seems reasonable to ask why this should be so, as endurance exercise induces comparable increases in the capaci· ties of skeletal muscle to oxidize fat and carbohydrate (5, 56, 84). The answer probably lies in certain of the control mechanisms that regulate carbohydrate metab­olism. Among these is the rate of fatty acid oxidation.

At a given metabolic rate, the rate of fatty acid oxidation by a tissue appears to be determined by two factors; the concentration of fatty acids (i.e. substrate avail­ability) and the capacity of the tissue to oxidize fat. When the metabolic rate is constant at rest, or during steady-state exercise, the rate of fat oxidation increases linearly with fatty acid concentration; saturating concentrations of free fatty acids do not appear to have been attained in vivo (93, 95). Thus the availability of fatty acids to the mitochondria is probably the rate-limiting factor for fatty acid oxidation at any given respiratory rate, in vivo. However, at any concentration of fatty acids, the rate of fatty acid oxidation will be highest in those tissues with the greatest

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ADAPTATIONS TO EXERCISE 281

capacity to oxidize fat. For example, at the same fatty acid concentration, the heart will oxidize fatty acids more rapidly than will skeletal muscle, and red muscle will oxidize fat more rapidly than white. Because the rate at which a substrate is utilized is a function of the level of enzyme activity, regardless of whether or not substrate concentration is at a saturating level, the muscles of trained individuals, with their greater capacity for fat oxidation, could be expected to oxidize more fat at the same fatty acid concentration than those of untrained individuals. An increase in the oxidation of fatty acids results in a decrease in carbohydrate utilization brought about, in part, by a reduction in the rate of glycolysis and pyruvate oxidation (87, 94). This may be mediated by an increase in the concentration of citrate with inhibition of phosphofructokinase (87, 91). As plasma FF A levels tend to be lower in the trained than in the untrained state during submaximal exercise (69, 70, 102, 127), the greater rate of fat oxidation in trained individuals appears to be due entirely to the increase in the capacity of their muscles to oxidize fatty acids.

One factor implicated in the development of muscle fatigue during prolonged exercise, which forces an individual to stop or slow his pace, is depletion of muscle glycogen stores (1, 17). The adaptations induced in skeletal muscle by endurance exercise could, by the above mechanisms, be responsible for postponing depletion of muscle glycogen and thus increasing endurance. The accumulation of a high concentration of lactate may also play a role in the development of fatigue during brief, strenuous exercise (55, 72). Lactic acid concentrations are lower in skeletal muscle and blood in the trained, as compared to the untrained, state at the same submaximal work rate (35, 54, 105, 110, Ill). This difference may be explained by a decrease in the rate of glycolysis by the mechanisms discussed earlier and perhaps by the increases in the capacities of alanine transaminase and of the malate-aspartate shuttle to compete with lactate dehydrogenase for pyruvate and NADH, respec­tively. A third factor that may limit endurance during prolonged exercise is the development of hypoglycemia (25, 100). Trained animals deplete their liver glycogen more slowly than untrained animals during submaximal exercise and are therefore protected against hypoglycemia (4, 40). Decreased utilization of glucose by trained muscle, probably as a result of increased fat oxidation, must play the major role in accounting for the slower depletion of liver glycogen (4, 40).

Maximum O2 Uptake

Endurance exercise-training induces an adaptive increase in maximum cardiac out­put (34, 35, 107, 109), which implies an increase in the maximum capacity to supply O2 to the working muscles. However, studies using the 133Xe clearance method have shown that maximum flow to the working muscles, expressed as milliliters per gram of muscle per minute, is not increased in the trained state (16,27, 50, 124). It would therefore appear that any increase in V02 max brought about by an increase in maximum cardiac output is the result of delivery of O2 to a larger mass of working muscle rather than to delivery of more O2 to the individual muscle cells. Although considerable variability in response has been noted among individuals, on the average, an increase in maximum cardiac output appears to account for appro x-

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282 HOLLOSZY & BOOTH

imately 50% of the rise in V02max that occurs in response to training (34, 35, 107-109).

The other 50% of the increase is accounted for by increased extraction of O2 by the working muscles; this is reflected in an increased arteriovenous O2 difference and a lower O2 tension in venous blood (34, 35, 107-109). It is therefore not surprising that a number of investigators have found a good correlation between skeletal muscle respiratory capacity and V02max (20, 65, 75).

There is no experimental information regarding the mechanism by which trained muscle ceIJs extract more O2 from the blood. However, if delivery of O2 to the muscle cells during maximal exercise is the same in the trained and untrained states, as suggested by the 133Xe clearance data, it seems reasonable to assume that O2 tension in the muscle cells and, secondarily, in the capillaries must be lower in the trained state as a result of the greater number of muscle mitochondria and of the higher work rate required to attain V02max.

ENZYMES OF GLYCOLYSIS, GLYCOGENOLYSIS, AND GLYCOGEN SYNTHESIS

Hexokinase is unique among the glycolytic enzymes in that its activity in different types of muscles varies with respiratory capacity (I5), which is highest in the heart and lowest in white skeletal muscle. It is interesting in this context that exercise, which induces an increase in the mitochondrial content of skeletal muscle, also results in an increase in hexokinase activity (8, 13, 63, 77, 98). In rats subjected to a strenuous program of running, hexokinase activity increased 170% in fast-twitch red muscle, 52% in slow-twitch red muscle, and 30% in white muscle (8). As is discussed later, these differences in response may reflect the extent of involvement of the different types of muscle fibers in prolonged, submaximal exercise. Like exercise, insulin administration increases hexokinase to supernormal levels, whereas insulin deprivation decreases hexokinase in skeletal muscle (73). The effect of insulin on hexokinase may be mediated by increased entry of glucose into muscle (73). In keeping with this hypothesis is the finding that repeated muscle contraction also markedly increases muscle cell permeability to sugar (5t, 59, 60). In contrast to the mitochondrial enzymes, which increase in response to prolonged bouts of exercise over a period of weeks, hexokinase activity increases in response to single bouts of prolonged exercise (77) or a few brief bouts of exercise (13).

In the rat, the other glycolytic enzymes undergo rather small changes in response to endurance exercise. In fast-red muscle, which has a high glycolytic capacity, a decrease of approximately 20% occurred in the levels of glycogen phosphorylase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, cytoplasmic a-glycerophosphate dehydrogenase, and lactate dehydrogenase in re­sponse to the running program described earlier (8). It has been reported that the decrease in lactate dehydrogenase is limited to the �'M" isozyme of the enzyme (132). Slow-red muscle, which has a low glycogenolytic capacity, in contrast to fast-red muscle, underwent small increases in the above enzymes (8). In white muscle, the only change found in the glycolytic enzymes, other than hexokinase, was

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ADAPTATIONS TO EXERCISE 283

a 15% decrease in lactate dehydrogenase (8). In studies on mixed muscles such as the gastrocnemius and quadriceps, the exercise-induced decreases in the glycolytic enzymes in fast-red muscle are obscured by the increases in slow-red and the unchanged glycolytic enzyme levels in the white fibers (8, 63). Like endurance running, a program of sprinting has been reported to produce a small increase in glycolytic enzymes in soleus muscle; however, no changes were found in fast-red or fast-white muscle ( l12).

Some confusion exists regarding the response of phosphofructokinase (PFK) activity in human skeletal muscle to exercise-training. In a study in which subjects trained one leg by means of bicycle exercise while the other leg served as an untrained control, no significant changes were found in phosphorylase, PFK, aldo­lase, or pyruvate kinase in quadriceps muscle (86). In contrast, another group of investigators reported that a strenuous program of bicycle exercise results in a greater than twofold increase in PFK activity in the quadriceps (47). However, the same investigators reported that PFK activity in skeletal muscles of highly trained competitive bicyclists, distance runners, and swimmers is no higher than in seden­tary controls (45).

The finding that muscle glycogen concentration is generally elevated in trained individuals (45, 47) has led to investigation of the effects of training on enzymes involved in glycogen synthesis. Total glycogen synthetase activity was increased in skeletal muscles of humans and rodents in response to exercise training (67,86, 120). The magnitude of this increase varied between 30% and 100%. Glycogen branching enzyme activity was also increased in muscle with training (119). These findings suggest that there is an increased capacity for glycogen synthesis in trained muscles.

ADENINE NUCLEOTIDE CYCLE ENZYMES

Ammonia formed by muscle during work arises from deamination of AMP in the AMP deaminase reaction; the IMP thus formed may be reconverted to AMP by the adenylosuccinate synthetase and adenylosuccinase reactions (80). AMP deaminase closely parallels the activities of the glycolytic enzymes in the different types of muscle fibers; the highest AMP deaminase activity is seen in the white fibers and in the fast-red fibers; the slow-twitch red fibers have the lowest AMP deaminase activity of the three fiber types (130). In the rat, the fast-twitch red muscle, the only fiber type that undergoes a decrease in glycogenolytic capacity (8), is also the only type that shows a decrease in AMP deaminase activity ( 40%) in response to the running program described earlier (130). Adenylosuccinase activity is much lower than AMP deaminase activity in all three muscle fiber types and does not change significantly (130).

One suggestion regarding the physiological role of AMP deaminase is that this enzyme may regulate the rate of glycolysis by controlling the activity of PFK, as the ammonium ion activates this enzyme (80). The finding that there is a very high correlation between AMP deaminase activity and of PFK activity lends credence to the idea that one function of AMP deaminase may be to regulate flux through the glycolytic pathway (80, 130).

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284 HOLLOSZY & BOOTH

ACTOMYOSIN ATPASE

Slow muscle myosin and fast muscle myosin have different biochemical properties (l0, 115). When a slow muscle and a fast muscle are cross-innervated, a reversal of contractile properties occurs if sufficient time is permitted to elapse (10). Concom­itantly, the biochemical properties of myosin change as slow muscle myosin is replaced by fast myosin and vice versa (10). Conflicting reports have appeared regarding the adaptive response of skeletal muscle actomyosin to exercise. One group reported a 44% increase in actomyosin ATPase activity in gastrocnemius muscle of rats subjected to a program of exhausting bouts of swimming (125). Others have reported that no change occurs in actomyosin ATPase in mixed muscles of rats trained by means of either swimming (117) or running (3), or in Go/ago senega/ensis (lesser bush baby) trained by means of running (31). The contractile properties of mixed muscles were also unchanged in trained animals (12, 31). On the other hand, a small but significant increase in actomyosin ATPase activity was found in soleus muscles of young rats subjected to a swimming program (17). In rats trained by means of the running program described earlier, actomyosin ATPase activity de- . creased 20% in fast-twitch red muscle and increased about 20% in slow-red soleus;

. no significant change in actomyosin occurred in white muscle (9). These responses are very similar in magnitude and direction to the changes seen in PFK, phosphory­lase, and a number of other glycolytic enzymes (8, 9). There is also a remarkably close parallel between actomyosin ATPase activity and the capacity of the glycogenolytic-glycolytic pathway as reflected in PFK activity in the different types of muscle in trained and untrained animals (9). Thus there appears to be a constant relationship between the capacities of these major pathways for ATP generation and A TP utilization in muscle (9).

ADAPTIVE RESPONSES TO ENDURANCE EXERCISE IN THE DIFFERENT TYPES OF MUSCLE

In histochemical studies in which the staining intensities of succinate dehydroge­nase, DPNH diaphorase, or malate dehydrogenase were used to distinguish the fiber types in rodent skeletal muscle, it appeared that the percentage of fibers with the staining characteristics of white muscle decreased, while the percentage of red­appearing fibers increased (11, 32, 37). This finding suggested that endurance exer­cise brings about the conversion of some white fibers to red. However, as reviewed above, biochemical studies have shown that although the respiratory capacity of white muscle increases, white fibers are not converted to red. On the contrary, some of the differences between the white and the red muscle fibers are accentuated in exercise-trained rodents (5, 8, 9, 126, 130). The stains for the respiratory enzymes, as generally used, are relatively insensitive and are inappropriate for quantitation of enzyme activity. They serve to distinguish fibers with a mitochondrial content above some critical level which makes them appear "red" from "white" fibers with a mitochondrial content below this level. Training apparently increases respiratory enzymes sufficiently in certain white fibers, perhaps those with the highest respira-

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ADAPTATIONS TO EXERCISE 285

tory capacity initially, to reach the critical staining intensity needed to give a red appearance. The differences between red and white fibers are, however, maintained. Histochemical studies on the effects of exercise on human skeletal muscle, in which the fibers have been characterized only as fast or slow on the basis of actomyosin ATPase staining characteristics, have shown no evidence of interconversion of fiber types (47).

Information from cross-innervation studies (10, 106) and studies involving chronic electrical stimulation (99) suggest that skeletal muscle fibers may have the potential for conversion from one type to another. However, as reviewed above, normal endurance exercise does not appear to result in interconversion of fiber types. The white fibers (which have the lowest respiratory capacity and hexokinase activ­ity, the highest glycogenolytic capacity and actomyosin ATPase activity, and there­fore the greatest potential for exercise-induced adaptive change) undergo the smallest increase in respiratory capacity and hexokinase activity and little or no change in actomyosin ATPase or glycolytic enzyme activity. On the other hand, the fast-twitch red fibers, which have the highest respiratory capacity and hexokinase activity, somewhat surprisingly. undergo the largest, absolute increase in oxidative capacity and hexokinase.

Because the extent of an adaptive response is usually related to the magnitude of the inducing stimulus, the small changes in enzyme levels in white muscles relative to red could reflect a lesser participation in endurance exercise. This possibility has been investigated using muscle glycogen depietion as an indicator of prior con­tractile activity. In rats subjected to a two-hour long bout of running such as was used in training studies that resulted in a twofold increase in muscle mitochondria (5, 126), glycogen concentration decreased approximately 5.6 mg per gram of mus­cle in fast-twitch red muscle, 2.7 mg per grain in slow-twitch red muscle, and only 0.3 mg per gram in white muscle (6). Muscle biopsy studies on humans have shown that exercise of an intensity that can be maintained continuously for prolonged periods results in glycogen depletion primarily in slow-twitch fibers with little involvement of the white fibers (46). It seems reasonable to conclude that a positive relationship exists between the magnitude of the habitual level of contractile activ­ity, and the extent of the adaptive response. The finding that white muscle is minimally involved in endurance exercise, as evidenced by minimal glycogen deple­tion, helps to explain why the absolute increases in respiratory capacity and hexoki­nase activity are so much smaller in white than in red muscle. With a different exercise program that results in greater recruitment of the white fibers, larger adaptive responses may occur in white skeletal muscle.

In contrast to skeletal muscle, heart muscle does not undergo an adaptive increase in respiratory capacity in response to endurance exercise (89, 90,113). The activities of a number of mitochondrial enzymes and the concentrations of cytochrome c and mitochondrial protein, expressed per gram of heart, are unchanged in hearts of trained animals (89, 90, 113). The heart hypertrophies in response to endurance exercise, so that trained animals have heavier hearts than untrained controls of the same body weight. There is evidence that myocardial contractility is enchanced by training (28, 96). The specific activity of actomyosin ATPase is increased in hearts

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286 HOLLOSZY & BOOTH

of rats subjected to programs of swimming, this adaptation could play a role in increasing myocardial contracility (18, 125). There is also evidence that the hearts of trained animals have increased resistance to hypoxia (114).

Because heart muscle contracts continuously and has the highest capacity for aerobic metabolism of any mammalian muscle, it seems reasonable that the levels of activity of the enzymes for the generation of A TP and for the hydrolysis of A TP during muscle contraction are the optimal ones for continuous, vigorous contractile activity. The heart appears to obtain its energy essentially completely from aerobic metabolism, taking up lactate rather than forming it (53, 74). Skeletal muscle has specialized functions (such as maintenance of posture in the case of slow-muscle fibers and the performance of short bursts of intense work that exceed the muscles' capacity for aerobic metabolism in the case of fast-twitch muscle fibers) that pre­clude an enzyme pattern identical to that in heart. However, in response to endur­ance exercise-training, the slow-red and the fast-red types of skeletal muscle fibers become more like heart muscle in their enzyme patterns, with respect to (a) mito­chondrial enzymes involved in the generation of ATP via aerobic metabolism (126), (b) glycolytic and glycogenolytic enzymes (8), and (c ) actomyosin ATPase (9).

ACKNOWLEDGMENTS

We are grateful to Ms. Sandra Zigler for help in the preparation of this manuscript. Research in the authors' laboratory was supported by NIH Grants HD 01613 and AM 05341, and by a grant from the Muscular Dystrophy Associations of America. Dr. Booth was supported by a Research Fellowship from the Muscular Dystrophy Associations of America.

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