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Clinical Science (1999) 97, 557–567 (Printed in Great Britain)

Muscle protein degradation and amino acid metabolism during prolonged knee-extensor exercise in humans G. VAN HALL*, B. SALTIN† and A. J. M. WAGENMAKERS* *Department of Human Biology, Maastricht University, Maastricht, The Netherlands, and †Copenhagen Muscle Research Centre, Copenhagen, Denmark

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The aim of this study was to investigate whether prolonged one-leg knee-extensor exercise enhances net protein degradation in muscle with a normal or low glycogen content. Net amino acid production, as a measure of net protein degradation, was estimated from leg exchange and from changes in the concentrations of amino acids that are not metabolized in skeletal muscle. Experiments were performed at rest and during one-leg knee-extensor exercise in six subjects having one leg with a normal glycogen content and the other with a low glycogen content. Exercise was performed for 90 min at a workload of 60–65 % of maximal one-leg power output, starting either with the normal-glycogen or the low-glycogen leg, at random. The net production of threonine, lysine and tyrosine and the sum of the non-metabolized amino acids were 9–20-fold higher (P 0.05) during exercise of the normal-glycogen leg than at rest. Total amino acid production was also 10-fold higher during exercise compared with that at rest (difference not significant). The net production rates of threonine, glycine and tyrosine and of the sum of the non-metabolized amino acids were about 1.5–2.5-fold higher during exercise with the leg with a low glycogen content compared with the leg with a normal glycogen content (P 0.05). Total amino acid production was 1.5-fold higher during exercise for the low-glycogen leg compared with the normal-glycogen leg (difference not significant). These data indicate that prolonged one-leg knee-extensor exercise leads to a substantial increase in net muscle protein degradation, and that a lowering of the starting muscle glycogen content leads to a further increase. The carbon atoms of the branched-chain amino acids (BCAA), glutamate, aspartate and asparagine, liberated by protein degradation, and the BCAA and glutamate extracted in increased amounts from the blood during exercise, are used for the synthesis of glutamine and for tricarboxylic-acid cycle anaplerosis.

INTRODUCTION In the 1840s, the German physiologist Von Liebig hypothesized that muscle protein is the main fuel used to achieve muscular contraction [1]. Fick and Wislecenus, in their classic study [2], raised considerable doubts about

Von Liebig’s proposal, as they failed to show an increase in urinary nitrogen excretion during and after the ascent of the Faulhorn, a Swiss mountain. Many investigators have confirmed their conclusions in more recent controlled nitrogen balance studies, although conflicting reports have appeared due to both methodological

Key words : amino acids, knee-extensor, protein turnover. Abbreviations : BCAA, branched-chain amino acids ; TCA cycle, tricarboxylic acid cycle ; VO , maximal oxygen uptake ; W , max #max maximal one-leg power output. Correspondence : G. van Hall, Copenhagen Muscle Research Centre, Rigshospitalet, section 7652, Tagens vej 20, 2200 Copenhagen N, Denmark (e-mail RH01769!RH.DK).

# 1999 The Biochemical Society and the Medical Research Society

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problems and the chosen conditions under which exercise was performed. Since the early 1980s, stable isotope tracer methodology has been used to investigate whether exercise leads to net protein catabolism at the wholebody level and in the exercising muscles. Treadmill exercise for 3.75 h at 50 % of maximal oxygen uptake (VO max) resulted in increases in whole# body protein turnover, urea excretion and amino acid oxidation [3]. Furthermore, a positive relationship was observed between [1-"$C]leucine oxidation and cycle exercise intensity [4]. In contrast, cycle exercise at relatively low intensities (30–40 % of VO max) for 1–3 h # decreased whole-body protein synthesis ([1-"$C]leucine incorporation) by 40 % [5], but subsequent studies did not show changes in whole-body protein turnover and protein degradation [6], or whole-body urea production [7]. No effect was seen either on a direct estimate of muscle protein synthesis measured as ["$C]leucine incorporation [6]. In an 1 h study at 70 % of VO max, # Carraro et al. [6] also did not observe any increase in whole-body urea production, indicating that these types of exercise do not lead to substantial increases in net protein degradation or protein oxidation. However, during competitive endurance events (ultra-marathons, triathlons), athletes exercise for periods of 5–15 h at moderate-to-high intensities (50–60 % of VO max). The # few field studies that have been carried out under extreme conditions [8] have shown increased urinary excretion during and after ultra-distance running. Lemon and Mullin [9] have also shown that, in addition to urinary losses, a substantial amount of urea is lost in sweat, especially during endurance exercise leading to glycogen depletion. These data together suggest that ultra-endurance exercise of moderate-to-high intensity, leading to glycogen depletion (as occurs in the field during competitive ultra-endurance events), results in net protein degradation. During one-leg knee-extensor exercise, approx. 3 kg of skeletal muscle is actively contracting [10]. The maximal heart rate that can be achieved during one-leg kneeextensor exercise is about 140 beats:min−", compared with 190 beats:min−" with whole-body exercise. The maximal oxygen consumption of the active muscle is about 800 ml:min−":kg−" muscle, which is 2–3-fold higher than with whole-body exercise. This implies that the metabolic rate of the active muscle during one-leg knee-extensor exercise is greater than during whole-body exercise. High metabolic rates have also been reached in electrically stimulated rat muscles, which show a marked decrease in the rate of muscle protein synthesis [11]. Good estimates of muscle protein degradation rates during whole-body dynamic exercise in humans are not available in the literature, due to a lack of suitable methods for the measurement of protein degradation in vivo. Estimates of the arterio–venous balance of amino acids that are not metabolized in muscle is the most direct # 1999 The Biochemical Society and the Medical Research Society

method of measuring net protein degradation. However, during exercise, muscle blood flow may increase by 10fold or more, and this leads to a decrease in the measured differences between the arterial and venous amino acid concentrations. The percentage error associated with the measurement of the arterio–venous balance is therefore increased, unless the exercise results in an increase in net protein degradation that is proportional to the increase in blood flow. Therefore the first aim of the present study was to estimate net muscle protein degradation using the arterio–venous amino acid balance method at rest and during prolonged one-leg knee-extensor exercise at 60–65 % of the one-leg power output. The second aim was to investigate whether a low pre-exercise muscle glycogen content results in increased net muscle protein degradation, as it has been suggested previously that protein catabolism is increased in glycogen-depleted subjects.

METHODS Subjects Six healthy male volunteers participated in the study. Their mean age, mass, height and maximal one-leg power output (Wmax) were 24p3 years, 75p4 kg, 1.88p0.03 m and 61p5 W respectively. All subjects were healthy and physically active, and participated regularly in leisure sport. The subjects were informed about possible risks and discomfort involved in the experiment before giving their consent to participate. The study was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association, and was approved by the Ethical Committee of the Karolinska Institute, Stockholm, Sweden.

Protocol The protocol used in the present study has been described in detail previously [12]. In brief, subjects performed one-leg exercise in the upright position on an ergometer that permits the exercise to be confined to the quadriceps femoralis muscle group [10]. At 3 days before the actual experiment, subjects performed a graded exercise test to determine their Wmax value. The exercise protocol consisted of 90 min of one-leg knee-extensor exercise at a workload of 60–65 % of Wmax. After the 90 min exercise period, the subjects were not completely exhausted, but experienced intense exertion localized in the exercised leg. The mean heart rate was 53p6, 103p7 and 107p6 beats:min−" at rest and after 30 and 90 min of exercise respectively. The subjects were studied under two experimental conditions : with a normal glycogen content and a low glycogen content of the quadriceps femoralis muscles. The two tests were performed on the same day. In the morning, subjects exercised with the leg with the

Protein breakdown during exercise

normal or low glycogen content (randomly chosen), and in the afternoon (after a resting period of 2 h) they exercised with the other leg. In order to obtain one leg with a normal glycogen content and one leg with a low glycogen content, the subjects had to undergo a glycogen depletion protocol on the evening before the actual test (see below). On the experimental day, subjects reported to the laboratory in the morning. Catheters for blood sampling were placed in the femoral artery and vein in the inguinal region of the leg to be exercised, and advanced with the tip placed 5–6 cm proximal to the inguinal ligament. A thermistor was inserted through the venous catheter for measurement of blood flow by the constant-infusion thermodilution technique. After placement of the catheters, the subjects rested for 30 min in the supine position. During the resting period between the two tests, a femoral venous catheter was placed in the leg to be exercised in the afternoon. Local anaesthesia was applied to the skin and percutaneous incisions were made before exercise for subsequent muscle biopsies using the needle biopsy technique [13]. Muscle biopsies were taken from the lateral part of the quadriceps femoralis muscle before exercise, and after 10 and 90 min of exercise. Arterial and venous blood was sampled simultaneously before exercise and after 10, 30, 60 and 90 min of exercise. Blood flow was measured just before the blood samples were taken in the morning exercise period, since preliminary experiments in the present study and previous work [14] had shown that no measurable differences in blood flow occurred between the normal- and low-glycogen legs when performing work at the same intensity.

femoral vein for 10–15 s, in order to achieve a drop in femoral venous blood temperature of 0.5–1.0 mC. At rest blood flow is low, and can be measured using a 3 ml bolus injection of ice-cold saline [15].

Blood and muscle analysis Blood samples were obtained using heparinized syringes and centrifuged immediately (3200 g for 10 min) to obtain plasma. Plasma was deproteinized with sulphosalicylic acid (6 mg:100 µl−" plasma), vortex-mixed and frozen in liquid nitrogen until analysis by HPLC [16]. Muscle biopsies obtained using the needle biopsy technique were directly frozen in liquid nitrogen. The biopsies were freeze-dried and freed from adherent blood and connective tissue. The water content of the biopsies was calculated from the weight difference before and after freeze-drying, and used for conversion from dry to wet weight. Glycogen was determined in samples of 5 mg of dried muscle by a fluorimetric enzymic method [17]. For amino acid analysis, 2–5 mg of dried muscle was extracted with 5 % sulphosalicylic acid under vigorous vortex-mixing, and analysed by HPLC [16].

Muscle mass Thigh volume (V) was calculated using the thigh length, three circumferences and three skin-fold measurements [18]. The active muscle mass (M) was calculated as M l 0.307iVj0.353 [19]. This anthropometric method gave similar values as were obtained from multiple CAT scans [20].

Calculations Glycogen depletion protocol In order to obtain one leg with a normal glycogen content and one leg with a low glycogen content, subjects underwent a combination of upper-body exercise and one-leg knee-extensor exercise on the night before the experiment. Muscle glycogen returned to near normal when the glycogen depletion protocol was limited to one-leg knee-extensor exercise. The glycogen depletion protocol consisted of 1 h of arm cranking (Monark ; Rehab trainer) and 1 h of one-leg knee-extensor exercise, both for 40 min at 60–70 % of Wmax, followed by five exercise bouts of 3 min at maximal power output. After the glycogen depletion protocol, subjects were instructed to eat only a boiled egg as an evening snack. From the evening until the end of the experiment on the next day, subjects did not ingest food, and the only fluid allowed was water ad libitum.

Blood flow Femoral venous blood flow was measured by the thermodilution technique [10]. Briefly, ice-cold saline (0.9 % NaCl) was infused at a constant rate into the

Amino acid net release\uptake rates (µmol:min−") were calculated as the femoral arterial–venous difference (µmol:l−") in the plasma concentration of the amino acid multiplied by the plasma flow (l:min−"). The plasma flow was used, as no substantial amino acid transport is expected to take place via the blood cells (plasma flow is derived from blood flow using the haematocrit). The total net release\uptake of amino acids between 10 and 90 min of exercise was determined as the area under the curve. The release\uptake rates of two consecutive time points were averaged and multiplied by the time span, followed by summing these for the whole exercise period. Net production\consumption of amino acids at rest (µmol:min−":kg−" dry muscle) is equal to the net release\uptake rates of the amino acids, assuming that at rest the intramuscular amino acid pool does not change and that the total leg muscle weight is " 9 kg (" 2.1 kg dry weight). However, net amino acid production and consumption between 10 and 90 min of exercise was estimated as the sum of total net release\uptake and the change in the intramuscular amino acid pool expressed per kg of active muscle. The active muscle mass involved in knee-extensor exercise is restricted to the quadriceps # 1999 The Biochemical Society and the Medical Research Society

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Table 1

Arterial amino acid concentrations at rest and femoral arterial–venous differences at rest and during exercise

NG, normal glycogen content ; LG, low glycogen content ;. n-m AA, sum of the non-metabolized amino acids (Thr, Met, Phe, Lys, Gly and Tyr) ; Tot. AA, sum of all amino acids. Values are meanspS.E.M. from six subjects. Femoral arterial–venous difference (µmol:l−1) Exercise Arterial concn. (µmol/l)

Amino acid

Leg

Threonine

NG LG NG LG NG LG NG LG NG LG NG LG

104p11 105p8 26p6 23p2 52p4 48p5 146p16 145p11 181p13 184p11 50p5 50p6

n-m AA

NG LG

Arginine

Methionine Phenylalanine Lysine Glycine Tyrosine

Asparagine Serine Histidine Glutamine Glutamate Alanine BCAA Tot. AA

Rest

10 min

30 min

60 min

90 min

k34p6 k32p6 k3.7p3.2 k6.5p3.1 k6.3p2.2 k5.8p2.7 k20.5p8.2 k30.1p17.9 k39.1p15.0 k29.8p20.3 k6.1p2.9 k4.6p1.9

k2.4p0.6 k0.2p2.5 k0.2p1.6 k1.4p1.7 k1.4p0.5 k1.1p1.8 k11.9p4.3 1.3p6.4 k9.9p2.0 k5.9p5.2 k2.2p2.3 k2.9p1.3

k2.1p0.6 k2.7p2.4 k1.3p1.5 1.7p0.8 0.4p0.5 k0.3p1.4 k7.9p2.8 k3.9p3.7 k9.6p2.8 k7.1p3.9 k4.1p2.0 k2.8p3.5

k3.9p1.8 k8.6p2.6 k1.1p1.4 2.7p1.4 0.1p0.9 k0.6p0.8 k3.9p3.7 k5.5p5.7 k0.9p4.7 k17.8p4.6 k0.2p0.9 k4.5p1.6

k2.7p1.6 k5.5p1.3 k2.1p1.0 k2.5p1.5 1.6p0.4 k1.9p0.6 k2.0p1.4 k3.8p1.8 k3.4p1.8 k7.0p3.6 1.9p2.6 k0.5p2.2

532p19 531p19

k94p26 k80p21

k28p4 k10p16

k25p5 k15p11

k10p11 k34p12

k7p7 k23p9

NG LG NG LG NG LG NG LG NG LG NG LG NG LG

61p6 58p6 42p6 43p3 107p14 109p7 82p4 82p3 507p22 517p10 77p19 85p19 213p18 198p21

k10.5p1.1 k12.2p2.7 k12.8p4.4 k8.3p1.7 1.1p5.5 3.3p3.3 k16.7p2.2 k7.2p2.2 k78p13 k36p25 44p15 34p2 k68p14 k60p13

k1.1p1.0 k0.7p1.1 k1.4p0.2 0.4p0.9 2.7p0.7 6.3p2.1 k3.1p0.5 k1.7p1.4 k23p4 k16p10 23p8 18p9 k25p7 k24p6

k2.0p0.9 k1.4p1.0 k0.9p0.2 k0.4p0.5 2.1p1.6 1.1p2.4 k1.5p0.9 k1.0p1.6 k20p6 k11p7 6p10 14p10 k22p6 k19p4

k0.8p0.5 k2.0p1.2 0.4p1.3 k1.1p0.7 2.5p1.8 k5.3p2.0 k0.5p0.8 k3.8p1.8 k13p8 k19p6 9p2 5p5 k15p5 k19p4

0.9p0.8 k2.2p1.2 k0.9p0.4 k0.8p0.7 0.1p1.2 k1.4p1.7 k0.1p0.7 k0.6p1.0 k1p5 k16p7 6p2 8p7 k7p3 k25p6

NG LG NG LG

641p36 573p48 2421p102 2344p118

2.7p9.4 k11.2p11.2 k351p52 k277p49

k2.1p6.9 3.5p6.9 k87p27 k67p66

2.7p4.5 5.0p7.0 k62p24 k53p60

8.4p6.1 0.5p2.5 k34p24 k98p35

16.0p6.0 14.4p4.2 k68p20 k72p48

femoris muscles, which weigh " 2.5 kg (" 0.6 kg dry weight). The first 10 min of exercise was excluded for the estimation of net production and consumption. This is because the large changes in leg amino acid release\uptake and muscle amino acid concentrations (mainly glutamate and alanine) on moving from rest to exercise may have a pronounced effect on the calculations in the more steadystate period between 10 and 90 min of exercise. The net rate of protein degradation (g protein:day−":kg−" dry muscle) is estimated from the net rate of production of the amino acids that are not metabolized in muscle (threonine, methionine, phenyl# 1999 The Biochemical Society and the Medical Research Society

alanine, lysine, glycine and tyrosine) [21]. These amino acids are only used for protein synthesis ; therefore net production can only occur via net protein degradation. The rate of net total muscle protein degradation can then be estimated from the increase in the net rate of production from rest to exercise of a non-metabolized amino acid multiplied by its relative concentration in muscle [22].

Statistical analysis All data are meanspS.E.M. Statistical analysis of the data was carried out using one-way repeated-measures analy-

Protein breakdown during exercise

sis of variance. Differences between amino acid production\consumption at rest and during exercise were checked for statistical significance using the Fisher’s protected least-significant-difference test. The non-parametric Wilcoxon signed-rank test was used to determine differences between data obtained from the legs with the low and normal glycogen content respectively, and for determining whether the rate of amino acid production\consumption was different from zero. Statistical significance was set at P 0.05.

RESULTS The muscle glycogen depletion protocol, using a combination of upper-body and one-leg exercise the night before the experiment, resulted in a significantly lower resting glycogen content in the leg that underwent the depletion protocol, i.e. 174p24 glycosyl units:kg−" dry muscle, compared with 415p22 units:kg−" in the other leg. The arterial concentrations of amino acids at rest and

Table 2

the arterio-venous differences at rest and during exercise are presented in Table 1. The changes in arterial concentrations during exercise have been published previously [12]. Blood flow was 0.3p0.03 l:min−" at rest and 3.8p0.6 l:min−" during exercise. The 13-fold increase in blood flow going from rest to exercise resulted in a considerable decrease in the arterio–venous amino acid concentration differences. Net production and consumption rates for amino acids are given in Table 2, both at rest and between 10 and 90 min of the exercise period. Except for methionine, all non-metabolized amino acids were released by the leg at rest in significant amounts (P 0.05) (Table 2). However, the net production of the sum of all amino acids was not significantly different from zero (Table 2), due to the large variation between individuals. Exercise caused significant increases in the net muscle production of threonine, lysine, tyrosine and the sum of the non-metabolized amino acids. The mean production of the sum of all amino acids increased 11fold during exercise compared with that at rest, even though this difference was not significant. At rest, the muscle glycogen content did not influence

Net production or consumption of amino acids at rest and during one-leg knee-extensor exercise

n-m AA, sum of non-metabolized amino acids (Thr, Met, Phe, Lys, Gly and Tyr) ; Tot. AA, sum of all amino acids. Net amino acid production or consumption at rest and during exercise is expressed in µmol:min−1:kg−1 dry muscle. A negative value indicates net production. Net production or consumption is calculated from the net release/uptake and the dry muscle mass of the leg (" 9 kg of muscle ; " 2.1 kg dry weight) at rest and from the release/uptake and change in muscle concentration after 10 and 90 min of exercise normalized using the estimated quadriceps dry weight involved in exercise (" 2.5 kg muscle ; " 0.62 kg dry weight). Values are meanspS.E.M. from six subjects. Significance (all P 0.05) : *significant production/consumption ; †significant difference from value at rest ; ‡significant difference from the leg with normal glycogen content. Production/consumption (µmol:min−1:kg−1) Rest

Amino acid

Normal glycogen

Exercise Low glycogen

Low/normal

Normal glycogen

Low glycogen

Low/normal

Threonine Methionine Phenylalanine Lysine Glycine Tyrosine

k1.1p0.1* k0.2p0.1 k0.3p0.1* k1.0p0.4* k2.0p0.4* k0.3p0.1*

k1.0p0.2* k0.3p0.1* k0.3p0.1* k1.0p0.2* k1.7p0.3* k0.2p0.1*

0.9 1.5 1.0 1.0 0.9 0.7

k11p4*† k4p2 1p1 k19p4*† k11p5 k5p1*†

k18p3*†‡ 3p1 k2p1* k19p4*† k34p7*†‡ k12p3*†‡

1.0 3.0 2.5

n-m AA

k4.8p0.9*

k4.1p0.9*

0.9

k51p14*†

k81p13*†‡

1.6

Arginine Asparagine Serine Histidine Glutamine Glutamate Alanine

k0.5p0.05* k0.7p0.2* 0.1p0.3 k0.6p0.1* k7.3p1.0* 3.4p1.3* k6.3p1.3*

k0.6p0.1* k0.4p0.1* 0.3p0.2 k0.4p0.1* k5.9p0.9* 3.4p1.8 k5.6p1.4*

1.2 0.6 6.0 0.7 0.8 1.0 0.9

k5p2*† k4p2 4p4 k4p2 k65p42 26p3*† k8p23

k7p2*†‡ k6p1*† k10p4*†‡ k10p3*† k83p21* 43p4*†‡ k35p37

1.4 1.7

BCAA Tot. AA

0.1p0.4 k13p3.2

k0.5p0.6 k12p4.1

0.9

16p12 k91p105

13p13 k180p65

1.7

2.8 1.2 0.8 2.3 0.8 1.9

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Table 3

Net amino acid uptake/release and change in the muscle free amino acid pool during exercise

n-m AA, sum of non-metabolized amino acids (Thr, Met, Phe, Lys, Gly and Tyr) ; Tot. AA, sum of all measured amino acids. Net amino acid uptake/release between 10 and 90 min of exercise (net exchange) and the change in the intramuscular amino acid concentration (δ muscle) are both given in units of µmol:min−1:kg−1 dry muscle. The change in the intramuscular amino acid concentration is also given as a percentage of the muscle free amino acid pool present in the resting biopsy ( % pool). A negative value indicates net release or a decrease in the muscle free amino acid pool. Values are meanspS.E.M. from six subjects. Normal glycogen

Low glycogen

Net exchange (µmol:min−1:kg−1) Threonine Methionine Phenylalanine Lysine Glycine Tyrosine n-m AA Arginine Aspargine Serine Histidine Glutamine Glutamate Alanine BCAA Tot. AA

k10p3 k4p3 k1p1 k17p5 k9p5 k4p2 k45p14 k3p2 k2p2 7p3 k4p1 k43p13 13p9 k46p17 14p11 k109p36

Table 4

δ muscle (µmol:min−1:kg−1) 1.3p1.4 0p0.1 0.2p0.3 1.5p2.0 2.1p0.8 1.0p1.2 6.0p1.6 1.5p0.3 2.2p0.6 3.0p1.8 k0.1p1 22p11 k13p3 k38p11 k2p2 k18p29

% pool

Net exchange (µmol:min−1:kg−1)

6 0 5 6 4 26 7 16 22 14 1 7 7 56 7 1

k17p2 3p1 k3p1 k17p3 k29p8 k10p4 k73p13 k6p1 k3.2p0.8 k4p4 k7p2 k62p9 21p5 k71p12 13p7 k193p91

δ muscle (µmol:min−1:kg−1) 0.6p2.8 0.3p0.1 k0.4p0.3 1.5p2.0 4.8p1.4 1.3p1.1 8.5p2.3 1.7p0.8 3.2p0.6 6.6p1.6 2.8p0.8 20p12 k21p8 k36p11 k1p1 k13p26

% pool 3 16 11 5 10 29 8 14 33 35 13 5 9 74 8 4

Net rates of protein degradation caused by exercise

NG, normal glycogen content ; LG, low glycogen content ;. n-m AA, sum of non-metabolized amino acids (Thr, Met, Phe, Lys, Gly and Tyr) ; Tot. AA, sum of all amino acids. Net rates of protein degradation (in g of protein:day−1:kg−1 dry muscle) are calculated from the net production of non-metabolized amino acids (in µmol:min−1:kg−1 dry muscle ; Table 2) divided by the relative occurrence of the individual amino acids in human muscle protein (in µmol:g−1 protein). Values for the relative occurrence of amino acids are from [22], and were converted from mol % composition to µmol:g−1 dry muscle for the individual amino acids. The ratio exercise/rest was calculated from the rates of net protein degradation at rest and during exercise. Net protein degradation (g of protein:day−1:kg−1 dry muscle) Rest

Amino acid

Occurrence (mol % of muscle protein)

NG

LG

NG

LG

NG

LG

Threonine Methionine Phenylalanine Lysine Glycine Tyrosine n-m AA Tot. AA

5.4 1.8 3.3 8.1 9.7 2.1 30.4 100

k3.5 k2.4 k2.2 k2.6 k2.2 k3.7 k3.2 k2.8

k3.2 k3.6 k2.2 k2.6 k1.9 k2.5 k2.7 k2.6

k31 k45 7 k47 k20 k58 k32 k29

k54 39 k12 k47 k30 k146 k51 k43

9 19

16

net production rates of the non-metabolized and other amino acids (Table 2). However, prolonged exercise by muscles with a low glycogen content appeared to result in # 1999 The Biochemical Society and the Medical Research Society

Exercise

Exercise/rest

18 9 16 10 10

5 18 16 58 19 17

a further increase in net protein degradation in comparison with muscles with a normal glycogen content. The rates of production of threonine, glycine, tyrosine

Protein breakdown during exercise

Table 5 Production and consumption of individual amino acids expressed as a percentage of the expected total production from net protein degradation

The net production or consumption of the individual amino acids (in µmol:min−1:kg−1 dry muscle ; Table 2) is divided by the production of the individual amino acids by net protein degradation (in µmol:day−1:kg−1 dry muscle). The latter value is obtained from the net protein degradation rates, as calculated from the non-metabolized amino acids (in g of protein:day−1:kg−1 dry muscle ; Table 4) and the relative occurrence of the individual amino acids in muscle protein. A positive value indicates production ; a negative value indicates consumption. When values exceed the mol % of muscle protein, then there is net synthesis of that amino acid in muscle ; when values are smaller there is net metabolism ; and when values are negative there is uptake and subsequent metabolism. Production expected by net protein degradation (%) Exercise

Rest

Amino acid

Occurrence (mol % of muscle protein)

Normal glycogen

Low glycogen

Normal glycogen

Low glycogen

Threonine Methionine Phenylalanine Lysine Glycine Tyrosine Arginine Asparagine Serine Histidine Glutamine Glutamate Alanine BCAA

5.4 1.8 3.3 8.1 9.7 2.1 4.3 " 5* 5 2.5 " 8* " 7* 8.7 18.8

5.9 1.3 2.2 6.6 6.8 2.4 3.9 4.2 k0.2 4.2 48 k22 25 k1

6.3 2.4 2.6 7.8 6.8 1.9 5.6 2.8 k1.7 3.3 46 k26 27 4

5.9 2.7 k0.5 12.5 3.7 4.1 5.9 2.4 k1.9 2.8 49 k17 3 k10

6 k1.3 0.9 7.8 6.1 6.1 3.4 2.2 3 4.4 34 k18 9 k5

* Values are estimates based on mixed muscle protein amino acid content.

and the sum of the non-metabolized amino acids were 1.5–2.5-fold higher under exercise conditions when measured in the leg with a low glycogen content (P 0.05) (Table 2). The sum of all amino acids was 1.5-fold higher for the low-glycogen leg, but this difference failed to reach statistical significance. The net production rate was estimated from the net amino acid uptake\release and the change in the concentration of that amino acid in the intracellular free amino acid pool. The absolute contribution of the change in the intramuscular free amino acid pool to the amino acid production rate was between 8 and 25 % for the nonmetabolized amino acids (Table 3), and the changes in the size of the intramuscular free amino acid pools were small, except for glycine, which showed a considerable decrease (Table 3). This implies that the increased net protein degradation does not lead to an expansion of the free amino acid pool, and that most amino acids produced by net protein degradation are released from the muscle into the circulation. From the net production of the non-metabolized amino acids and the relative occurrence of the individual amino acids in human muscle protein, net rates of protein

degradation have been calculated in units of g protein:day−":kg−" dry muscle (Table 4), both for values obtained for the individual non-metabolized amino acids and for the sum of these amino acids. For most of the individual non-metabolized amino acids, quite similar estimates are obtained, especially at rest. Deviations do occur during exercise, especially for those amino acids with the lowest relative occurrence and, therefore, the smallest arterio–venous difference. The contributions that individual amino acids make to total amino acid production by the muscle have been calculated (Table 5) from the observed net production (Table 2) and the total net protein breakdown rate calculated from the release of the sum of the nonmetabolized amino acids (Table 4). Amino acids that are released in proportion to their occurrence in human skeletal muscle protein (Table 4) are not metabolized. Amino acids released in smaller amounts are either oxidized or used for the synthesis of other amino acids or muscle metabolites, and amino acids released in larger quantities are synthesized in muscle. The interpretation of the data in Table 5 is presented in the Discussion section. # 1999 The Biochemical Society and the Medical Research Society

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DISCUSSION The major finding of the present study is that voluntary one-leg knee-extensor exercise in the post-absorptive state leads to an increase in net protein degradation compared with at rest, and that this exercise-induced increase in net protein degradation is augmented in muscles with a low glycogen content. The latter finding is in agreement with earlier reports that urea excretion is higher in glycogen-depleted subjects during two-legged cycling [9]. Workload and oxygen consumption per kg of muscle are higher during one-leg knee-extensor exercise than during two-legged cycling (see the Introduction section) and, therefore, the results cannot be directly extrapolated to whole-body dynamic exercise. However, the increased net leg protein degradation measured in the present study seems to be in agreement with the study of Rennie and co-workers [3]. They observed an increase in wholebody protein turnover, [1-"$C]leucine oxidation and urea excretion during 3.75 h of treadmill exercise at 50 % of VO max. In contrast, other workers did not observe # changes in whole-body protein turnover or protein degradation during two-legged cycle exercise at 40 % and 70 % of VO max [6,23], or changes in urea production [7]. # In addition, no effect was seen on a direct estimate of muscle protein synthesis [6]. A high rate of oxygen consumption per kg of muscle has also been observed in electrically stimulated rat muscles, which showed a marked decrease in muscle protein synthesis during contraction [11]. A decrease in protein synthesis may contribute to the production of non-metabolized amino acids observed in the present study during one-leg kneeextensor exercise. The conclusion that net protein degradation is increased during one-leg cycle exercise is based on the release of threonine, methionine, phenylalanine, lysine, glycine and tyrosine, as it has been shown previously that these amino acids are not metabolized in rat muscle [24,25]. However, little information is available on the metabolism of these and other amino acids in human skeletal muscle. As one-leg knee-extensor exercise appears to lead to the largest physiological imbalance between muscle protein synthesis and degradation observed in the scientific literature so far, the data obtained are also suited to investigating whether the amino acids released by net protein degradation are metabolized (totally or in part) in muscle, or are released in proportion to their relative occurrence in human muscle protein. The data in Table 5 indicate that threonine, lysine, arginine and histidine are subject to little or no metabolism. In future experiments it therefore seems best to base estimates of net protein degradation on these four amino acids. Threonine and lysine seem most suited to this purpose, as they also have a high relative occurrence in muscle protein, and therefore show the largest arterio– # 1999 The Biochemical Society and the Medical Research Society

venous differences during exercise. However, using the sum of the six amino acids indicated in the present study as primary markers of net protein degradation leads to similar conclusions. In the case of glycine and asparagine, 30–60 % of the amino acid released by net protein degradation is metabolized in the muscle. Data for methionine are inconsistent, most probably due to the fact that arterio–venous differences are small and variable (Table 2). It has been suggested that methionine is partly metabolized in rat muscle [26]. During exercise, tyrosine is released in larger amounts and phenylalanine in smaller amounts than their relative occurrence in muscle protein. This seems to suggest that phenylalanine is converted into tyrosine. In liver and gut this conversion is catalysed by phenylalanine hydroxylase. The possibility that phenylalanine is converted into tyrosine, along with their low occurrence in muscle protein, may make phenylalanine and tyrosine less suited as markers of net protein degradation in muscle. This is important, as phenylalanine and tyrosine have been used most frequently to estimate net muscle protein degradation in animal and human studies both in vitro and in vivo. All these conclusions depend on the assumption that only skeletal muscle proteins are broken down, rather than collagen and skin ; the contribution of the latter has been suggested to be small at rest [27]. It is clear that branched-chain amino acids (BCAA) and glutamate are metabolized in skeletal muscle, as they are taken up from the circulation during exercise despite a high occurrence in muscle and net production by net protein degradation. There is no uptake of BCAA at rest, but during exercise the uptake is substantial (Table 5). Glutamate is taken up both at rest and during exercise, but the rate of glutamate uptake increases 10-fold when going from rest to exercise (Table 2). We do not have data on aspartate, as it coincides with the sulphosalicylic acid peak in the HPLC chromatogram [16]. In rat muscle, aspartate is rapidly transaminated to oxaloacetate and is not released [24]. Alanine is produced during exercise in an amount about equal to its relative occurrence, while glutamine is released in amounts that are much larger (Table 5). This seems to indicate that the carbon skeletons of the BCAA, glutamate, aspartate and other amino acids that are partly metabolized in muscle are not used for the synthesis of alanine, but are used primarily for conversion into tricarboxylic acid (TCA)-cycle intermediates and glutamine (Scheme 1). A similar conclusion was drawn using rat muscle incubated in vitro [24,25,28]. The radioactive label of [U-"%C]valine was only recovered in glutamine, and not in alanine, pyruvate or lactate. This also implies that the amino acids that are metabolized in muscle to TCA-cycle intermediates (Scheme 1) are not used for complete oxidation, as conversion into pyruvate and acetyl-CoA is required before complete oxidation is possible. Therefore only leucine and part of the isoleucine molecule are metabolized to acetyl-CoA (Scheme 1) and

Protein breakdown during exercise

Scheme 1

Schematic presentation of the various interactions of amino acids with the TCA cycle

The oxo acids from leucine, isoleucine and valine are α-oxoisocaproic acid (α-KIC), α-oxomethylvaleric acid (α-KMV) and α-oxoisovaleric acid (α-KIV). Glucose 6-P, glucose 6-phosphate ; α-ketoglutarate l 2-oxoglutarate. can help to provide fuel to the exercising muscle for ATP synthesis. Tracer experiments in humans in vivo [29,30] also suggest that, in overnight-fasted non-exercising humans, the carbon atoms of glutamine originate primarily from protein-derived amino acids, whereas the carbon atoms of alanine are derived from blood glucose and muscle glycogen. Glutamine, therefore, seems to be much more important than alanine for transport of protein-derived carbon from muscle to liver and kidney, where the carbon skeletons can be used for gluconeogenesis. This conclusion does not confirm the original formulation of the glucose\alanine cycle [31]. Amino acid production observed during the first 10 min of exercise in the present study has been described and interpreted before [12]. Here we focus on the changes in amino acid production that occurred between 10 and 90 min of the exercise period, as changes occur in the first 10 min in the muscle free amino acid pool that are not related to an imbalance between protein synthesis and protein degradation. The intramuscular glutamate pool decreased by 60 % within the first 10 min, while alanine increased by 30 %. These changes are the consequence of a shift to the right of the alanine aminotransferase reaction (pyruvatejglutamate  alaninejα-oxoglutarate). This shift most probably functions to increase the concentrations of TCA-cycle intermediates [32] when going from rest to exercise, hence increasing flux in the TCA cycle and ATP production in proportion to energy demand [12]. Most of the alanine released from muscle between 10 and 90 min of exercise was accounted for by the decrease in the muscle free pool back to the preexercise concentration [12]. Graham et al. [33] reported a

much higher rate of alanine production during 3 h of one-leg knee-extensor exercise at 60 % of Wmax, as they did not correct for the initial change in the amino acid pool size due to the alanine aminotransferase reaction. As indicated above, the alanine aminotransferase reaction is the main anaplerotic reaction (‘ filling up ’ the TCA cycle) at the beginning of exercise, when ample amounts of glycogen are present to maintain a high pyruvate concentration in the muscle [12]. The increase in the muscle pyruvate concentration at the beginning of exercise, in fact, is the driving force leading to net conversion of pyruvate and glutamate into alanine and TCA-cycle intermediates. During prolonged exercise and in glycogen-depleted muscles, other mechanisms apparently take over. Removal of amino groups from muscle in the form of glutamine provides one alternative mechanism for the net synthesis of TCA-cycle intermediates, as illustrated by the following net reactions (see Scheme 1 for the complete metabolic pathways) : 2Glutamate

glutaminejα-oxoglutarate

Valinejisoleucine Aspartatejisoleucine

succinyl-CoAjglutamine oxaloacetatejglutamine.

Therefore BCAA and glutamate released by the net breakdown of muscle protein and taken up from the circulation seem to be used for the net synthesis of TCAcycle intermediates and glutamine during prolonged cycle exercise. Previously, we also observed that deamination of the indicated amino acids gradually increases during prolonged exercise, and this also provides a mechanism for TCA-cycle anaplerosis [12]. # 1999 The Biochemical Society and the Medical Research Society

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The net rate of protein degradation observed at rest is about 3 g of protein:day−":kg−" dry muscle (Table 4). From tracer studies it is known that the mean fractional synthesis rate of muscle protein at rest in the postabsorptive state is about 1.5 % per day, or 15 g of protein:day−":kg−" dry muscle [34–36]. As net protein degradation equals protein synthesis minus protein degradation, the data indicate that there is a 20 % imbalance between protein synthesis and degradation at rest in the post-absorptive state, as previously indicated by tracer studies [34–36]. During exercise, however, the net rate of protein degradation in the leg with a normal glycogen content is estimated at 32 g of protein:day−":kg−" dry muscle (Table 4), and that in the glycogen-depleted leg is even higher, This is a major disturbance in the balance between protein synthesis and protein degradation. Even if the mean protein synthesis rate fell to zero during exercise, net protein degradation would maximally increase to 15 g per day. This implies that the absolute mean rate of protein degradation must increase 2-fold or more during one-leg knee-extensor exercise. Such extreme values have not been reported previously under any physiological or pathophysiological conditions [37]. Alternatively, a small specific muscle protein pool with a higher turnover rate may be rapidly degraded by an imbalance between protein synthesis and degradation. In conclusion, the data from the present study clearly indicate that net muscle protein degradation is substantially increased by prolonged moderate-to-high intensity one-leg knee-extensor exercise. Quantitative estimates suggest that both a decrease in protein synthesis and an increase in protein degradation contribute to the large negative amino acid balance. Furthermore, a low initial muscle glycogen content augments the exerciseinduced increase in net muscle protein degradation. It is suggested that the functionality of the negative amino acid balance is provision of protein-derived carbon for the synthesis of TCA-cycle intermediates and glutamine in muscle, and release of gluconeogenic precursor amino acids to the liver and kidney.

ACKNOWLEDGMENT We thank H. van Eijk and colleagues from the Department of Surgery, Maastricht University, for professional support with the amino acid analysis and for helpful discussions.

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31 Felig, P., Pozefski, T., Marliss, E. and Cahil, G. F. (1970) Alanine : a key role in gluconeogenesis. Science 167, 1003–1004 32 Gibala, M. J., Tarnopolsky, M. A. and Graham, T. E. (1997) Tricarboxylic acid cycle intermediates in human muscle at rest and during prolonged cycling. Am. J. Physiol. 272, E239–E244 33 Graham, T. E., Turcotte, L. P., Kiens, B. and Richter, E. A. (1995) Training and muscle ammonia and amino acid metaolism during prolonged exercise. J. Appl. Physiol. 78, 725–735 34 Halliday, D., Pacy, P. J., Cheng, K. N., Dworzak, F., Gibson, J. N. A. and Rennie, M. J. (1988) Rate of protein synthesis in skeletal muscle of normal man and patients with muscular dystrophy : a reassessment. Clin. Sci. 74, 237–240 35 Bennet, W. M., Connacher, A. A., Scrimgeour, C. M. and Rennie, M. J. (1990) The effect of amino acid infusion on leg protein turnover assessed by L-["&N]phenylalanine and L-[1-"$C]leucine exchange. Eur. J. Clin. Invest. 20, 41–50 36 Chesley, A., MacDougall, J. D., Tarnopolsky, M. A., Atkinson, S. A. and Smith, K. (1992) Changes in human muscle protein synthesis after resistance exercise. J. Appl. Physiol. 73, 1383–1388 37 Rennie, M. J. (1985) Muscle protein turnover and the wasting due to injury and disease. Br. Med. Bull. 41, 257–264 Received 17 December 1998/1 June 1999; accepted 22 July 1999

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