Amino acid metabolism
after intense exercise
JOHN T. DEVLIN, IRWIN BRODSKY, ANGUS SUSAN FULLER, AND DENNIS M. BIER
SCRIMGEOUR,
Metabolic Unit, University of Vermont College of Medicine, Burlington, Vermont 05405; and Metabolism Division, Washington University School of Medicine, St. Louis, Missouri 63110 DEVLIN, JOHN T., IRWIN BRODSKY, ANGUS SCRIMGEOUR, SUSAN FULLER, AND DENNIS M. BIER. Amino acid metabolism after intensive exercise. Am. J. Physiol. 258 (Endocrinol. Metab.
21: E249-E255, 1990.-We studied postexercise amino acid metabolism, in the whole body and across the forearm. Seven volunteers were infused with L- [a(-15N]lysine and L- [ l-13C] leucine twice [one time during 3 h after cycle exercise (75% h max),and one time in the resting state]. Whole body protein breakdown was estimated from dilution of L-[&‘N]lysine and L- [ l-13C] ketoisocaproic acid (KIC) enrichments in plasma. Leutine oxidation was calculated from 13C02 enrichments in expired air. Whole body protein breakdown was not increased above resting levels during the recovery period. Leucine oxidation was decreased after exercise (postexercise 13 t 2.3 vs. resting 19 t 3.2 prnol. kg-l. h-l; P < 0.02), while nonoxidative leucine disposal was increased (115 t 6.1 vs. 103 t 5.6 pg. kg-l min-l; P < 0.02). After exercise, forearm net lysine balance was unchanged (87 t 25 vs. 93 t 28 nmol. 100 ml-‘. min-l), but there were decreases in forearm muscle protein degradation (219 t 51 vs. 356 -C-85 nmol~lO0 ml-‘. min-‘; P < 0.05) and synthesis (132 & 41 vs. 255 t 69 nmollO0 ml-’ .min-‘; P < 0.01). In conclusion, after exercise 1) whole body protein degradation is not increased, 2) leucine disposal is directed away from oxidative and toward nonoxidative pathways, 3) forearm protein synthesis is decreased. Postexercise increases in whole body protein synthesis occur in tissues other than nonexercised muscle. l
protein synthesis; leucine oxidation; forearm muscle
ofphysicalexercise on amino acid metabolism have focused primarily on the exercise period itself. Leucine oxidation is known to be increased severalfold during exercise (20, 25), in part serving as an oxidative fuel for contracting muscles (14). Rates of whole body protein synthesis are reduced and protein degradation increased during exercise (20). After moderate-intensity exercise (at 50% of maximal aerobic capacity, VO 2 max), rates of protein synthesis increase and proteolysis decrease so that net protein balance is positive (20). However, estimates of proteolysis in this earlier study were made from calculations of leucine appearance measured by dilution of L-[ 1-13C] leucine in plasma. This approach measures the plasma leucine appearance rate reliably but, because proteolysis occurs intracellularly, plasma leucine appearance does not necessarily reflect the appearance of unlabeled leutine in the inaccessible intracellular compartments. Thus several authors (21,22, 24) have advocated using plasma enrichments of a-[ 1-13C] ketoisocaproic acid (KIC) as an PREVIOUS
STUDIES
OF THE EFFECTS
index of intracellular leucine enrichment to assess rates of whole body protein degradation. Wolfe et al. (26) have reported that different estimates of whole body proteolysis during exercise occur when different amino acid tracers are used (L- [ 13C]leucine and L- [ &‘N]lysine). Thus we also examined whether these same two tracers gave differing estimates for rates of whole body proteolysis during recovery when cy-[ l-13C]KIC enrichments were used as an index of intracellular leucine enrichment. Based on the previous report by Lemon and Muller (15) that exercise-induced proteolysis may be greater in states of glycogen-depletion, we hypothesized that whole body proteolysis would remain elevated throughout the 2- to 3-h recovery period after high-intensity, glycogendepleting exercise (at 75% Vo2 max). We further hypothesized that forearm muscle proteolysis would similarly be increased for a prolonged period after exercise, since nonexercised (forearm) muscle is insulin resistant 2-3 h after intense bicycle (leg) exercise (6) and continues to release gluconeogenic precursors (lactate and alanine) throughout this time period in amounts that cannot be accounted for by glucose uptake, suggesting ongoing forearm muscle glycogenolysis (2). We have adapted the technique described by Barrett et al. (4) and used the arterialized hand and deep forearm vein enrichments of L-[&5N]lysine to estimate rates of forearm muscle protein synthesis and degradation. METHODS
Subjects. Seven subjects (4 male and 3 female, ages 23-32 yr, and within 17% of Metropolitan ideal body weight) participated in the study. All subjects were in excellent health based on medical history, physical examination, and routine screening laboratory chemistries. No subject was taking medications regularly, nor were any engaged in a regular exercise training program. The design, purpose, and possible risks were carefully explained to each volunteer before obtaining written consent. The experimental protocol was approved by the University of Vermont Committee on Human Research. Prior to first admission to the University of Vermont Clinical Research Center (CRC), each subject underwent a maximum aerobic capacity (VO, max) test using a continuous exercise protocol on a Monarch bicycle (3). At the University of Vermont Clinical Research Center all subjects were fed a weight-maintaining diet containing 1.3 times their estimated daily resting energy expenditures, with protein constituting 20% of total calories for
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7 days prior to each study day. Subjects were fasted overnight at the CRC prior to each study day. Experimental design. On the morning of each study day, subjects were placed under the ventilated hood at 0630 h for measurement of resting metabolic rate using continuous indirect calorimetry as previously described (8). After a 3O- to 4O-min steady-state period subjects were removed from the hood. Expired air samples were collected using a mouthpiece connected via a J-valve to a &liter non-diffusing bag (Hans Rudolph, Kansas City, MO), and an aliquot was injected into a 20-ml Venoject Vacutainer tube for later analysis of 13C02 enrichment. Four base-line expired air samples were taken at 5-min intervals. On the “exercise” study days, subjects then performed continuous cycle exercise at a workload previously determined to represent 75% of their VOzmax. This was continued until subjects were subjectively exhausted and unable to maintain a pedaling frequency >5O revolutions/min. We have previously demonstrated that vastus lateralis skeletal muscle is uniformly glycogen depleted using this protocol (7, 8). After exercise subjects quickly showered and returned to bed in the study room. On the “nonexercise” study day subjects remained resting comfortably in bed during the same time period. Except for the period of bicycle exercise, the activity of the subjects was similar on the exercise and nonexercise days. At approximately 0900 h, intravenous catheters were inserted. A 19-gauge indwelling catheter was inserted in a left antecubital vein (infusion catheter). A 19-gauge indwelling needle was inserted in a left hand vein, and the hand was warmed in a heated box (70°C) for obtaining “arterialized” blood specimens (1). A 19-gauge indwelling catheter was placed retrogradely into a right antecubital vein and withdrawn l-2 cm from the “wedged” position for obtaining deep forearm venous blood samples. Two arterialized blood samples 5 min apart were taken for determining base-line amino acid enrichments. Four additional expired air samples were taken at 5-min intervals for postexercise base-line 13C02 enrichment. After base-line sampling the following primed continuous infusions were given at time 0 (-2030 min after exercise): NaH13C03, 0.08 pmol/kg bolus (99% 13C); L-[&-l5 Nllysine, 1.6 pmol/kg bolus and 1.8 pmol kg-’ . h-l infusion; and L- [ 1 -13C] leucine, 2 pmol/kg bolus and 2.4 prnol. kg-’ . h-l infusion (99% 15N and 13C, respectively; MSD Isotopes, Montreal, Canada). Subjects were again placed in the ventilated hood for continuous indirect calorimetry readings. Sampling procedures and steady-state period. Blood samples were taken from the arterialized hand and deep forearm vein catheters at 60 and 90 min, and every 15 min during the 120- to 180-min steady-state period. Forearm blood flow was simultaneously and noninvasively measured using impedance plethysmography (5). Leucine and lysine concentrations were determined on protein-free filtrates of whole blood obtained by adding an equal volume of 10% sulfosalicylic acid and mixing immediately. After separation in a refrigerated centrifuge the supernatants were frozen, stored at -8O”C, and later analyzed with a precolumn o’-phthalaldehyde high-perl
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formance liquid chromatography method (13). Samples for determining cu-KIC concentrations and enrichments of A!-[~-~~C]KIC, L-[l-13C]leucine, and L-[a-“Nllysine were centrifuged at 4”C, and plasma samples were stored at -80°C until later analysis. Rates of COn production were determined continuously with the subject in the ventilated hood, using an infrared CO2 analyzer (Applied Electrochemistry, Sunnyvale, CA) and a flowmeter (Vertek, Burlington, VT). Readings were corrected for standard temperature and pressure, dry (STPD) conditions. At 60 and 90 min and every 20 min throughout the 120- to 180-min steady-state period, the subject was briefly removed from the hood, and the mouthpiece was used to obtain an expired air sample for determining 13C02 enrichment. The steady-state period for our protocol design was verified by prolonged (5 h) isotope infusions in our initial two subjects. This control experiment demonstrated steady-state enrichments of CY-[ l-13C]KIC, L- [ 1-‘3C]leutine, and L+?N]lysine within 120 min of stopping exercise. In an additional control experiment, we also examined the 13C02 enrichment in expired air after exercise in two subjects who received no isotope infusions. The natural background 13C02 isotopic abundance remained unchanged during the entire experimental period. In two other subjects, we determined the rate of 13C02 recovery from a primed (890 pmol) constant (9.9 pmol/min) infusion of NaH13C03 during the postexercise recovery period (26). This value averaged 72%, which is within the range previously reported by Wolfe et al. (26) in resting subjects. Cakulations. Plasma leucine kinetics were determined using steady-state isotope dilution techniques as previously described (16,17). For the determination of leucine oxidation rates, steady-state plasma enrichments of CY[ l-13C]KIC were used to reflect the immediate precursor for the oxidative decarboxylation of leucine (17, 22). Estimates of the rates of whole body proteolysis and nonoxidative leucine disposal (an index of protein synthesis) were made with plasma enrichments of cy-[ 1-13C] KIC, taken to reflect the intracellular enrichment of leucine. Flux rates of lysine were also determined from steady-state plasma enrichments of L-[cx-15N]lysine and used as an indepe,ndent estimate of the rate of whole body proteolysis. Forearm balances of leucine and lysine were determined by the Fick principle, multiplying the arterialized hand deep forearm vein (A-V) gradients by the forearm blood flow that was determined by impedance plethysmography. The fractional extraction was calculated as (A-V) x 100/[AAla*,where [AA],, is the amino acid (lysine, leucine) concentration in the arterialized hand vein samples. The rate of forearm muscle protein breakdown was calculated using the formula D
=
[AA]art
x
[ ULlJLin)
-
11
x
BF
where E ah and Evein are the enrichments of [ 15N]lysine or [13C]leucine in arterialized hand vein and deep forearm vein samples, respectively, and BF is forearm blood flow determined by impedance plethysmography. Because forearm amino acid net balance equals synthesis minus
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PROTEIN TABLE
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Weight,
Height, cm
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1. Subject characteristics Subject
Sex
Age, Y*
AB LG RL TL MD JR JL Means t SE
F F F M M M M
32
29 26 24 24 12 24 2621.3
kg
VO 2
BMI*
kg-’
max9 l
ml
56 62 60 75 84 80 90
161 170 180 178 178 183 185
22 21 19 24 27 24 26
55 42 43 51 54 49 47
72t5.0
176k3.1
23kl.l
49k1.9
breakdown, forearm muscle protein synthesis can then be estimated as net balance plus breakdown. Although Barrett et al. (4) validated this method of calculating D with label phenylalanine in the dog, lysine is also an essential amino acid that is neither transaminated nor oxidized in muscle tissue and, therefore, exchange across the forearm reflects muscle protein synthesis and degradation. We also calculated the rate of appearance of unlabeled leucine across the forearm using the above formula to determine whether the results were qualitatively similar to those using lysine, as has been reported previously across the human forearm using phenylalanine and leucine tracers (11). This estimate of forearm leucine rate of appearance is not considered to accurately reflect forearm muscle proteolysis, since leucine can undergo transamination and oxidation in muscle tissues. Analytical methods. Plasma enrichments of stable isotopes were determined from the N-acetyl, n-propyl ester derivates of leucine and lysine and from the trimethylsilyl-quinoxalinol derivatives of KIC, using selected ion monitoring gas chromatography/mass spectrometry (16, 17). Expired air samples were collected in 20-ml Venoject containers and later analyzed for 13C02 enrichment using a VG-Isogas Siraisotope ratio mass spectrometer (Tracer Technologies, Somerville, MA). Statistical methods. All data are expressed as means t SE. Comparisons between the postexercise recovery period and the nonexercise (resting) day were made using analysis of variance (ANOVA; Kruskal-Wallis test for nonparametric data). When ANOVA indicated significant differences between the postexercise and nonexercise days, Student’s paired t test was used to indicate individual time points for which a difference was demonstrable.
cise, while concentrations decreased.
l
min-’
of glucogenic amino acids were
Isotope enrichments. In our initial two volunteers, we observed that plasma enrichments of amino acid isotopes plateaued within 120 min of infusion during the postexercise recovery period, and we selected the 120- to 180. min period of infusion as our steady-state plateau period. Figure 1 demonstrates the amino acid and KIC isotope enrichments in arterialized hand vein samples during this steady-state period after exercise. The slopes of the plasma enrichments were not significantly different from zero, using ANOVA (F ratios 0.069-0.098, P = 0.980.99). The forearm deep vein enrichments of lysine also reached a plateau during the 120- to 180-min steadystate period (F ratio 0.10, P = 0.98, for both resting and postexercise studies). The ratio of steady-state plasma enrichments of KIC to leucine was 0.68 t 0.019 in the resting state and 0.75 t 0.026 after exercise (P < 0.001; Fig. 2). Amino acid kinetics. Plasma leucine appearance was significantly higher after exercise than in the resting state (96 t 5.0 vs. 82 t 32. prnol. kg-‘. h-l, respectively; P c 0.001). Whole body leucine appearance, estimated from circulating ily-[ 1-‘3C]KIC dilution and a reflection of body protein breakdown, was not changed after exercise (128 t 6.3 vs. 122 t 5.3 prnol. kg-’ h-l), nor was lysine flux (92 t 8.4 vs. 85 If: 5.8, postexercise vs. resting) (Table 3). There was excellent agreement between these two tracers, which were selected to provide independent estimates of rates of whole body proteolysis. Leucine oxidation rates during the steady-state period were significantly lower after exercise, compared with l
TABLE
2. Arterial
amino acid concentrations
RESULTS
Patient
characteristics
(Table 1). Physical fitness, as-
sessed by maximal aerobic exercise testing (Vo2 max), fell within a relatively narrow range and confirmed that no volunteer was highly trained. We found no differences in rates of whole body or forearm amino acid fluxes between male and female subjects, and we have pooled their data in the subsequent analyses. Whole blood amino acid concentrations. Whole blood amino acid concentrations in arterialized hand vein samples are shown in Table 2. Branched-chain amino acid concentrations were significantlv increased after exer-
Resting
Postexercise
Leucine lOOk8 120t12* Isoleucine 40t3 51+5t Valine 134k14 152t14* Lysine 144t16 10724-t Glycine 17126 137+9t Alanine 159k12 121+5t Phenylalanine 40t2 40t2 Tyrosine 49*3 4723 Glutamine 437k36 374+17’f Glutamic acid 68t5 53+6t Values are means k SE in pmol/l. * P < 0.05, 7 P < 0.001, resting vs. postexercise.
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PROTEIN 4
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T
LEUCINE
0-o
Rest
e-----e o-0 *
.- - -@ Exedss
1
*
p
(0.001
t
I I
120
I 1
, I
135
150
I I
Exercise Rest 0.001
p< I I
165
-4
180
TIME (minutes)
4
T
FIG. 2. Ratio of plasma enrichments of a-ketoisocaproic acid (KIC) to leucine (EKIC/Eleucine)in resting and postexercise steady-state periods. * P < 0.001, resting vs. postexercise (ANOVA).
KIC
3. Whole bodjl and forearm amino acid metabolism
TABLE
Whole
Body Amino Acid Metabolism, h -1
Amino Acid Ra, protein breakdown [15N]Lys
4
-
T I
l
Leucine oxidation
NOLD
19t3.2 13t2.3*
103t5.6 115t6.1*
l
[‘“C]Leu
8525.8
122t5.3
9228.4
128t6.3 Forearm
LYSINE
.
Resting Postexercise
pmol kg-’
muscle amino acid metabolism, nmol - 100 ml-’ min-’ l
Net balance
1 1
120
I 1
135
1 v
150
I 1
I 1
165
4
180
FIG. 1. Plasma enrichments of leucine (top), ar-ketoisocaproic acid (KIC) (middl e) , and lysine (bottom) in arterialized hand vein samples, throughout the 120- to 180-min steady-state periods in the resting and postexercise studies. * P < 0.001, resting vs. postexercise (ANOVA).
the resting state (13 t 2.3 and 19 t 3.2 prnol. kg-’ h-l, respectively; P < 0.02; Table 3). Nonoxidative leucine disposal or estimated whole body protein synthesis, calculated as the difference between whole body leucine turnover, and leucine oxidation, was significantly increased after exercise compared with the resting state (115 t 6.1 and 103 t 5.6 prnol. kg-‘. h-‘, respectively; P < 0.02). Forearm amino acid exchange (Table 3). The net balance of lysine from the forearm was similar after exercise to the resting values (-87 t 25 vs. -93 t 28 nmol 100 ml-l . min-l, respectively), while forearm leucine balance was slightly less negative after exercise (postexercise -10 t 21 vs. resting -23 t 14 nmol 100 ml-’ min-‘) (0.05 < P < 0.10). Forearm KIC balance was not affected by exercise (postexercise -1 t 7 vs. resting -2 t 5 nmol l
l
Protein breakdown
Protein synthesis
Resting -93228 356285 255k69 Postexercise -87t25 219t51* 132*41* Values are means * SE. Ra, Rate of appearance is estimated as equivalent to the appearance rate of unlabeled amino acid measured by dilution of [Cy-15N]lysine and [ 1-13C]ketoisocaproic acid in plasma, since body protein breakdown is the only net source of unlabeled essential amino acid in the postabsorptive state. NOLD, Nonoxidative leucine disposal is taken as an index of whole body protein synthesis. Protein breakdown is estimated using formula described in METHODS section. Protein synthesis is the sum of net balance and protein breakdown (see METHODS). * P C 0.05, resting vs. postexercise using ANOVA (Kruskal- Wallis for nonparametric data).
100 ml-’ *minel). The fractional extraction of leucine was significantly greater (less negative) after exercise compared with the resting state (-3.9 t 11.5 and -7.3 t 3.7 nmol . 100 ml-l. min-l, respectively; P < 0.05). Forearm muscle protein degradation, calculated using the enrichments of [15N]lysine in arterialized hand and deep forearm veins (see METHODS) was significantly lower after exercise compared with the resting state (219 t 51 and 356 t 85 nmoLlO0 ml-‘. min-‘, respectively; P < 0.05). Qualitatively similar results were obtained using enrichments of [13C]leucine (postexercise 103 $- 56 vs. resting 156 t 40 nmol . 100 ml-‘. min-‘; 0.05 < P < 0.10). Forearm muscle protein synthesis, calculated as the sum of lysine net balance and degradation rates (4), was significantly lower after exercise compared with the resting state (132 t 40.8 and 255 t 69.0 nmol. 100 ml-‘. min-‘, respectively; P < 0.01). Correlations. The change in leucine oxidation rate between the resting and the postexercise study was significantly correlated with the subject’s VOW max(r = 0.72,
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PROTEIN
P < 0.05). Subjects with the lowest greatest inhibition
of leucine oxidation
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Vo2 max had the after exercise.
DISCUSSION
Previous studies of the effects of exercise on amino acid metabolism have focused primarily on the intraexercise period. These have demonstrated severalfold increases in leucine oxidation rate during exercise (20, 25), and increased rates of net proteolysis during exercise have been reported in some studies. We hypothesized that proteolysis would remain elevated for at least 2 h after high-intensity exercise using a protocol that has been shown to uniformly deplete vastus lateralis muscle glycogen content (7, 8), since previous work (15) suggests that protein catabolism is greater in the glycogen-depleted state. Rennie (20) has shown that during the recovery period after moderate intensity exercise (50% VOWmax) protein synthesis increases and exceeds degradation, and consequently net protein balance again becomes positive. However, in that study estimates of whole body protein turnover were made from calculations of leucine appearance measured by dilution of L-[ lJ3C]leucine in plasma. Wolfe et al. (26) have reported that the same two amino acid tracers used in our study, L- [ &‘N]lysine and L- [ lJ3C]leucine, gave disparate results when used to estimate whole body proteolysis during exercise. These discrepancies cannot be resolved by using the plasma enrichments of a[ 113C]KIC reported by Wolfe et al. (25) to reflect the enrichment of the precursor pool for intracellular leucine metabolism, as has been recommended (21, 22, 24). This suggests that there may be a limitation in the mathematical model for estimating rates of whole body proteolysis during exercise, since the models generally assume that different essential amino acid tracers will give comparable estimates of protein breakdown. Our data show that estimates of whole body proteolysis made from plasma enrichments of CY-[‘“C]KIC were in excellent agreement with those obtained using L- [ cwJ5N]lysine during the postexercise recovery period, with both tracers demonstrating an insignificant (5-S%) increase in proteolysis after exercise, compared with the nonexercised state. It may be that our study period was sufficiently delayed after exercise (2-3 h later) to allow steady-state estimates of whole body protein turnover to be made. The ratio of plasma enrichments of a-[ l-13C]KIC to L- [ l-13C]leucine has been relatively constant in the postabsorptive state and with varying levels of dietary protein intake (22). Our data suggest that this relationship is altered during early postexercise recovery and that there may be more complete equilibration of plasma and intracellular leucine pools during this period. The finding that the fractional extraction of leucine by forearm muscle was greater (less negative) after exercise suggests that this mechanism may at least be contributory. Previous work in the rat (27) has demonstrated increased amino acid transport into muscle after exercise. An alternative explanation is suggested by the data of Fielding et al. (10) demonstrating increased muscle KIC concentrations but delayed increases in plasma KIC immediately after intense exercise, possibly reflecting slow diffusion of KIC
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across the muscle membrane in this setting. This observation might limit the usefulness of plasma KIC enrichments as indicators of whole body proteolysis during and shortly after exercise. However, in Fielding’s study (10) muscle KIC determinations were only made immediately after exercise, and plasma KIC concentrations had returned to base line within 60 min of stopping exercise. Our steady-state period was 2-3 h after exercise at a time when plasma KIC enrichments were constant, making this an unlikely explanation for our findings. Using the rat hindlimb, Hood (14) has shown that the increase in leucine transamination (decarboxylation + KIC release) during muscle contractions can be directly attributed to the muscle tissues, suggesting that leucine is directly utilized by exercising muscle as a fuel. After exercise, leucine oxidation was significantly decreased in our study, suggesting that other oxidative fuels may be substituted for leucine at this time. Tessari et al. (23) have demonstrated that physiological elevations of free fatty acids (FFA), from 0.5 to 1.2 mM, resulted in a decreased rate of leucine oxidation. In a protocol identical to that of the present study with a separate group of volunteers, we found similar elevations in plasma FFA concentrations during the postexercise recovery period (resting 0.72, postexercise 1.26 mM) (6). Nair et al. (19) has shown that infusions of ,&hydroxybutyrate, which increase plasma concentrations to -2 mM, are capable of decreasing leucine oxidation rates. Although we previously reported increased plasma concentrations of ,& hydroxybutyrate after a bout of exercise similar to the one utilized in the present study (7), the degree of elevation was only -10% of that produced in Nair’s protocol. The nonoxidative disposal of leucine, calculated as the difference between whole body flux and oxidation rates, has been assumed to represent an index of protein synthesis. Nair et al. (18) has demonstrated a high degree of correlation between rates of whole body protein synthesis derived from tracer data with direct estimates of muscle protein synthesis derived from muscle biopsy samples measuring the incorporation of L- [ l-13C]leucine into skeletal muscle. Our data suggest that whole body protein synthesis is significantly increased after exercise, in contrast to the exercise period itself when protein synthesis is decreased and leucine oxidation is increased (12). This pattern is reversed during recovery, so that leucine carbons are redirected away from decarboxylation and toward incorporation into body protein. This finding is analogous to our previous data on whole body glucose metabolism in normal volunteers, in which the major effect of prior exercise was shown to be a shift away from oxidative and toward nonoxidative glucose disposal for glycogen resynthesis (8). In the present study, the magnitude of decrease in leucine oxidation after exercise, compared with the resting state, was significantly correlated with the subjects’ maximal aerobic capacity WO 2max). This suggests that the ability of a single bout of exercise to alter the pathway of leucine disposal during the recovery period may be attenuated in more highly trained subjects. Barrett (4) used infusions of L- [ring-2,6-3H]phenylal-
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anine in the dog to estimate limb muscle protein synthesis and degradation from arteriovenous differences in tracer specific activities. Because phenylalanine is neither synthesized nor degraded in muscle tissue, the net balance across a muscle bed must represent the difference between skeletal muscle protein synthesis and degradation. We used infusions of L- [ cGN]lysine for similar reasons because this is also an essential amino acid that is not degraded or transaminated in skeletal muscle. Using this approach, we found marked differences in forearm muscle protein synthesis and degradation between the resting and postexercise conditions. Qualitatively similar results were obtained when the rate of appearance of leucine across the forearm was calculated, although this is a less reliable estimate of forearm proteolysis because of leucine’s alternate metabolic fates in muscle tissue (transamination and oxidation). One limitation of the present study is the use of plasma enrichments of amino acids to estimate forearm muscle protein metabolism. Because red blood cells may participate in amino acid exchange across the forearm, Barrett et al. (4) have pointed out that whole blood measurements may be more appropriate for studies across the limb. However, similar estimates of the rates of hindlimb muscle proteolysis were obtained in their study using plasma and whole blood phenylalanine enrichments. It is of interest that estimated forearm muscle protein synthesis rates were decreased during postexercise recovery, at a time when whole body protein synthesis was increased. There may be several explanations for this finding. First, forearm muscle was not exercised during the bicycle exercise, and increases in muscle protein synthesis during recovery may be confined to the previously exercised muscle tissues. Alternatively, the increased protein synthesis during recovery might occur in the liver or other splanchnic tissues, since some previous studies have suggested that these tissues may contribute to proteolysis during the exercise period (9, 20). Thus it is possible that the increased whole body protein synthesis we observed during the recovery period was confined to those tissues which had increased protein degradation during exercise. In summary, whole body protein degradation returned to the basal resting level within 2 h after high-intensity exercise. The major effect of prior exercise on whole body leucine metabolism in the postexercise period was to decrease oxidative and to increase nonoxidative leucine disposal, the latter taken to represent increased protein synthesis. Forearm muscle protein synthesis was reduced after exercise, suggesting that the observed increases in whole body protein synthesis occurred either in the previously exercised muscles only or in other nonskeletal muscle tissues. We thank the University of Vermont Clinical Research Center nursing and dietary staffs, H. Christopherson for excellent technical assistance, and Dr. Naomi Fukagawa for assistance. This work was supported by a National Institutes of Health Clinical Investigator Award, K08-01554 (to J. T. Devlin), a Juvenile Diabetes Foundation Research Grant (to J. T. Devlin), Mass Spectrometry Resource Facility Award, RR-00954, and the University of Vermont Grant GCRC, RR 109.
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Address for reprint requests: J. T. Devlin, Metabolic Unit, University of Vermont College of Medicine, Burlington, VT 05405. Received 2 June 1989; accepted in final form 9 September 1989. REFERENCES N. N., D. RABIN, M. P. DIAMOND, AND W. W. LACY. Use of a heated superficial hand vein as an alternative site for measurement of amino acid concentrations and for the study of glucose and alanine kinetics in man. Metabolism 30: 936-940,198l. 2. AHLBORG, G., AND P. FELIG. Lactate and glucose exchange across the forearm, legs, and splanchnic bed during and after prolonged leg exercise. J. Clin. Inuest. 69: 45-54, 1982. 3. ASTRAND, P. O., AND K. RODAHL. Evaluation of physical performance on the basis of tests. In: Textbook of Work Physiology: Physiological Bases of Exercise. New York: McGraw-Hill, 1986, p. 354390. 4. BARRETT, E. J., J. H. REVKIN, L. H. YOUNG, B. L. ZARET, R. JACOB, AND R. A. GELFAND. An isotopic method for measurement of muscle protein synthesis and degradation in vivo. Biochem. J. 1. ABUMRAD,
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R. F. HELLON. Venous collection in forearm and hand measured by the strain gauge and volume plethysmographs. Clin. Sci. Lond. 16: 103-117, 1975. 6. DEVLIN, J. T., J. BARLOW, AND E. S. HORTON. Whole body and regional fuel metabolism during early postexercise recovery. Am. J. Physiol. 7. DEVLIN,
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E. S. HORTON. Effects of prior high-intensity exercise on glucose metabolism in normal and insulin-resistant men. Diabetes 34: 973-979, 1985. 9. DOHM, G. L., G. J. KASPAREK, E. B. TAPSCOTT, AND H. A. BARAKAT. Protein metabolism during endurance exercise. Federution Proc. 10. FIELDING, AND B.
44: 348-352,
1985.
R. A., W. J. EVANS, V. A. HUGHES, L. L. MOLDAWER, R. BISTRIAN. The effects of high intensity exercise on muscle and plasma levels of alpha-ketoisocaproic acid. Eur. J.
Appl. Physiol. Occup. Physiol. 55: 482-485, 1986. 11. GELFAND, R. A., AND E. J. BARRETT. Effect of
physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J. CZin. Invest. 80: l-6, 1987. 12. HAGG, S. A., E. L. MORSE, AND S. A. ADIBI. Effect of exercise on rates of oxidation, turnover, and plasma clearance of leucine in human subjects. Am. J. Physiol. 242 (Endocrinol. Metub. 5): E407E410,1982. 13. HILL, D.
W., F. H. WALTERS, T. D. WILSON, AND J. D. STUART. High performance liquid chromatography determination of amino acids in the picomole range. Anal. Chem. 51: 1339-1341,1979. 14. HOOD, D. A., AND R. L. TERJUNG. Leucine metabolism in perfused rat skeletal muscle during contractions. Am. J. Physiol. 253 (Endocrinol. Metub. 16): E636-E647, 1987. 15. LEMON, P. W. R., AND J. P. MULLIN. Effect of initial muscle glycogen levels on protein catabolism during exercise. J. AppZ. Physiol. 48: 624-629, 16. MATTHEWS, D. E., V. R. YOUNG, AND
1980.
K. J. MOTIL, D. K. ROHRBAUGH, J. F. BURKE, D. M. BIER. Measurement of leucine metabolism in man from a primed, continuous infusion of L- [ l-‘3C]leucine. Am. J. Physiol. 238 (Endocrinol. Metub. 1): E473-E479, 1980. 17. MATTHEWS, D. E., H. P. SCHWARZ, R. D. YANG, K. J. MOTIL, V. R. YOUNG, AND D. M. BIER. Relationship of plasma leucine and a-ketoisocaproate during a L-[l-‘3C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment. Metabolism 31: 1105-1112,1982. 18. NAIR, K. S., D. HALLIDAY, AND R. G. GRIGGS. Leucine incorporation into mixed skeletal muscle protein in humans. Am. J. Physiol. 19. NAIR,
254 (Endocrinol.
Metub.
17): E208-E213,
1988.
K. S., S. L. WELLE, D. HALLIDAY, AND R. G. CAMPBELL. Effect of ,&hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans. J.
CZin. Inuest. 82: 198-205, 20. RENNIE, M. J., R. H. DAVIES, D. HALLIDAY,
1988.
T. EDWARDS, S. KRYWAWYX=H, C. T. M. J. C. WATERLOW, AND D. J. MILLWARD.
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PROTEIN
TURNOVER
DURING
Effect of exercise on protein turnover in man. Clin. Sci. Lond.
61:
627-639,198l. 21. RENNIE, M. J., R. H. THEWS, S. L. WOLMAN,
synthesis of feeding 22.
SCHWENK,
T. EDWARDS, D. HALLIDAY, D. E. MATAND D. J. MILLWARD. Muscle protein measured by stable isotope technique in man: the effect and fasting. CZin. Sci. Lond. 63: 519-523, 1982. W. F., B. BEAUFRERE, AND M. W. HAYMOND. Use of pool specific activities to model leucine metabolism in
reciprocal humans. Am. J. Physiol. 1985. 23. TESSARI,
249 (Endocrinol.
Met&
12): E646-E650,
P., S. L. NISSEN, J. M. MILES, AND M. H. HAYMOND. Inverse relationship of leucine flux and oxidation to free fatty acid availability in vivo. J. Clin. Invest. 77: 575-581, 1986.
POSTEXERCISE
E255
RECOVERY
J. A., H. S. PAUL, AND S. A. ADIBI. Relation between plasma and tissue parameters of leucine metabolism in fed and starved rats. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E615-
24. VAZQUEZ, E621,
1986.
M. H. WOLFE, G. T. ROYLE, 25a WOLFE, R. R., R. D. GOODENOUGH, AND E. R. NADEL. Isotopic analysis of leucine and urea metabolism in exercising humans. J. AppZ. Physiol. 52: 458-466, 1982. 26 WOLFE, R. R., M. H. WOLFE, E. R. NADEL, AND J. H. F. SHAW. Isotopic determination of amino acid-urea interactions in exercise in humans. J. AppZ. Physiol. 56: 221-229, 1984. 27. ZORZANO, A., T. W. BALON, L. P. GARETTO, M. N. GOODMAN, AND N. B. RUDERMAN. Muscle cr-aminoisobutyric acid transport after exercise: enhanced stimulation by insulin. Am. J. Physiol. l
248 (Endocrinol.
Metab.
11): E546-E552,
1985.
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after intense exercise
JOHN T. DEVLIN, IRWIN BRODSKY, ANGUS SUSAN FULLER, AND DENNIS M. BIER
SCRIMGEOUR,
Metabolic Unit, University of Vermont College of Medicine, Burlington, Vermont 05405; and Metabolism Division, Washington University School of Medicine, St. Louis, Missouri 63110 DEVLIN, JOHN T., IRWIN BRODSKY, ANGUS SCRIMGEOUR, SUSAN FULLER, AND DENNIS M. BIER. Amino acid metabolism after intensive exercise. Am. J. Physiol. 258 (Endocrinol. Metab.
21: E249-E255, 1990.-We studied postexercise amino acid metabolism, in the whole body and across the forearm. Seven volunteers were infused with L- [a(-15N]lysine and L- [ l-13C] leucine twice [one time during 3 h after cycle exercise (75% h max),and one time in the resting state]. Whole body protein breakdown was estimated from dilution of L-[&‘N]lysine and L- [ l-13C] ketoisocaproic acid (KIC) enrichments in plasma. Leutine oxidation was calculated from 13C02 enrichments in expired air. Whole body protein breakdown was not increased above resting levels during the recovery period. Leucine oxidation was decreased after exercise (postexercise 13 t 2.3 vs. resting 19 t 3.2 prnol. kg-l. h-l; P < 0.02), while nonoxidative leucine disposal was increased (115 t 6.1 vs. 103 t 5.6 pg. kg-l min-l; P < 0.02). After exercise, forearm net lysine balance was unchanged (87 t 25 vs. 93 t 28 nmol. 100 ml-‘. min-l), but there were decreases in forearm muscle protein degradation (219 t 51 vs. 356 -C-85 nmol~lO0 ml-‘. min-‘; P < 0.05) and synthesis (132 & 41 vs. 255 t 69 nmollO0 ml-’ .min-‘; P < 0.01). In conclusion, after exercise 1) whole body protein degradation is not increased, 2) leucine disposal is directed away from oxidative and toward nonoxidative pathways, 3) forearm protein synthesis is decreased. Postexercise increases in whole body protein synthesis occur in tissues other than nonexercised muscle. l
protein synthesis; leucine oxidation; forearm muscle
ofphysicalexercise on amino acid metabolism have focused primarily on the exercise period itself. Leucine oxidation is known to be increased severalfold during exercise (20, 25), in part serving as an oxidative fuel for contracting muscles (14). Rates of whole body protein synthesis are reduced and protein degradation increased during exercise (20). After moderate-intensity exercise (at 50% of maximal aerobic capacity, VO 2 max), rates of protein synthesis increase and proteolysis decrease so that net protein balance is positive (20). However, estimates of proteolysis in this earlier study were made from calculations of leucine appearance measured by dilution of L-[ 1-13C] leucine in plasma. This approach measures the plasma leucine appearance rate reliably but, because proteolysis occurs intracellularly, plasma leucine appearance does not necessarily reflect the appearance of unlabeled leutine in the inaccessible intracellular compartments. Thus several authors (21,22, 24) have advocated using plasma enrichments of a-[ 1-13C] ketoisocaproic acid (KIC) as an PREVIOUS
STUDIES
OF THE EFFECTS
index of intracellular leucine enrichment to assess rates of whole body protein degradation. Wolfe et al. (26) have reported that different estimates of whole body proteolysis during exercise occur when different amino acid tracers are used (L- [ 13C]leucine and L- [ &‘N]lysine). Thus we also examined whether these same two tracers gave differing estimates for rates of whole body proteolysis during recovery when cy-[ l-13C]KIC enrichments were used as an index of intracellular leucine enrichment. Based on the previous report by Lemon and Muller (15) that exercise-induced proteolysis may be greater in states of glycogen-depletion, we hypothesized that whole body proteolysis would remain elevated throughout the 2- to 3-h recovery period after high-intensity, glycogendepleting exercise (at 75% Vo2 max). We further hypothesized that forearm muscle proteolysis would similarly be increased for a prolonged period after exercise, since nonexercised (forearm) muscle is insulin resistant 2-3 h after intense bicycle (leg) exercise (6) and continues to release gluconeogenic precursors (lactate and alanine) throughout this time period in amounts that cannot be accounted for by glucose uptake, suggesting ongoing forearm muscle glycogenolysis (2). We have adapted the technique described by Barrett et al. (4) and used the arterialized hand and deep forearm vein enrichments of L-[&5N]lysine to estimate rates of forearm muscle protein synthesis and degradation. METHODS
Subjects. Seven subjects (4 male and 3 female, ages 23-32 yr, and within 17% of Metropolitan ideal body weight) participated in the study. All subjects were in excellent health based on medical history, physical examination, and routine screening laboratory chemistries. No subject was taking medications regularly, nor were any engaged in a regular exercise training program. The design, purpose, and possible risks were carefully explained to each volunteer before obtaining written consent. The experimental protocol was approved by the University of Vermont Committee on Human Research. Prior to first admission to the University of Vermont Clinical Research Center (CRC), each subject underwent a maximum aerobic capacity (VO, max) test using a continuous exercise protocol on a Monarch bicycle (3). At the University of Vermont Clinical Research Center all subjects were fed a weight-maintaining diet containing 1.3 times their estimated daily resting energy expenditures, with protein constituting 20% of total calories for
0193-1849/90 $1.50 Copyright 0 1990 the American Physiological
Society
E249
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E250
PROTEIN
TURNOVER
DURING
7 days prior to each study day. Subjects were fasted overnight at the CRC prior to each study day. Experimental design. On the morning of each study day, subjects were placed under the ventilated hood at 0630 h for measurement of resting metabolic rate using continuous indirect calorimetry as previously described (8). After a 3O- to 4O-min steady-state period subjects were removed from the hood. Expired air samples were collected using a mouthpiece connected via a J-valve to a &liter non-diffusing bag (Hans Rudolph, Kansas City, MO), and an aliquot was injected into a 20-ml Venoject Vacutainer tube for later analysis of 13C02 enrichment. Four base-line expired air samples were taken at 5-min intervals. On the “exercise” study days, subjects then performed continuous cycle exercise at a workload previously determined to represent 75% of their VOzmax. This was continued until subjects were subjectively exhausted and unable to maintain a pedaling frequency >5O revolutions/min. We have previously demonstrated that vastus lateralis skeletal muscle is uniformly glycogen depleted using this protocol (7, 8). After exercise subjects quickly showered and returned to bed in the study room. On the “nonexercise” study day subjects remained resting comfortably in bed during the same time period. Except for the period of bicycle exercise, the activity of the subjects was similar on the exercise and nonexercise days. At approximately 0900 h, intravenous catheters were inserted. A 19-gauge indwelling catheter was inserted in a left antecubital vein (infusion catheter). A 19-gauge indwelling needle was inserted in a left hand vein, and the hand was warmed in a heated box (70°C) for obtaining “arterialized” blood specimens (1). A 19-gauge indwelling catheter was placed retrogradely into a right antecubital vein and withdrawn l-2 cm from the “wedged” position for obtaining deep forearm venous blood samples. Two arterialized blood samples 5 min apart were taken for determining base-line amino acid enrichments. Four additional expired air samples were taken at 5-min intervals for postexercise base-line 13C02 enrichment. After base-line sampling the following primed continuous infusions were given at time 0 (-2030 min after exercise): NaH13C03, 0.08 pmol/kg bolus (99% 13C); L-[&-l5 Nllysine, 1.6 pmol/kg bolus and 1.8 pmol kg-’ . h-l infusion; and L- [ 1 -13C] leucine, 2 pmol/kg bolus and 2.4 prnol. kg-’ . h-l infusion (99% 15N and 13C, respectively; MSD Isotopes, Montreal, Canada). Subjects were again placed in the ventilated hood for continuous indirect calorimetry readings. Sampling procedures and steady-state period. Blood samples were taken from the arterialized hand and deep forearm vein catheters at 60 and 90 min, and every 15 min during the 120- to 180-min steady-state period. Forearm blood flow was simultaneously and noninvasively measured using impedance plethysmography (5). Leucine and lysine concentrations were determined on protein-free filtrates of whole blood obtained by adding an equal volume of 10% sulfosalicylic acid and mixing immediately. After separation in a refrigerated centrifuge the supernatants were frozen, stored at -8O”C, and later analyzed with a precolumn o’-phthalaldehyde high-perl
POSTEXERCISE
RECOVERY
formance liquid chromatography method (13). Samples for determining cu-KIC concentrations and enrichments of A!-[~-~~C]KIC, L-[l-13C]leucine, and L-[a-“Nllysine were centrifuged at 4”C, and plasma samples were stored at -80°C until later analysis. Rates of COn production were determined continuously with the subject in the ventilated hood, using an infrared CO2 analyzer (Applied Electrochemistry, Sunnyvale, CA) and a flowmeter (Vertek, Burlington, VT). Readings were corrected for standard temperature and pressure, dry (STPD) conditions. At 60 and 90 min and every 20 min throughout the 120- to 180-min steady-state period, the subject was briefly removed from the hood, and the mouthpiece was used to obtain an expired air sample for determining 13C02 enrichment. The steady-state period for our protocol design was verified by prolonged (5 h) isotope infusions in our initial two subjects. This control experiment demonstrated steady-state enrichments of CY-[ l-13C]KIC, L- [ 1-‘3C]leutine, and L+?N]lysine within 120 min of stopping exercise. In an additional control experiment, we also examined the 13C02 enrichment in expired air after exercise in two subjects who received no isotope infusions. The natural background 13C02 isotopic abundance remained unchanged during the entire experimental period. In two other subjects, we determined the rate of 13C02 recovery from a primed (890 pmol) constant (9.9 pmol/min) infusion of NaH13C03 during the postexercise recovery period (26). This value averaged 72%, which is within the range previously reported by Wolfe et al. (26) in resting subjects. Cakulations. Plasma leucine kinetics were determined using steady-state isotope dilution techniques as previously described (16,17). For the determination of leucine oxidation rates, steady-state plasma enrichments of CY[ l-13C]KIC were used to reflect the immediate precursor for the oxidative decarboxylation of leucine (17, 22). Estimates of the rates of whole body proteolysis and nonoxidative leucine disposal (an index of protein synthesis) were made with plasma enrichments of cy-[ 1-13C] KIC, taken to reflect the intracellular enrichment of leucine. Flux rates of lysine were also determined from steady-state plasma enrichments of L-[cx-15N]lysine and used as an indepe,ndent estimate of the rate of whole body proteolysis. Forearm balances of leucine and lysine were determined by the Fick principle, multiplying the arterialized hand deep forearm vein (A-V) gradients by the forearm blood flow that was determined by impedance plethysmography. The fractional extraction was calculated as (A-V) x 100/[AAla*,where [AA],, is the amino acid (lysine, leucine) concentration in the arterialized hand vein samples. The rate of forearm muscle protein breakdown was calculated using the formula D
=
[AA]art
x
[ ULlJLin)
-
11
x
BF
where E ah and Evein are the enrichments of [ 15N]lysine or [13C]leucine in arterialized hand vein and deep forearm vein samples, respectively, and BF is forearm blood flow determined by impedance plethysmography. Because forearm amino acid net balance equals synthesis minus
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PROTEIN TABLE
TURNOVER
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POSTEXERCISE
Weight,
Height, cm
E251
RECOVERY
1. Subject characteristics Subject
Sex
Age, Y*
AB LG RL TL MD JR JL Means t SE
F F F M M M M
32
29 26 24 24 12 24 2621.3
kg
VO 2
BMI*
kg-’
max9 l
ml
56 62 60 75 84 80 90
161 170 180 178 178 183 185
22 21 19 24 27 24 26
55 42 43 51 54 49 47
72t5.0
176k3.1
23kl.l
49k1.9
breakdown, forearm muscle protein synthesis can then be estimated as net balance plus breakdown. Although Barrett et al. (4) validated this method of calculating D with label phenylalanine in the dog, lysine is also an essential amino acid that is neither transaminated nor oxidized in muscle tissue and, therefore, exchange across the forearm reflects muscle protein synthesis and degradation. We also calculated the rate of appearance of unlabeled leucine across the forearm using the above formula to determine whether the results were qualitatively similar to those using lysine, as has been reported previously across the human forearm using phenylalanine and leucine tracers (11). This estimate of forearm leucine rate of appearance is not considered to accurately reflect forearm muscle proteolysis, since leucine can undergo transamination and oxidation in muscle tissues. Analytical methods. Plasma enrichments of stable isotopes were determined from the N-acetyl, n-propyl ester derivates of leucine and lysine and from the trimethylsilyl-quinoxalinol derivatives of KIC, using selected ion monitoring gas chromatography/mass spectrometry (16, 17). Expired air samples were collected in 20-ml Venoject containers and later analyzed for 13C02 enrichment using a VG-Isogas Siraisotope ratio mass spectrometer (Tracer Technologies, Somerville, MA). Statistical methods. All data are expressed as means t SE. Comparisons between the postexercise recovery period and the nonexercise (resting) day were made using analysis of variance (ANOVA; Kruskal-Wallis test for nonparametric data). When ANOVA indicated significant differences between the postexercise and nonexercise days, Student’s paired t test was used to indicate individual time points for which a difference was demonstrable.
cise, while concentrations decreased.
l
min-’
of glucogenic amino acids were
Isotope enrichments. In our initial two volunteers, we observed that plasma enrichments of amino acid isotopes plateaued within 120 min of infusion during the postexercise recovery period, and we selected the 120- to 180. min period of infusion as our steady-state plateau period. Figure 1 demonstrates the amino acid and KIC isotope enrichments in arterialized hand vein samples during this steady-state period after exercise. The slopes of the plasma enrichments were not significantly different from zero, using ANOVA (F ratios 0.069-0.098, P = 0.980.99). The forearm deep vein enrichments of lysine also reached a plateau during the 120- to 180-min steadystate period (F ratio 0.10, P = 0.98, for both resting and postexercise studies). The ratio of steady-state plasma enrichments of KIC to leucine was 0.68 t 0.019 in the resting state and 0.75 t 0.026 after exercise (P < 0.001; Fig. 2). Amino acid kinetics. Plasma leucine appearance was significantly higher after exercise than in the resting state (96 t 5.0 vs. 82 t 32. prnol. kg-‘. h-l, respectively; P c 0.001). Whole body leucine appearance, estimated from circulating ily-[ 1-‘3C]KIC dilution and a reflection of body protein breakdown, was not changed after exercise (128 t 6.3 vs. 122 t 5.3 prnol. kg-’ h-l), nor was lysine flux (92 t 8.4 vs. 85 If: 5.8, postexercise vs. resting) (Table 3). There was excellent agreement between these two tracers, which were selected to provide independent estimates of rates of whole body proteolysis. Leucine oxidation rates during the steady-state period were significantly lower after exercise, compared with l
TABLE
2. Arterial
amino acid concentrations
RESULTS
Patient
characteristics
(Table 1). Physical fitness, as-
sessed by maximal aerobic exercise testing (Vo2 max), fell within a relatively narrow range and confirmed that no volunteer was highly trained. We found no differences in rates of whole body or forearm amino acid fluxes between male and female subjects, and we have pooled their data in the subsequent analyses. Whole blood amino acid concentrations. Whole blood amino acid concentrations in arterialized hand vein samples are shown in Table 2. Branched-chain amino acid concentrations were significantlv increased after exer-
Resting
Postexercise
Leucine lOOk8 120t12* Isoleucine 40t3 51+5t Valine 134k14 152t14* Lysine 144t16 10724-t Glycine 17126 137+9t Alanine 159k12 121+5t Phenylalanine 40t2 40t2 Tyrosine 49*3 4723 Glutamine 437k36 374+17’f Glutamic acid 68t5 53+6t Values are means k SE in pmol/l. * P < 0.05, 7 P < 0.001, resting vs. postexercise.
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E252
PROTEIN 4
TURNOVER
DURING
POSTEXERCISE
RECOVERY
T
LEUCINE
0-o
Rest
e-----e o-0 *
.- - -@ Exedss
1
*
p
(0.001
t
I I
120
I 1
, I
135
150
I I
Exercise Rest 0.001
p< I I
165
-4
180
TIME (minutes)
4
T
FIG. 2. Ratio of plasma enrichments of a-ketoisocaproic acid (KIC) to leucine (EKIC/Eleucine)in resting and postexercise steady-state periods. * P < 0.001, resting vs. postexercise (ANOVA).
KIC
3. Whole bodjl and forearm amino acid metabolism
TABLE
Whole
Body Amino Acid Metabolism, h -1
Amino Acid Ra, protein breakdown [15N]Lys
4
-
T I
l
Leucine oxidation
NOLD
19t3.2 13t2.3*
103t5.6 115t6.1*
l
[‘“C]Leu
8525.8
122t5.3
9228.4
128t6.3 Forearm
LYSINE
.
Resting Postexercise
pmol kg-’
muscle amino acid metabolism, nmol - 100 ml-’ min-’ l
Net balance
1 1
120
I 1
135
1 v
150
I 1
I 1
165
4
180
FIG. 1. Plasma enrichments of leucine (top), ar-ketoisocaproic acid (KIC) (middl e) , and lysine (bottom) in arterialized hand vein samples, throughout the 120- to 180-min steady-state periods in the resting and postexercise studies. * P < 0.001, resting vs. postexercise (ANOVA).
the resting state (13 t 2.3 and 19 t 3.2 prnol. kg-’ h-l, respectively; P < 0.02; Table 3). Nonoxidative leucine disposal or estimated whole body protein synthesis, calculated as the difference between whole body leucine turnover, and leucine oxidation, was significantly increased after exercise compared with the resting state (115 t 6.1 and 103 t 5.6 prnol. kg-‘. h-‘, respectively; P < 0.02). Forearm amino acid exchange (Table 3). The net balance of lysine from the forearm was similar after exercise to the resting values (-87 t 25 vs. -93 t 28 nmol 100 ml-l . min-l, respectively), while forearm leucine balance was slightly less negative after exercise (postexercise -10 t 21 vs. resting -23 t 14 nmol 100 ml-’ min-‘) (0.05 < P < 0.10). Forearm KIC balance was not affected by exercise (postexercise -1 t 7 vs. resting -2 t 5 nmol l
l
Protein breakdown
Protein synthesis
Resting -93228 356285 255k69 Postexercise -87t25 219t51* 132*41* Values are means * SE. Ra, Rate of appearance is estimated as equivalent to the appearance rate of unlabeled amino acid measured by dilution of [Cy-15N]lysine and [ 1-13C]ketoisocaproic acid in plasma, since body protein breakdown is the only net source of unlabeled essential amino acid in the postabsorptive state. NOLD, Nonoxidative leucine disposal is taken as an index of whole body protein synthesis. Protein breakdown is estimated using formula described in METHODS section. Protein synthesis is the sum of net balance and protein breakdown (see METHODS). * P C 0.05, resting vs. postexercise using ANOVA (Kruskal- Wallis for nonparametric data).
100 ml-’ *minel). The fractional extraction of leucine was significantly greater (less negative) after exercise compared with the resting state (-3.9 t 11.5 and -7.3 t 3.7 nmol . 100 ml-l. min-l, respectively; P < 0.05). Forearm muscle protein degradation, calculated using the enrichments of [15N]lysine in arterialized hand and deep forearm veins (see METHODS) was significantly lower after exercise compared with the resting state (219 t 51 and 356 t 85 nmoLlO0 ml-‘. min-‘, respectively; P < 0.05). Qualitatively similar results were obtained using enrichments of [13C]leucine (postexercise 103 $- 56 vs. resting 156 t 40 nmol . 100 ml-‘. min-‘; 0.05 < P < 0.10). Forearm muscle protein synthesis, calculated as the sum of lysine net balance and degradation rates (4), was significantly lower after exercise compared with the resting state (132 t 40.8 and 255 t 69.0 nmol. 100 ml-‘. min-‘, respectively; P < 0.01). Correlations. The change in leucine oxidation rate between the resting and the postexercise study was significantly correlated with the subject’s VOW max(r = 0.72,
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PROTEIN
P < 0.05). Subjects with the lowest greatest inhibition
of leucine oxidation
TURNOVER
DURING
Vo2 max had the after exercise.
DISCUSSION
Previous studies of the effects of exercise on amino acid metabolism have focused primarily on the intraexercise period. These have demonstrated severalfold increases in leucine oxidation rate during exercise (20, 25), and increased rates of net proteolysis during exercise have been reported in some studies. We hypothesized that proteolysis would remain elevated for at least 2 h after high-intensity exercise using a protocol that has been shown to uniformly deplete vastus lateralis muscle glycogen content (7, 8), since previous work (15) suggests that protein catabolism is greater in the glycogen-depleted state. Rennie (20) has shown that during the recovery period after moderate intensity exercise (50% VOWmax) protein synthesis increases and exceeds degradation, and consequently net protein balance again becomes positive. However, in that study estimates of whole body protein turnover were made from calculations of leucine appearance measured by dilution of L-[ lJ3C]leucine in plasma. Wolfe et al. (26) have reported that the same two amino acid tracers used in our study, L- [ &‘N]lysine and L- [ lJ3C]leucine, gave disparate results when used to estimate whole body proteolysis during exercise. These discrepancies cannot be resolved by using the plasma enrichments of a[ 113C]KIC reported by Wolfe et al. (25) to reflect the enrichment of the precursor pool for intracellular leucine metabolism, as has been recommended (21, 22, 24). This suggests that there may be a limitation in the mathematical model for estimating rates of whole body proteolysis during exercise, since the models generally assume that different essential amino acid tracers will give comparable estimates of protein breakdown. Our data show that estimates of whole body proteolysis made from plasma enrichments of CY-[‘“C]KIC were in excellent agreement with those obtained using L- [ cwJ5N]lysine during the postexercise recovery period, with both tracers demonstrating an insignificant (5-S%) increase in proteolysis after exercise, compared with the nonexercised state. It may be that our study period was sufficiently delayed after exercise (2-3 h later) to allow steady-state estimates of whole body protein turnover to be made. The ratio of plasma enrichments of a-[ l-13C]KIC to L- [ l-13C]leucine has been relatively constant in the postabsorptive state and with varying levels of dietary protein intake (22). Our data suggest that this relationship is altered during early postexercise recovery and that there may be more complete equilibration of plasma and intracellular leucine pools during this period. The finding that the fractional extraction of leucine by forearm muscle was greater (less negative) after exercise suggests that this mechanism may at least be contributory. Previous work in the rat (27) has demonstrated increased amino acid transport into muscle after exercise. An alternative explanation is suggested by the data of Fielding et al. (10) demonstrating increased muscle KIC concentrations but delayed increases in plasma KIC immediately after intense exercise, possibly reflecting slow diffusion of KIC
POSTEXERCISE
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across the muscle membrane in this setting. This observation might limit the usefulness of plasma KIC enrichments as indicators of whole body proteolysis during and shortly after exercise. However, in Fielding’s study (10) muscle KIC determinations were only made immediately after exercise, and plasma KIC concentrations had returned to base line within 60 min of stopping exercise. Our steady-state period was 2-3 h after exercise at a time when plasma KIC enrichments were constant, making this an unlikely explanation for our findings. Using the rat hindlimb, Hood (14) has shown that the increase in leucine transamination (decarboxylation + KIC release) during muscle contractions can be directly attributed to the muscle tissues, suggesting that leucine is directly utilized by exercising muscle as a fuel. After exercise, leucine oxidation was significantly decreased in our study, suggesting that other oxidative fuels may be substituted for leucine at this time. Tessari et al. (23) have demonstrated that physiological elevations of free fatty acids (FFA), from 0.5 to 1.2 mM, resulted in a decreased rate of leucine oxidation. In a protocol identical to that of the present study with a separate group of volunteers, we found similar elevations in plasma FFA concentrations during the postexercise recovery period (resting 0.72, postexercise 1.26 mM) (6). Nair et al. (19) has shown that infusions of ,&hydroxybutyrate, which increase plasma concentrations to -2 mM, are capable of decreasing leucine oxidation rates. Although we previously reported increased plasma concentrations of ,& hydroxybutyrate after a bout of exercise similar to the one utilized in the present study (7), the degree of elevation was only -10% of that produced in Nair’s protocol. The nonoxidative disposal of leucine, calculated as the difference between whole body flux and oxidation rates, has been assumed to represent an index of protein synthesis. Nair et al. (18) has demonstrated a high degree of correlation between rates of whole body protein synthesis derived from tracer data with direct estimates of muscle protein synthesis derived from muscle biopsy samples measuring the incorporation of L- [ l-13C]leucine into skeletal muscle. Our data suggest that whole body protein synthesis is significantly increased after exercise, in contrast to the exercise period itself when protein synthesis is decreased and leucine oxidation is increased (12). This pattern is reversed during recovery, so that leucine carbons are redirected away from decarboxylation and toward incorporation into body protein. This finding is analogous to our previous data on whole body glucose metabolism in normal volunteers, in which the major effect of prior exercise was shown to be a shift away from oxidative and toward nonoxidative glucose disposal for glycogen resynthesis (8). In the present study, the magnitude of decrease in leucine oxidation after exercise, compared with the resting state, was significantly correlated with the subjects’ maximal aerobic capacity WO 2max). This suggests that the ability of a single bout of exercise to alter the pathway of leucine disposal during the recovery period may be attenuated in more highly trained subjects. Barrett (4) used infusions of L- [ring-2,6-3H]phenylal-
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anine in the dog to estimate limb muscle protein synthesis and degradation from arteriovenous differences in tracer specific activities. Because phenylalanine is neither synthesized nor degraded in muscle tissue, the net balance across a muscle bed must represent the difference between skeletal muscle protein synthesis and degradation. We used infusions of L- [ cGN]lysine for similar reasons because this is also an essential amino acid that is not degraded or transaminated in skeletal muscle. Using this approach, we found marked differences in forearm muscle protein synthesis and degradation between the resting and postexercise conditions. Qualitatively similar results were obtained when the rate of appearance of leucine across the forearm was calculated, although this is a less reliable estimate of forearm proteolysis because of leucine’s alternate metabolic fates in muscle tissue (transamination and oxidation). One limitation of the present study is the use of plasma enrichments of amino acids to estimate forearm muscle protein metabolism. Because red blood cells may participate in amino acid exchange across the forearm, Barrett et al. (4) have pointed out that whole blood measurements may be more appropriate for studies across the limb. However, similar estimates of the rates of hindlimb muscle proteolysis were obtained in their study using plasma and whole blood phenylalanine enrichments. It is of interest that estimated forearm muscle protein synthesis rates were decreased during postexercise recovery, at a time when whole body protein synthesis was increased. There may be several explanations for this finding. First, forearm muscle was not exercised during the bicycle exercise, and increases in muscle protein synthesis during recovery may be confined to the previously exercised muscle tissues. Alternatively, the increased protein synthesis during recovery might occur in the liver or other splanchnic tissues, since some previous studies have suggested that these tissues may contribute to proteolysis during the exercise period (9, 20). Thus it is possible that the increased whole body protein synthesis we observed during the recovery period was confined to those tissues which had increased protein degradation during exercise. In summary, whole body protein degradation returned to the basal resting level within 2 h after high-intensity exercise. The major effect of prior exercise on whole body leucine metabolism in the postexercise period was to decrease oxidative and to increase nonoxidative leucine disposal, the latter taken to represent increased protein synthesis. Forearm muscle protein synthesis was reduced after exercise, suggesting that the observed increases in whole body protein synthesis occurred either in the previously exercised muscles only or in other nonskeletal muscle tissues. We thank the University of Vermont Clinical Research Center nursing and dietary staffs, H. Christopherson for excellent technical assistance, and Dr. Naomi Fukagawa for assistance. This work was supported by a National Institutes of Health Clinical Investigator Award, K08-01554 (to J. T. Devlin), a Juvenile Diabetes Foundation Research Grant (to J. T. Devlin), Mass Spectrometry Resource Facility Award, RR-00954, and the University of Vermont Grant GCRC, RR 109.
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