BIOCHEMICAL MEDICINk. 21, 196-X1 (1979)

Amino Acid Uptake and Levels: influence of Endurance Training

GARY R. BEECHER,” FERNANDO R. PUENTE,?

AND G. LYNIS DOHMt

*Protein Nutrition Laboratory. Nutrition Institute, Science and Education Administration,

U. S. Department qf Agriculture, Beltsville, Maryland 20705, and

TDepartment of Biochemistry, School of Medicine, East Carolina University.

Greenville, North Carolina 27834

Received January 23. 1979

We recently reported that endurance training increased amino acid oxidation and urea excretion and decreased protein synthesis in liver, heart, and the stromal fraction of muscle (1,2). Endurance training also decreased nitrogen balance 48% yet the nitrogen balance of trained ani- mals remained positive (1). In continuing this line of research, we were particularly interested in elucidating the factors that control protein me- tabolism in response to training. High concentrations of the branched- chain amino acids have been reported to stimulate muscle protein synthe- sis and inhibit breakdown during in vitro experiments (3,4). Thus, it seemed important to investigate whether changes in amino acid levels might be responsible for the changes in protein metabolism we had ob- served.

Goldberg and Goodman (5) found that amino acid transport into hyper- trophing muscle was increased at a time when protein synthesis was enhanced. Since amino acid transport into a muscle may be related to altered amino acid levels and changes in protein metabolism, we have also investigated the effect of endurance training on muscle uptake of a-aminoisobutyric acid (AIB).

METHODS

Male Holtzman’ rats (Holtzman, Madison, Wis.) weighing approxi- mately 200 g at the start of the experiment were housed in individual cages

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ENDURANCE TRAINING AND AMINO ACIDS 197

and given water and commercial lab chow (Wayne Lab Blox, Allied Mills, Inc., Chicago, Ill.) ad libitum. Rats were divided into two groups: un- trained, which remained sedentary in their cages; and trained, which were subjected to daily treadmill running at 35 m/min, 1 hr/day, 6 days/week, for 6 weeks as previously described (6).

During the seventh week of training, trained and untrained rats were anesthesized with an intraperitoneal injection of Nembutal(50 mg/kg), the abdominal cavity was opened by a midline incision, and blood was drawn from the vena cava into a heparinized syringe. Plasma and red blood cells were separated and frozen. The gastrocnemius muscle, heart, and liver were quickly excised and frozen; all samples were stored at -70°C for subsequent analyses.

Sulfosalicyclic acid extracts of muscle, heart, liver, and plasma were prepared as previously described (7). Amino acids were measured quan- titatively on a microcolumn amino acid analyzer (7).

Extracellular water was determined in trained and untrained rats by injecting hydroxy [methylJ4C]inulin (1 $X/100 g body weight, 18.2 mCi/mmole, Schwarz/Mann, Orangeburg, N.Y.) into the tail vein. The animals were anesthesized and tissue samples excised as described above. All tissue samples were treated with NCS solubilizer (Amersham/Searle, Arlington Heights, Ill.) and counted in a toluene cocktail (6 g PPOlliter toluene) with a Beckman LS-233 liquid scintillation counter. Quench was corrected by the method of external standard ratios.

To estimate the rate of uptake of AIB into tissues, [c$~C] aminoisobutyric acid (1.0 &i/100 g body weight, 60 mCi/mmole, Schwarz/Mann, Orangeburg, N.Y.) was injected into the tail veins of trained and untrained rats. The animals were sacrificed 1 hr later; samples were excised and prepared, and radioactivity was determined as de- scribed above.

Statistical analysis of the data consisted of one-way analysis of variance and, when significance was indicated, Neuman-Keuls analysis as de- scribed by Weiner (8).

RESULTS AND DISCUSSION

Endurance training had little influence on the total water content of muscle, heart, and liver (Table 1). Training increased the extracellular water in muscle but not in heart or liver (Table 1). This adaptation may be related to the increased capillary blood supply found after training (9).

The data in Table 1 also show that training decreased the uptake of AIB into heart but not into muscle or liver. Oxender et al. (10) observed a correlation between the rate of transport of specific amino acids and cell growth. When cell growth was arrested with inhibitors of synthesis, the activity of transport system A, which has a high afflnity for alanine,

198 BEECHER. PUENTE. AND DOHM

TABLE I EFFECT OF ENDURANCE TRAINING ON TOTAL WATER. EXTRACELLULAR WATER

AND a-AMINOISOBUTYRATE (AIB) UPTAKE BY MIJS~LE, HEART, AND LIVER

Tissue water Extracellular water (ml/g tissue) (ml/g tissue)

AIB uptake (dpm x IO-Yg/hr)

Muscle (gastrocnemius) Untrained Trained

Heart Untrained Trained

Liver Untrained Trained

0.761 2 .003(9)” 0.0% -e .005(8)b 11.8 c 1.2(8) 0.758 -t .003(9) 0.116 t .006(7)b 13.3 t 0.7(9)

0.734 t .002(9) 0.187 -t .007(9) 17.5 2 0.7(9)b 0.729 t .005(9) 0.184 2 .008(7) 13.8 + 0.8(7)”

0.704 + .005(9) 0.175 It .007(9) 45.1 + 3.9(9) 0.710 2 .00.5(9) 0.181 it .013(6) 45.8 2 2.6(9)

LI Values in the table are means k SE with the number of observations in parenthesis. b Trained and untrained are significantly different (P < 0.05).

decreased and the activity of system L, which has a high affinity for leucine, increased. Concurrent changes in muscle cell growth, protein synthesis, and amino acid (AIB) uptake in an in viva system were demon- strated by Goldberg and Goodman (5). The present results, in conjunction with those previously published (2), suggest that AIB uptake and protein synthesis in heart are decreased in concert by endurance training. How- ever, it appears that there is not an obligatory link between the two processes in liver and muscle, for training lowered incorporation of [14Cl leucine into protein of liver and stromal fraction of muscle but did not change AIB uptake in either tissue.

Amino acid levels in muscle, heart, and liver are shown in Table 2 and levels in plasma and red blood cells are shown in Table 3. Comparison of results from trained and untrained rats shows that the levels of 10 of the amino acids were affected by the endurance training regimen employed in these studies. In muscle, the level of camosine was depressed by training; anserine likewise tended to be somewhat lower in trained rats (17.8 + 1.4, P > 0.05). This seems to be consistent with the report by Christman (11) who found that an acute bout of exercise lowered carnosine in muscle of guinea pigs. Although the physiological functions of carnosine and an- serine are not known with certainty, evidence suggests that they may be involved in the regulation of myosin ATPase activity and muscle contrac- tion (12). Either or both of these physiological functions are important during muscular activity and thus the alteration in the camosine level may have significance in the adaptation of the muscle to endurance training.

Muscle levels of phenylalanine were depressed and levels of serine were increased as a result of exercise training. In heart, training increased

ENDURANCE TRAINING AND AMINO ACIDS 199

TABLE 2 EFFECT OF ENDURANCE TRAINING ON LEVELS OF FREE AMINO ACIDS

IN SKELETAL MUSCLE, HEART, AND LIVER

Amino acid content= &mole/g dry tissue)

Muscle Heart Liver

Alanine 21.0 r 0.9 17.1 2 1.9 &Alanine 1.03 c 0.17 0.46 k 0.07 a-Amino-n-butyric acid* 1.23 2 0.05 1.26 t 0.04 Anserine 22.9 ” 1.0 ND Asparagine 1.04 -t 0.05 2.68 + 0.09 Aspartic acid 1.45 2 0.10 8.08 2 0.77 Arginine 0.90 2 0.07 1.29 + 0.04(+25%) Camosine 12.7 t 0.75(-2%) ND Glutamic acid 14.7 + 1.0 53.6 2 2.2 Glutamine 16.0 2 0.6 37.5 + 0.8 Glycine 13.2 k 0.8 3.08 2 0.11 Histidine 0.95 ‘- 0.02 0.88 2 0.03 Isoleucine 0.29 f 0.08 0.55 +- 0.08 Leucine 0.76 2 0.08 1.56 k 0.14 Lysine 2.41 + 0.11 2.57 + 0.08(+20%) Methionine 0.32 + 0.03 0.49 k 0.03 Omithine 0.27 k 0.05 0.28 t 0.05 Phenylalanine 0.37 + 0.03(-42%) 0.43 2 0.02 Proline 3.04 f 0.17 1.61 -t 0.12 Phosphoserine 2.41 f 0.24 2.79 + 0.31 Serine 3.92 ” 0.20(+21%) 3.16 ” 0.17 Taurine’ - - Threonine 3.48 2 0.17 2.38 f 0.12 Tyrosine 0.56 2 0.05 0.39 2 0.05 Urea 159 t 21 195 ” 33 Valine 1.39 + 0.07 1.17 2 0.06

22.5 f 0.6 0.83 k 0.35

- ND

0.46 t 0.04(+84%) 4.42 + 0.23

ND ND

14.6 rf: 0.5 12.0 f 1.0 7.26 + 0.40 2.16 + 0.07 1.18 f 0.15 2.90 f. 0.38 2.62 -e 0.17 0.67 _+ 0.08 1.76 5 0.12 1.12 + 0.15 1.93 + 0.18 19.8 2 0.9 2.23 + 0.24 43.4 2 2.q-24%) 2.32 f 0.26 0.73 2 0.08 117 2 17

2.55 + 0.16

a The mean 2 SE for six samples of the untrained group is shown for each amino acid except when the concentration was too low to detect (ND). When the amino acid level in the trained group was significantly diierent (P < 0.05) from the untrained group, the direction and percentage change in the level of the amino acid for the trained group, compared to the untrained group, are shown in parenthesis.

* cY-Amino-n-butyric acid and citrulline coeluted from the ion-exchange column under the conditions used for these analyses; data are reported as a-amino-n-butyric acid equivalents. In liver, a-amino-n-butyric acid was observed but could not be accurately measured.

r Taurine was observed in muscle and heart preparations but was not accurately quanti- tated.

only the levels of the basic amino acids arginine and lysine. Asparagine was increased in liver whereas the level of taurine was depressed.

The data in Table 3 show that the concentrations of all amino acids, except glutamine, were greater in red blood cells than in plasma. These observations are consistent with previous reports of higher levels of

200 BEECHER, PUENTE. AND DOHM

TABLE 3 EFFECT OF ENDURANCE TRAINING ON THE LEVELS OF FREE AMINO ACIDS

IN PLASMA AND RED BLOOD CELLS

Amino acid content”

Plasma Red blood cells (fimole/ml) (@mole/g cell pack)

Alanine Asparagine Aspartic acid Arginine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Omithine Phenylalanine Proline Phosphoserine Serine Tatuine Threonine Tyrosine Urea Valine

0.495 + 0.025 0.038 t 0.007 0.022 2 0.032 0.069 k 0.012 0.190 2 0.012(-25%) 0.534 2 0.027(-l%) 0.206 t 0.014 0.040 + 0.003 0.091 2 0.011 0.151 ? 0.008 0.228 IT 0.009 0.062 It 0.003(-17%) 0.094 2 0.009 0.063 -c 0.005 0.249 r 0.022 0.029 2 0.006 0.175 f 0.012 0.164 2 0.018(-28%) 0.232 k 0.011 0.056 + 0.006

9.33 + 0.15 0.222 2 0.015

1.053 2 0.074 0.163 ? 0.005 0.188 2 0.026 0.438 +- 0.021(+2%) 1.144 2 0.048 0.168 ) 0.006 1.170 2 0.065 0.117 r 0.013 0.191 -c 0.017 0.354 t 0.032 0.757 t 0.034 0.082 t 0.015 0.112 -+ 0.021 0.142 + 0.009 0.376 + 0.068 0.163 + 0.030 0.513 2 0.026 0.484 iz 0.098 0.468 2 0.034 0.144 r 0.016

15.9 t 1.8(+33%) 0.380 t 0.019

0 The mean ? SE for six samples of the untrained group are reported for each amino acid. When the amino acid level in the trained group was significantly different (P < 0.05) from the untrained group, the direction and percentage change in the level of the amino acid for the trained group, compared to the untrained group, are shown in parenthesis. a-Amino-n- butyric acid was observed in all preparations but was not accurately measured (see Table 2, footnote b). Similarly, glutathione was observed in red blood cell preparations but could not be accurately determined.

amino acids in red blood cells than plasma from dogs (13) and humans (14). Elwyn ef al. (15) have proposed that plasma carries free amino acids from peripheral tissues to the liver, while red blood cells carry free amino acids from the liver to peripheral tissues. Thus, the decreased levels of amino acids in the plasma of endurance-trained animals (Table 3) may indicate a greater usage of these amino acids for energy or synthesis of essential metabolites in peripheral tissues (muscle) even though the levels of these amino acids in muscle or heart are not altered by training (Table 2). On the other hand, the increased levels of arginine and urea in red blood cells of endurance-trained animals indicate an increased catabolism

ENDURANCE TRAINING AND AMINO ACIDS 201

of amino acids in the livers of trained animals. In addition, arginine and urea may serve to transport “excess” nitrogen from the liver to the kidneys for subsequent excretion.

SUMMARY

The influence of endurance training on the rate of uptake and levels of free amino acids in plasma and various tissues was investigated. Training decreased the uptake of cu-aminoisobutyric acid into heart but not into muscle and liver. Total tissue water of muscle, heart, and liver was similar in trained and untrained animals. Training increased extracellular water in muscle but not in heart or liver. Endurance training decreased the concen- trations of carnosine and phenylalanine in muscle, taurine in liver, and glutamic acid, glutamine, methionine, and taurine in plasma. However, the levels of serine in muscle, arginine, and lysine in heart, asparagine in liver, and arginine and urea in red blood cells were increased in trained animals.

ACKNOWLEDGMENTS This research was supported in part by cooperative agreement No. 12-14-1001-429 from

the Protein Nutrition Laboratory, Nutrition Institute, SEA, USDA, Belt&he, Maryland, and U.S. Public Health Service Grant AM 19116.

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