Life Sciences, Vol . 23, pp .

845-850 Pargamon Press

Printed in the U .S .A .

CHANGES IN TISSUE PROTEIN LEVELS AS A RESULT OF ENDURANCE EXERCISE

G. Lynis Dohm, Fernando R. Puente,

Carlton P. Smith, and Ave Edge

Department of Biochemistry, School of Medicine

East Carolina University, Greenville, N. C. 27834

(Received is final form June 29, 1978)

Summary

We previously reported that endurance training increases amino

acid catabolism . In this study, the effects of an acute endurance
exercise bout on tissue protein levels and urea excretion have been
investigated . Exhaustive exercising of trained rats resulted in an
increase in anmonia excretion but there was no significant change in
urea excretion. Protein levels of muscle and liver were signifi-
cantly decreased by an exhaustive bout of exercise . In muscle, both
the soluble and myofibrillar protein fractions were decreased in
exhausted rats . These results demonstrate that during exercise
there is a net loss of protein in muscle and liver.

A previous study (1) demonstrated that training increases urea excretion

and leucine oxidation in rats . We suggested that the increase in protein
metabolism could have supplied an appreciable portion of the energy expended
during exercise but we had no evidence to suggest that protein catabolism was
actually increased during the exercise bout .

In a preliminary report, White and Brooks (2) reported that 14 C-alanine

and 1 4C-leucine were oxidized at more rapid rates in exercising rats than in
rested rats . In addition, alanine release from muscle is increased during
exercise (3) as is release of ammonia as a result of the purine nucleotide
cycle (4) . In the present study, we have investigated whether an acute bout
of endurance exercise results in altered protein levels and whether urea and
ammonia excretion are changed after exercise .

Methods

In the first experiment (see Tables I and III) male Holtzman rats weighing
approximately 200 g at the start of the study were housed in individual cages
and given water and commercial lab chow (Wayne Lab Blox, Allied Mills, Inc .,
Chicago, I11 .) ad libitum. The rats were divided into two groups : untrained,
which remained sédentary in their cages ; and trained, which were subjected to
daily treadmill running at 35 m/min, 8~ grade, 1 hr/day, 6 days/week for 6
weeks as previously described (5) . During the last week of training a 24 hour
urine collection was taken on 16 trained rats that had been rested 24 hours
before the urine collection was started. A 24 hour urine collection was also
taken for a second group of 7 trained rats that were exhaustively exercised
inmediately before the urine collection was taken. Urine was frozen for analy-
sis of ammonia and urea at a later time . Ammonia was determined by the method
of Bescher and Whitten (6) and urea was analyzed using urease and glutamic
dehydrogenase in coupled enzyme assay essentially as described by Talks and

0300-9653/78/0828-0845$02 .00/0
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846 Tissue Proteia Levels after Scercisa Vol . 23, No . 8, 1978

Shubert (7) . The reagents were purchased from Sigma Chemical Co ., St . Louis,
Mo ., as the single reagent BUN assay system .

During the seventh week of the experiment, untrained, trained rested, and
trained exhausted rats were sacrificed and the gastrocnemius muscles were ex-
cised and fractionated into soluble (sarcoplasmic), myofibrillar, and insoluble
(stromal) protein fractions as previously described (8) . Protein concentration
was assayed by the biuret method (9) using bovine serum albumin (Sigma Chemical
Co .) as a standard .

For the second experiment (see Table II), male rats weighing 300 g at the
start of the experiment were trained for only 3 weeks on regimen II, as previ-
ously described (5) and at the end of the third week were running 1 hour/day at
30 m/min . Half of the rats were sacrificed in the rested condition and the
remainder killed after an exhaustive bout of exercise (82+5 min at 30 m/min) .
Heart, liver, and gastrocnemius muscle were quickly excised and protein concen-
trations determined as described above .

Results

Concentrati ons of soluble and rt~yofi bri 11 ar protein were decreased by

training (untrained vs . trained rested, Table I) . In addition, an acute exhaus-
tive bout of exercise also caused a lowering of muscle protein concentration .

We previously reported (5) that training reduced body weight and this was
confirmed in this study (untrained, 384+8 g ; trained rested, 292+10 g ; trained
exhausted, 303+12 g) . We have previously reported (5) that training does not
alter the muscle weight to lean body mass ratio .

The effect of exhaustive exercise on protein levels was further investi-

gated in rats trained only three weeks and the results demonstrated that pro-
tein concentration was depressed in muscle and liver (Table II) . The decrease
in muscle protein was primarily in the soluble and rt~yofibrillar fractions.

The results shown in Table III demonstrate that ammonia excretion was
elevated in exhausted rats compared to rested rats but urea excretion was not
significantly changed as a result of exhaustive exercise . This experiment was
subsequently repeated with the same results .

Discussion

The effects of acute endurance exercise on muscle and liver are quite
striking since there is a loss of 10-15% of the protein in these two tissues in
a period of only about 80 min . We had speculated that there might be a small
loss from muscle to provide amino acids for transamination of pyruvate, but the
loss of protein from liver was unexpected and we can offer no explanation as to
the advantage such a loss of protein could have for the exercising rat .

There appear to be at least three possible mechanisms that may be involved

in the loss of muscle protein during exercise : a decrease in protein synthesis,
an increase 1n protein degradation, and "leakage" of protein from the muscle .
Zimmer and Gerlach (10) have reported that acute exercise depresses muscle pro-
tein synthesis and this effect could lead to a net loss of protein . Increased
degradation of muscle protein to amino acids during exercise was suggested by
the results of Haralambie and Berg (11) who danonstrated that plasma tyrosine
levels increase progressively with exercise in human subjects . Another possi-
ble cause of muscle protein loss could be the "leakage" of protein from the
muscle during exercise . There have been reports that several soluble en~yrties
(i .e ., LDH and CPK) are released from the muscle as a result of exercise (12)
Vol . 23, No . 8, 1978 Tissue Protein Leoels after Baercise 847

and this could cause muscle protein to decrease . This mechanism might account
for the loss of some soluble protein ; however, it seems unlikely that proteins
as large and structurally organized as the contractile proteins would leak from
the cell .

TABLE I

Effect of Training and Exhaustive Exercise on Water

and Protein Confient of Gastrocnemius Muscle
Trained Trained
Untrained Rested Exhausted

Muscle Water Content (~) 76 .02+,11(18) ° 76 .30±.12(9) 77 .24+,35(8)

Muscle Protein Fractions

(mg protein/g muscle)
c
Soluble Protein 68+1(18) 64±1(9) b 60±2(8)

Myofibrillar Protein 131±1(18) 122±2(9) b 115+4(8)

Insoluble Protein 34±1(18) 34±1(9) 32±1(8)

a Values are mean + SEM with the number of observations in parentheses .

b Trained rested are different (P<,05) than untrained .
c Trained exhausted are different (P<,05) than trained rested .

There are a number of hormones that have been shown to influence protein
degradation . For example, insulin has been shown to decrease muscle protein
degradation (13) and glucocorticolds seem to stimulate muscle proteolysis (14) .
Since exercise has been shown to decrease plasma insulin and increase cortisol
(15), these hormones may be instrumental in causing the loss of muscle protein
during exercise .

In an earlier study we had reported that exhaustively exercising trained

rats resulted in a decrease in mltochondrial yield (16) and decreases in the
activities of several enzymes of the citric acid cycle (17) . We concluded from
those results that mitochondria were lost during the exercise . In light of the
present results, it seems possible that such loss of mitochondria might be the
result of increased proroteolysic activity during exercise .

If the protein loss is due to increased proteolysis, the question arises as

to the fate of the amino acids that are 11be~~ted . In a preliminary report,
White and Brooks (2) reported that injected C-alanine and 4C-leucine were
oxidized to 14C02 more rapidly in exercising rats than in rested rats . Thus,
the amino acids may be oxidized and serve as an energy source . An increase in
the oxidation of amino acids should be reflected in an increased urinary excre-
tion of ammonia and/or urea . We did observe an increase in ammonia excretion
but found no significant change in urea excretion . This finding may indicate
that urea synthesis from ammonia is depressed by exercise . Other investigators
have observed an increase in blood ammonia during exercise (18,19), but it is
not known with certainty whether the increase in blood ammonia is caused by
increased deamination of amino acids or by increased activity of the purine
nucleotide cycle (4) .
848 Tissue Protein Levels after Fzerciae Vol . 23, No . 8, 1978

TABLE II

Effect of Exhaustive Exercise on Organ Weight to

Body Weight Ratio and Tissue Protein Levels

Parameter Trained Rested Trained Exhausted

Body Weight (g) 406±4(16) 396±6(16)

Organ Weight/Body Weight (g/Kg)

fiastrocnemius 6.61+ .08(15) 6.76+ .12(16)

Heart 3.28+ .07(16) 3.20±.06(16)

Liver 34 .4+.6(14) 30 .4+ .4(16) b

Tissue Protein
(g/organ/Kg body weight)

6astrocnemius 1 .40+ .06(15) 1 .18+ .04(16) b

Heart 1 .16+ .03(15) 1 .11+ .03(13)

Liver 5.35+ .20(14) 4.68±.11(16) b

Muscle Protein Fractions

(g/organ/Kg body weight)
b
Soluble Protein 0.410+.015(15) 0 .349±.008(16)

Myofibrillar Protein 0.749±.021(15) 0.691+.017(15)b

Insoluble Protein 0.132+.005(14) 0 .126+.004(15)

a Values are mean + SEM with the number of observations in parentheses .

b
Significant difference (P<.05) between rested and exhausted .

TABLE III

Effect of Exercise on Urea and Ammonia

Excretion in Trained Rats

Trained Rested Trained Exhausted

Urea Excreti on
(g N/24 hr/Kg body weight) 0.84+ .05(16)a 0 .94±.11(7)

Ammonia Excretion
(mg N/24 hr/Kg body weight) 8.2±0 .7(16) 21 :6+2 .9(7)b

a
Values are mean + SEM with the number of observations in parentheses .
b
Significant différence (P<.05) between exhausted and rested .
Vol . 23, No . 8, 1978 Tieeue Protaia Levels attar Szerciee 849

It is now well established that alanine release from the muscle is

increased during exercise and the alanine is then taken up b the liver as
part of the "glucose-alanine cycle" (3) . Odessey et al . (20~ found that a1a
nine release was increased when leucine was provided in the medium bathing the
muscle, and suggested that branched-chain amino acids contribute a large por-
tion of thé amino groups for traRSamination of pyruvate . From the results of
the present study 1t appears that protein may be broken down at an increased
rate during exercise, and the amino acids released could be available from
transamination of pyruvate .

Acknowledgements

This research was supported by Grant 1 RO1 AM 19116-02 from the U .S . Public
Health Service .

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