- Email: [email protected]
Effects of administration of branched-chain amino acids vs. glucose during acute exercise in the rat
Physiology& Behavior,Vol. 55, No. 3, pp. 523-526, 1994 Copyright© 1994ElsevierSciencel.,td Printedin the USA.All rightsreserved 0031-9384/94$6.00 + .00
Pergamon
Effects of Administration of Branched-Chain Amino Acids vs. Glucose During Acute Exercise in the Rat PH. V E R G E R , .1 P. A Y M A R D , * L. CYNOBERT,I" G. A N T O N * A N D R. LUIGI*
*Laboratoire de Nutrition du Service Central d'Etudes et de R~alisation du Commissariat de l'Arm~e de T e r r e - - l , Bd Louis Loucheur 92211, St. Cloud, France ?~Laboratoire de Biochimie, Hopital St. Antoine, Paris, France Received 23 N o v e m b e r 1992 VERGER, PH., P. AYMARD, L. CYNOBERG, G. ANTON AND R. LUIGI. Effects of administration of branched-chain amino acids vs. glucose during acute exercisein the rat. PHYSIOL BEHAV 55(3) 523-526, 1994.--Several studies of the relationship of food intake to physical exercise strongly suggest that the two are closely linked. Serotonintransmissionseems to be particularly important in the regulation of both appetite and central fatigue. Thus, the ingestion of branched-chain amino acids has been reported to decrease serotoninproductionand to improve physical performance. In the present study, the effects of three treatments (water, glucose, and branched-chainamino acids) were compared in 34 male Wistar rats. Maximalexercise duration,blood insulin, and glucose levels were measured. Results showed that following the ingestion of branched-chainamino acids, physical performance is lower and blood insulin level is higher than after glucose administration. Exercise
Performance
Branched-chainamino acids
Glucose
PHYSICAL endurance can be substantialy extended by dietary manipulation of the body's carbohydrate stores (21). Fatigue occurs during sustained physical activity when blood glucose or muscle glycogen levels are lowered. However, fatigue can also emanate from changes within the central nervous system (1,3,4). One hypothesis explaining central fatigue is based on an increase in the concentration of 5-hydroxytryptamine in the brain during exercise (5,14,22). There is evidence that 5-HT is the neurotransmitter responsible for causing a state of tiredness and sleep in man and experimental animals through an increase in the rate of synthesis and, hence, an increase in the concentration of 5-hydroxytryptamine (5-HT) in one or more specific areas of the brain (2,5,8,18,22). Tryptophan is the precursor of 5-HT, which undergoes hydroxylation in 5-hydroxy tryptophan (5-HTP) by means of tryptophan hydrolase activity. 5-HTP is then converted to 5-HT by the aromatic amino acid decarboxylase. The existence of a saturable, neutral aminoacid carrier system has been clearly documented (11,15,17). Most neutral aminoacids compete with tryptophan for transport at the carrier site. BCAA compete with free tryptophan for entry to the brain (16,17,23), and this competition has been demonstrated to be of physiological importance in regulating tryptophan uptake in the brain and, thus, brain tryptophan concentration (11,12). Furthermore, the brain serotonin concentration and metabolism is sensitive to nutrient intake (13). For instance, carbohy-
Bloodglucose level
Seruminsulin level
drate ingestion or insulin administration has been shown to decrease the level of the neutral competing amino acids so that brain TRP (and, consequently, brain 5-HT synthesis) is increased (12). The increase in plasma concentration of BCAA during exercise, would be expected to decrease or to maintain at an almost constant level the ratio between free tryptophan and other large neutral amino acids. This, in turn, means that the rate of transport of tryptophan into the brain and, therefore, the rate of synthesis of 5-HT would remain approximately constant or decrease. If this neurotransmitter plays a part in causing physical fatigue, then the BCAA supplement would be expected to delay fatigue. A recent study demonstrated that in man, running performance (time taken to run a marathon) was improved when a supplement of BCAA was ingested during the race (7). This result may reflect the beneficial effect of BCAA on central fatigue; however, another possibility is that such an intake could have met protein need. There is probably an increased need for protein due to muscular fiber degradation during exercise. This hypothesis seems to be confirmed by protein intake after exercise. Various experiments [for review see (19)] have shown that rats submitted to physical exercise increase their protein intake. A recent study in man shows that, just after exercise, spontaneous food intake shows a preference for proteins (20). Could a judicious intake of amino acids prevent fatigue and provide material for muscular protein replacement?
To whom requests for reprints should be addressed. 523
524
VERGER ET AL.
g
lo
[]
,-,,,,•,,,,,,: ,,;,;,7,7,;,;.
,',,',-,,',',,v, -10
,;,;,;,;,;,;,
Body Weight
.,~,~,,,,,,,,,
Experimental Procedure
:~, BCAA'~,
Rats were adapted to the treadmill in the morning on the 2 days immediately preceding the experiment for 15 min per day. All rats were submitted to the same experimental schedule. Every day in a random order, six rats were exercised. They were run at the beginning of the dark period at 0900 h. During exercise, rats were denied access to their usual food. Just before the beginninng of the run and every 30 min during the run, two rats were tube-fed with solution 1 (water), two rats were tube-fed with solution 2 (glucose solution), and two rats were tube fed with solution 3 (BCAA solution) in a random order. Total volume for one administration was 1 ml and total caloric content was 0.2 kcal per rat. The end of the running session and, consequently, the point of exhaution, was defined as being when the rat had remained on the electrified grid for 1 min. Then a blood sample was taken and the rats were replaced in their home cages. All rats were submitted to three tests at intervals of 4 weeks. They received the three gastric loads (water, glucose, and BCAA) in random order. In each session, exercise duration to exhaustion was measured.
,,,,,,,,,,,,,.
,,~,:,~,,,,,,,
3
4
~1 • r s x , ' J " ,
-20'
-30
1
2
Plasma samples were assayed for glucose concentration with a YSI Glucose Analyser (YSI, 23A, Bioblock, France). The standard error with this method was ± 1%. Amino acid concentrations were determined using a Beckman 6300 amino acid analyser.
FIG. 1. Average weight gain during a rest day compared to average weights loss during exercise for the three treatments (n = 34). Vertical bars represent the SEM. The aim of the present study was to examine in rats the hypothesis that an increased intake of branched chain amino acids (valine, leucine, isoleucine) as compared to water and glucose, can improve exercise performance because of an increased need for protein or by influencing brain serotonin.
StatisticalAnalysis METHOD
Animals Thirty-four male adult Wistar rats weighing 379.6 ± 27.4 g at the beginning of the experiment and 432.4 + 25.7 g at the end, were used. Animals were placed in a quiet, temperature-controlled room (20 ± I°C) with a 12:12 h light:dark cycle (21000900 h light). Rats had continuous access to water and food except when otherwise indicated.
Exercise Exercise was performed during the first hours of darkness on a motor-driven treadmill at 5% gradient and at a moderate intensity (0.9 km/h or 16 m/min) to the point of exhaution. An electrified grid (50 volts; 60 re.A), located at the rear of each compartment, prevented the animals from stopping running.
Results are expressed as means ± standard errors. Data were compared using a one-way analysis of variance (n = 34 and three treatments) for repeated measures. When significant effects were found (p < 0.05), comparison of group means were made using post hoc analysis by Scheffe F-test. RESULTS
Body Weight (See Fig. 1) There were no significant differences in loss of body weight between the three tests. Mean body weight of the rats immediately before exercise was 410 ___ 25 g, 409 _+ 29, 409 ± 26 for water, glucose, and amino acid tests, respectively. Immediately after exercise, mean
Feeding
[]
EXERCISED U R A ~
Outside training periods, rats were fed ad lib with standard food (UAR 113). Water was also available ad lib. During the training periods, rats received, every 30 min, a gastric load of three different solutions• Intubation were effected by hand. The first solution was demineralised water. The second was a solution of glucose (5%). The last solution contained valine (1.6 g/100 ml), leucine (1.4 g/100 ml), isoleucine (0.9 g/100 ml). Total caloric density for solutions except water was 20 kcal/100 ml. •
P < 0,05
Blood Analysis At the end of each exercise session, blood was collected from the retro-orbital sinus and serum was separated out. Serum immunoreactive insulin was determined using an insulin radioimmunoassay kit (CIS Bio International, ref.: SB-INSI-5).
I
o
,Go
~Go
300 min
FIG. 2. Exercise duration for the three treatments (n = 34). Horizontal bars represent the SEM.
A M I N O A C I D S VS. G L U C O S E IN EXPERIENCE
525
TABLE 1 EFFECT OF DIFFERENT SOLUTIONS ON PLASMA AMINO ACID LEVELS Amino Acids
Glucose Mean +- SEM 0zmol/l)
Water Mean _+ SEM (#tool/l)
BCAA Mean _+ SEM ~mol/l)
253 ___ 9 222 __. 6 131 ± 15 341 ___22 500 ± 56 250 ± 26 109 __. 14 194±26 114±25 9 6 ± 11 429 _+ 39 64± 6
254 ___11 219 ___15 123 ± 12 325 ± 21 437 ± 64 279 _.+ 5 116 ± 1 209± 2 114± 8 96___ 6 453 __. 38 57± 7
239 ___ 15 223 ± 8 111 ± 5 286 ___ 8 646 ± 30 924 _ 124 283 ___ 36 4 5 7 ± 58 79± 7 79± 5 365 ± 4 58+ 4
THR SER PRO GLY ALA VAt, ILE LEU TYR PHE LYS HIS
Plasma amino acid levels measured for the three treatments after exercise session (n = 34). body weight was 389 ___ 23 g, 386 ___ 29, 390 ___ 24 for the same rats. Figure 1 compares the mean daily body weight increase with body weight loss after each test.
Physical Performance (Results are Plotted in Fig. 2) A N O V A results showed the treatment had a significant effect, F = 3.26, p < 0.05. Time courses for water, glucose, and amino acids were, respectively, 191 _+ 9 min, 208 _+ 11 min, 179 __ 10 min. The difference between glucose and amino acid treatment was significant using Scheffe F-test (F = 3.23, p < 0,05).
Blood Analysis Amino acids. Results (means and SEM) are plotted in Table 1. They confirm that blood valine, leucine, and isoleucine levels were higher after B C A A administration than after both water and glucose administration. Blood glucose. Serum glucose concentration in rats after the three tests did not differ, respectively, 109 _ 9 mg/100 ml, 132 _+ 12 rag/100 ml, and 128 _+ 10 rag/100 ml for water, glucose, and amino acid administration (F = 1.41, p = 0.25) (see Fig. 3).
Serum insulin. At the end of the exercise, insulin levels were, respectively, 50 _+ 1.3 #U/ml, 47 _+ 1.8 #U/ml, 61 + 1.9 #U/ml for water, glucose, and amino acid administration. The A N O V A analysis showed significant differences between the three treatments (F = 3.77, p < 0.05). A Scheffe F-test confirmed that insulin level was significantly higher after B C A A administration than after glucose (F = 3.36, p < 0.05). The difference between insulin levels after water and amino acid administration just failed to reach significance (F = 2.16) (see Fig. 4). DISCUSSION
The question adressed by this experiment was: can dietary amino acid supplementation affect exercise performance? The results showed that maximal exercise duration is shorter when rats are intubated with a B C A A solution than with a glucose solution. This contradicts the previous study made by Bloomstrand (7). Nevertheless, the comparison between experimental ( B C A A administration) and reference groups (glucose administration) showed that in the Bloomstrand study (7), as in the present work, branched chain amino acid levels after effort were significantly higher in the group submitted to amino acid supplementation.
B-OODGLUCO~m ~ .
A
[]
A
BLO00 I~.~JUN LEVEL
ns A
• P < 0,05
I
0
~00
2~o
mg 1 100 ml
FIG. 3. Blood glucose levels for the three treatments (n = 34). Horizontal bars represent the SEM.
I
0
20
40
5'0
8'o
.U/ml
FIG. 4. Blood insulin levels for the three treatments (n = 34). Horizontal bars represent the SEM.
526
V E R G E R ET AL.
Moreover, the ratio experimental A A level/placebo A A level for each branched-chain amino acid was similar (2.6 vs. 2.4 for isoleucine level) or identical (respectively, 2.2 and 3.3 for leucine and valine levels). Thus, there are apparantly no differences between the two protocols. It is important to note that at the end of exercise in both groups, blood glucose levels were similar in spite of different duration of exercise, which suggests another possible explanation of these results: the difference in plasma insulin concentration between the B C A A group and both other groups. The higher level of insulin in the B C A A group could explain the earlier onset of exhaustion by a peripheral effect of insulin; an accelerated decrease in blood glucose level. This hypothesis is consistent with the well established role of branched chain amino acids on insulin production and on the inhibition of glycogenolysis.
On the other hand, measurement of performance here was very different from Bloomstrand's method (7): his study consisting of measuring speed during a marathon race (a time as short as possible) whereas in the present study, the total duration of a running session until rats reached exhaustion (a time as longer as possible), was measured. It is tentatively concluded that a secretion of insulin and an inhibition of glycogenolysis occurs in response to amino acid ingestion and a decrease in glucose production and plasma glucose level occurs in spite of an increase of gluconeogenesis induced by branched chain amino acids. ACKNOWLEDGEMENTS The authors thank J. Louis Sylvestre and C. Larue Achagiotis for their assistance during manuscript preparation and J. Blet and C. Coudray-Lucas for their technical participation.
REFERENCES 1. Asmussen, E. Muscle fatigue. Med. Sci. Sports 11:313-321; 1979. 2. Barchas, J. D.; Freedman, D. Brain amines: Response to physiological stress. Biochem. Pharmacol. 12:1232-1235; 1963. 3. Bigland-Ritchie, B. EMG and fatigue of human voluntary and stimulated contractions. Ciba Found. Symp. 82:130-156; 1981. 4. Bigland-Ritchie, B.; Furbush, F.; Woods, J. J. Fatigue of intermittent submaximal voluntary contractions: Central and peripheral factors. J. Appl. Physiol. 61:421-429; 1986. 5. Blomstrand, E.; Celsing, F.; Newsholme, E. A. Changes in plasma concentrations of aromatic and branched-chain amino acids during sustained exercise in man and their possible role in fatigue. Acta Physiol. Scand. 133:115-121; 1988. 6. Blomstrand, E.; Perrett, D.; Parry-Billings, M.; Newsholme, E. A. Effect of sustained exercise on plasma amino acids concentrations and on 5-hydroxytryptamine metabolism in six different brain regions in the rat. Acta Physiol. Scand. 136:473-481; 1989. 7. Blomstrand, E.; Hassmen, P.; Ekblom, B.; Newsholme, E. A. Administration of branched-chain amino acids during sustained exercise-effects on performance and on plasma concentration of some amino acids. Eur. J. Appl. Physiol. 63:83-88; 1991. 8. Chaouloff, F.; Elghozi, J. L.; Guezennec, Y.; Laude, D. Effects of conditioned running on plasma, liver and brain tryptophan and on brain 5-hydroxytryptamine metabolism of the rat. Br. J. Pharmacol. 86:33-41; 1985. 9. Conlay, L. A.; Wurtman, R. J.; Lopez, I.; Blusztajn, J. K.; Vacanti, C. A.; Logue, M.; During, M.; Caballero, B.; Maher, T. J.; Evoniuk, G. Effect of running the Boston marathon on plasma concentration of large neutral amino acids. J. Neural Transm. 76:65-71; 1989. 10. Curzon, G.; Friedel, J.; Knott, P. J. The effect of fatty acids on the binding of tryptophan to plasma protein. Nature 242:198-200; 1973. 11. Fernstrom, J. D.; Wurtman, R. J. Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178:414416; 1972.
12. Fernstrom, J. D.; Wurtman, R. J. Elevation of plasma tryptophan by insulin in rat. Metabolism 21:337-342; 1972. 13. Green, H.; Greenberg, S. M.; Erickson, R. W.; Sawyer, J. L.; Ellison, T. Effect of dietary phenylalanine and tryptophan upon rat brain amine levels. J. Pharmacol. Exp. Ther. 136:174-178; 1962. 14. Kandel, E. R.; Schwartz, J. H. Principles of neural science. New York: Elsevier/North Holland; 1982:482-484. 15. Oldendorf, W. H. Brain Uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. Am. J. Physiol. 221:1629-1639; 1971. 16. Pardridge, W. M.; Oldendorf, W. H. Kinetic analysis of blood-brain barrier transport of amino acids. Biochem. Biophys. Aeta 401:128136; 1975. 17. Pardridge, W. M. Kinetics of competitive inhibition of neutral amino acid transport across the blood-brain barrier. J. Neurochem. 28:103-108; 1977. 18. Romanowski, W.; Grabiec, S. The role of serotonin in the mechanism of central fatigue. Acta Physiol. Pol. 25:127-134; 1974. 19. Titchenal, C. A. Exercise and food intake, what is the relationship? Sports Med. 6:135-145; 1988. 20. Verger, P.; Lanteaume, M. T.; Louis-Sylvestre J. Human intake and choice of foods at intervals after exercise. Appetite 18:9399; 1992. 21. Williams C. The influence of starch and sugar intake on physical performance. In: Dobbing, J., ed. Dietary starches and sugars in man: A comparison. New York: Springer Verlag; 1989:193212. 22. Young, S. N. The clinical psycho pharmacology of tryptophan. In: Wurtman, R. J.; Wurtman, J. J., eds. Nutrition and the brain, vol. 7. New York: Raven Press: 1986:49-88. 23. Yuwiler, A.; Oldendoff, W. H.; Geller, E.; Braun, L. Effect of albumin binding and amino acid competition on tryptophan uptake into brain. J. Neurochem. 28:1015-1023; 1977.
Pergamon
Effects of Administration of Branched-Chain Amino Acids vs. Glucose During Acute Exercise in the Rat PH. V E R G E R , .1 P. A Y M A R D , * L. CYNOBERT,I" G. A N T O N * A N D R. LUIGI*
*Laboratoire de Nutrition du Service Central d'Etudes et de R~alisation du Commissariat de l'Arm~e de T e r r e - - l , Bd Louis Loucheur 92211, St. Cloud, France ?~Laboratoire de Biochimie, Hopital St. Antoine, Paris, France Received 23 N o v e m b e r 1992 VERGER, PH., P. AYMARD, L. CYNOBERG, G. ANTON AND R. LUIGI. Effects of administration of branched-chain amino acids vs. glucose during acute exercisein the rat. PHYSIOL BEHAV 55(3) 523-526, 1994.--Several studies of the relationship of food intake to physical exercise strongly suggest that the two are closely linked. Serotonintransmissionseems to be particularly important in the regulation of both appetite and central fatigue. Thus, the ingestion of branched-chain amino acids has been reported to decrease serotoninproductionand to improve physical performance. In the present study, the effects of three treatments (water, glucose, and branched-chainamino acids) were compared in 34 male Wistar rats. Maximalexercise duration,blood insulin, and glucose levels were measured. Results showed that following the ingestion of branched-chainamino acids, physical performance is lower and blood insulin level is higher than after glucose administration. Exercise
Performance
Branched-chainamino acids
Glucose
PHYSICAL endurance can be substantialy extended by dietary manipulation of the body's carbohydrate stores (21). Fatigue occurs during sustained physical activity when blood glucose or muscle glycogen levels are lowered. However, fatigue can also emanate from changes within the central nervous system (1,3,4). One hypothesis explaining central fatigue is based on an increase in the concentration of 5-hydroxytryptamine in the brain during exercise (5,14,22). There is evidence that 5-HT is the neurotransmitter responsible for causing a state of tiredness and sleep in man and experimental animals through an increase in the rate of synthesis and, hence, an increase in the concentration of 5-hydroxytryptamine (5-HT) in one or more specific areas of the brain (2,5,8,18,22). Tryptophan is the precursor of 5-HT, which undergoes hydroxylation in 5-hydroxy tryptophan (5-HTP) by means of tryptophan hydrolase activity. 5-HTP is then converted to 5-HT by the aromatic amino acid decarboxylase. The existence of a saturable, neutral aminoacid carrier system has been clearly documented (11,15,17). Most neutral aminoacids compete with tryptophan for transport at the carrier site. BCAA compete with free tryptophan for entry to the brain (16,17,23), and this competition has been demonstrated to be of physiological importance in regulating tryptophan uptake in the brain and, thus, brain tryptophan concentration (11,12). Furthermore, the brain serotonin concentration and metabolism is sensitive to nutrient intake (13). For instance, carbohy-
Bloodglucose level
Seruminsulin level
drate ingestion or insulin administration has been shown to decrease the level of the neutral competing amino acids so that brain TRP (and, consequently, brain 5-HT synthesis) is increased (12). The increase in plasma concentration of BCAA during exercise, would be expected to decrease or to maintain at an almost constant level the ratio between free tryptophan and other large neutral amino acids. This, in turn, means that the rate of transport of tryptophan into the brain and, therefore, the rate of synthesis of 5-HT would remain approximately constant or decrease. If this neurotransmitter plays a part in causing physical fatigue, then the BCAA supplement would be expected to delay fatigue. A recent study demonstrated that in man, running performance (time taken to run a marathon) was improved when a supplement of BCAA was ingested during the race (7). This result may reflect the beneficial effect of BCAA on central fatigue; however, another possibility is that such an intake could have met protein need. There is probably an increased need for protein due to muscular fiber degradation during exercise. This hypothesis seems to be confirmed by protein intake after exercise. Various experiments [for review see (19)] have shown that rats submitted to physical exercise increase their protein intake. A recent study in man shows that, just after exercise, spontaneous food intake shows a preference for proteins (20). Could a judicious intake of amino acids prevent fatigue and provide material for muscular protein replacement?
To whom requests for reprints should be addressed. 523
524
VERGER ET AL.
g
lo
[]
,-,,,,•,,,,,,: ,,;,;,7,7,;,;.
,',,',-,,',',,v, -10
,;,;,;,;,;,;,
Body Weight
.,~,~,,,,,,,,,
Experimental Procedure
:~, BCAA'~,
Rats were adapted to the treadmill in the morning on the 2 days immediately preceding the experiment for 15 min per day. All rats were submitted to the same experimental schedule. Every day in a random order, six rats were exercised. They were run at the beginning of the dark period at 0900 h. During exercise, rats were denied access to their usual food. Just before the beginninng of the run and every 30 min during the run, two rats were tube-fed with solution 1 (water), two rats were tube-fed with solution 2 (glucose solution), and two rats were tube fed with solution 3 (BCAA solution) in a random order. Total volume for one administration was 1 ml and total caloric content was 0.2 kcal per rat. The end of the running session and, consequently, the point of exhaution, was defined as being when the rat had remained on the electrified grid for 1 min. Then a blood sample was taken and the rats were replaced in their home cages. All rats were submitted to three tests at intervals of 4 weeks. They received the three gastric loads (water, glucose, and BCAA) in random order. In each session, exercise duration to exhaustion was measured.
,,,,,,,,,,,,,.
,,~,:,~,,,,,,,
3
4
~1 • r s x , ' J " ,
-20'
-30
1
2
Plasma samples were assayed for glucose concentration with a YSI Glucose Analyser (YSI, 23A, Bioblock, France). The standard error with this method was ± 1%. Amino acid concentrations were determined using a Beckman 6300 amino acid analyser.
FIG. 1. Average weight gain during a rest day compared to average weights loss during exercise for the three treatments (n = 34). Vertical bars represent the SEM. The aim of the present study was to examine in rats the hypothesis that an increased intake of branched chain amino acids (valine, leucine, isoleucine) as compared to water and glucose, can improve exercise performance because of an increased need for protein or by influencing brain serotonin.
StatisticalAnalysis METHOD
Animals Thirty-four male adult Wistar rats weighing 379.6 ± 27.4 g at the beginning of the experiment and 432.4 + 25.7 g at the end, were used. Animals were placed in a quiet, temperature-controlled room (20 ± I°C) with a 12:12 h light:dark cycle (21000900 h light). Rats had continuous access to water and food except when otherwise indicated.
Exercise Exercise was performed during the first hours of darkness on a motor-driven treadmill at 5% gradient and at a moderate intensity (0.9 km/h or 16 m/min) to the point of exhaution. An electrified grid (50 volts; 60 re.A), located at the rear of each compartment, prevented the animals from stopping running.
Results are expressed as means ± standard errors. Data were compared using a one-way analysis of variance (n = 34 and three treatments) for repeated measures. When significant effects were found (p < 0.05), comparison of group means were made using post hoc analysis by Scheffe F-test. RESULTS
Body Weight (See Fig. 1) There were no significant differences in loss of body weight between the three tests. Mean body weight of the rats immediately before exercise was 410 ___ 25 g, 409 _+ 29, 409 ± 26 for water, glucose, and amino acid tests, respectively. Immediately after exercise, mean
Feeding
[]
EXERCISED U R A ~
Outside training periods, rats were fed ad lib with standard food (UAR 113). Water was also available ad lib. During the training periods, rats received, every 30 min, a gastric load of three different solutions• Intubation were effected by hand. The first solution was demineralised water. The second was a solution of glucose (5%). The last solution contained valine (1.6 g/100 ml), leucine (1.4 g/100 ml), isoleucine (0.9 g/100 ml). Total caloric density for solutions except water was 20 kcal/100 ml. •
P < 0,05
Blood Analysis At the end of each exercise session, blood was collected from the retro-orbital sinus and serum was separated out. Serum immunoreactive insulin was determined using an insulin radioimmunoassay kit (CIS Bio International, ref.: SB-INSI-5).
I
o
,Go
~Go
300 min
FIG. 2. Exercise duration for the three treatments (n = 34). Horizontal bars represent the SEM.
A M I N O A C I D S VS. G L U C O S E IN EXPERIENCE
525
TABLE 1 EFFECT OF DIFFERENT SOLUTIONS ON PLASMA AMINO ACID LEVELS Amino Acids
Glucose Mean +- SEM 0zmol/l)
Water Mean _+ SEM (#tool/l)
BCAA Mean _+ SEM ~mol/l)
253 ___ 9 222 __. 6 131 ± 15 341 ___22 500 ± 56 250 ± 26 109 __. 14 194±26 114±25 9 6 ± 11 429 _+ 39 64± 6
254 ___11 219 ___15 123 ± 12 325 ± 21 437 ± 64 279 _.+ 5 116 ± 1 209± 2 114± 8 96___ 6 453 __. 38 57± 7
239 ___ 15 223 ± 8 111 ± 5 286 ___ 8 646 ± 30 924 _ 124 283 ___ 36 4 5 7 ± 58 79± 7 79± 5 365 ± 4 58+ 4
THR SER PRO GLY ALA VAt, ILE LEU TYR PHE LYS HIS
Plasma amino acid levels measured for the three treatments after exercise session (n = 34). body weight was 389 ___ 23 g, 386 ___ 29, 390 ___ 24 for the same rats. Figure 1 compares the mean daily body weight increase with body weight loss after each test.
Physical Performance (Results are Plotted in Fig. 2) A N O V A results showed the treatment had a significant effect, F = 3.26, p < 0.05. Time courses for water, glucose, and amino acids were, respectively, 191 _+ 9 min, 208 _+ 11 min, 179 __ 10 min. The difference between glucose and amino acid treatment was significant using Scheffe F-test (F = 3.23, p < 0,05).
Blood Analysis Amino acids. Results (means and SEM) are plotted in Table 1. They confirm that blood valine, leucine, and isoleucine levels were higher after B C A A administration than after both water and glucose administration. Blood glucose. Serum glucose concentration in rats after the three tests did not differ, respectively, 109 _ 9 mg/100 ml, 132 _+ 12 rag/100 ml, and 128 _+ 10 rag/100 ml for water, glucose, and amino acid administration (F = 1.41, p = 0.25) (see Fig. 3).
Serum insulin. At the end of the exercise, insulin levels were, respectively, 50 _+ 1.3 #U/ml, 47 _+ 1.8 #U/ml, 61 + 1.9 #U/ml for water, glucose, and amino acid administration. The A N O V A analysis showed significant differences between the three treatments (F = 3.77, p < 0.05). A Scheffe F-test confirmed that insulin level was significantly higher after B C A A administration than after glucose (F = 3.36, p < 0.05). The difference between insulin levels after water and amino acid administration just failed to reach significance (F = 2.16) (see Fig. 4). DISCUSSION
The question adressed by this experiment was: can dietary amino acid supplementation affect exercise performance? The results showed that maximal exercise duration is shorter when rats are intubated with a B C A A solution than with a glucose solution. This contradicts the previous study made by Bloomstrand (7). Nevertheless, the comparison between experimental ( B C A A administration) and reference groups (glucose administration) showed that in the Bloomstrand study (7), as in the present work, branched chain amino acid levels after effort were significantly higher in the group submitted to amino acid supplementation.
B-OODGLUCO~m ~ .
A
[]
A
BLO00 I~.~JUN LEVEL
ns A
• P < 0,05
I
0
~00
2~o
mg 1 100 ml
FIG. 3. Blood glucose levels for the three treatments (n = 34). Horizontal bars represent the SEM.
I
0
20
40
5'0
8'o
.U/ml
FIG. 4. Blood insulin levels for the three treatments (n = 34). Horizontal bars represent the SEM.
526
V E R G E R ET AL.
Moreover, the ratio experimental A A level/placebo A A level for each branched-chain amino acid was similar (2.6 vs. 2.4 for isoleucine level) or identical (respectively, 2.2 and 3.3 for leucine and valine levels). Thus, there are apparantly no differences between the two protocols. It is important to note that at the end of exercise in both groups, blood glucose levels were similar in spite of different duration of exercise, which suggests another possible explanation of these results: the difference in plasma insulin concentration between the B C A A group and both other groups. The higher level of insulin in the B C A A group could explain the earlier onset of exhaustion by a peripheral effect of insulin; an accelerated decrease in blood glucose level. This hypothesis is consistent with the well established role of branched chain amino acids on insulin production and on the inhibition of glycogenolysis.
On the other hand, measurement of performance here was very different from Bloomstrand's method (7): his study consisting of measuring speed during a marathon race (a time as short as possible) whereas in the present study, the total duration of a running session until rats reached exhaustion (a time as longer as possible), was measured. It is tentatively concluded that a secretion of insulin and an inhibition of glycogenolysis occurs in response to amino acid ingestion and a decrease in glucose production and plasma glucose level occurs in spite of an increase of gluconeogenesis induced by branched chain amino acids. ACKNOWLEDGEMENTS The authors thank J. Louis Sylvestre and C. Larue Achagiotis for their assistance during manuscript preparation and J. Blet and C. Coudray-Lucas for their technical participation.
REFERENCES 1. Asmussen, E. Muscle fatigue. Med. Sci. Sports 11:313-321; 1979. 2. Barchas, J. D.; Freedman, D. Brain amines: Response to physiological stress. Biochem. Pharmacol. 12:1232-1235; 1963. 3. Bigland-Ritchie, B. EMG and fatigue of human voluntary and stimulated contractions. Ciba Found. Symp. 82:130-156; 1981. 4. Bigland-Ritchie, B.; Furbush, F.; Woods, J. J. Fatigue of intermittent submaximal voluntary contractions: Central and peripheral factors. J. Appl. Physiol. 61:421-429; 1986. 5. Blomstrand, E.; Celsing, F.; Newsholme, E. A. Changes in plasma concentrations of aromatic and branched-chain amino acids during sustained exercise in man and their possible role in fatigue. Acta Physiol. Scand. 133:115-121; 1988. 6. Blomstrand, E.; Perrett, D.; Parry-Billings, M.; Newsholme, E. A. Effect of sustained exercise on plasma amino acids concentrations and on 5-hydroxytryptamine metabolism in six different brain regions in the rat. Acta Physiol. Scand. 136:473-481; 1989. 7. Blomstrand, E.; Hassmen, P.; Ekblom, B.; Newsholme, E. A. Administration of branched-chain amino acids during sustained exercise-effects on performance and on plasma concentration of some amino acids. Eur. J. Appl. Physiol. 63:83-88; 1991. 8. Chaouloff, F.; Elghozi, J. L.; Guezennec, Y.; Laude, D. Effects of conditioned running on plasma, liver and brain tryptophan and on brain 5-hydroxytryptamine metabolism of the rat. Br. J. Pharmacol. 86:33-41; 1985. 9. Conlay, L. A.; Wurtman, R. J.; Lopez, I.; Blusztajn, J. K.; Vacanti, C. A.; Logue, M.; During, M.; Caballero, B.; Maher, T. J.; Evoniuk, G. Effect of running the Boston marathon on plasma concentration of large neutral amino acids. J. Neural Transm. 76:65-71; 1989. 10. Curzon, G.; Friedel, J.; Knott, P. J. The effect of fatty acids on the binding of tryptophan to plasma protein. Nature 242:198-200; 1973. 11. Fernstrom, J. D.; Wurtman, R. J. Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178:414416; 1972.
12. Fernstrom, J. D.; Wurtman, R. J. Elevation of plasma tryptophan by insulin in rat. Metabolism 21:337-342; 1972. 13. Green, H.; Greenberg, S. M.; Erickson, R. W.; Sawyer, J. L.; Ellison, T. Effect of dietary phenylalanine and tryptophan upon rat brain amine levels. J. Pharmacol. Exp. Ther. 136:174-178; 1962. 14. Kandel, E. R.; Schwartz, J. H. Principles of neural science. New York: Elsevier/North Holland; 1982:482-484. 15. Oldendorf, W. H. Brain Uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. Am. J. Physiol. 221:1629-1639; 1971. 16. Pardridge, W. M.; Oldendorf, W. H. Kinetic analysis of blood-brain barrier transport of amino acids. Biochem. Biophys. Aeta 401:128136; 1975. 17. Pardridge, W. M. Kinetics of competitive inhibition of neutral amino acid transport across the blood-brain barrier. J. Neurochem. 28:103-108; 1977. 18. Romanowski, W.; Grabiec, S. The role of serotonin in the mechanism of central fatigue. Acta Physiol. Pol. 25:127-134; 1974. 19. Titchenal, C. A. Exercise and food intake, what is the relationship? Sports Med. 6:135-145; 1988. 20. Verger, P.; Lanteaume, M. T.; Louis-Sylvestre J. Human intake and choice of foods at intervals after exercise. Appetite 18:9399; 1992. 21. Williams C. The influence of starch and sugar intake on physical performance. In: Dobbing, J., ed. Dietary starches and sugars in man: A comparison. New York: Springer Verlag; 1989:193212. 22. Young, S. N. The clinical psycho pharmacology of tryptophan. In: Wurtman, R. J.; Wurtman, J. J., eds. Nutrition and the brain, vol. 7. New York: Raven Press: 1986:49-88. 23. Yuwiler, A.; Oldendoff, W. H.; Geller, E.; Braun, L. Effect of albumin binding and amino acid competition on tryptophan uptake into brain. J. Neurochem. 28:1015-1023; 1977.