Research in Veterinary Science 1992, 53, 331-337
Does a single bout of exercise cause adaptation of amino acid metabolism in pigs? A. R. POSI3*, M. JENSEN-WAERN, Department of Medicine and Surgery, Swedish University of Agricultural Sciences, Box 7018, S-750 07 Uppsala, Sweden
Amino acid responses to exercise stress in welltrained racehorses and human athletes are well characterised, but the knowledge of amino acid metabolism during and after exercise in inactive animal species is limited. To study this, plasma amino acid concentrations were measured in previously unexereised pigs which performed two exercise tests on a treadmill with an interval of one week. In general, the changes in amino acids were more pronounced after the second than after the first exercise bout. Alanine, glutamine, phenylalanine and tyrosine were elevated for one hour only after the latter exercise. Twenty-four hours after the second exercise isoleucine, leucine, phenylalanine, tyrosine and valine were increased, but only isoleucine was increased after the first test. These differences between the two tests might be explained by adaptation of the amino acid metabolism after a single exercise bout and suggest that domestic pigs are well suited to study the early effects of exercise.
IN the animal kingdom physical activity appears to be a major determinant of muscle fibre composition. Muscles of trained athletes, racehorses and wild animals, like reindeer, are highly oxidative and well capillarised, whereas skeletal muscles of untrained domestic animals, like pigs, are not (Ess6n-Gustavsson 1986). Porcine muscle contains a high proportion of glycolytic, type IIB fibres. These fibres have a large transverse area, are poorly capillarised and have a low oxidative capacity. Furthermore, domestic pigs, which live a rather inactive life, are highly selected for rapid growth and a large muscle mass. Metabolic patterns differ among fibre types. Oxidative fibres, type I and type IIA, usually rely on aerobic metabolism of carbohydrates and fats in their energy production, whereas type IIB *Present address: Department of Biochemistry, College of Veterinary Medicine, Box 6, SF-00581 Fielsinki, Finland
fibres are dependent on anaerobic glycolysis. Owing to the fibre composition and husbandry of domestic pigs high lactate production is therefore to be expected during physical activity (Essrn-Gustavsson and Lindholm 1983). During and after both short- and long-term exercise the metabolism of protein and amino acids in skeletal muscle has been reported to be enhanced in many species, including man (Dohm 1986, P6s6 et al 1991), but whether all fibre types respond similarly is not known. During exercise, amino acids do not constitute a major fuel for energy production, but it has been suggested that through their participation in several metabolic pathways, such as gluconeogenesis in liver and kidney, they play an essential role in physical performance. The aim of this study was twofold: first, to study whether the exercise-induced changes in plasma amino acid concentrations are similar in inactive domestic pigs with a high proportion of glycolytic fibres to those previously found in other, more active and more oxidative species, such as trained athletes and racehorses; secondly, to study the effect of the exercise intensity on changes in plasma amino acid concentrations. The concentrations ofcortisol and lactate were measured as indicators of physical stress. To evaluate further the intensity of exercise, the concentrations of adenosine triphosphate breakdown products, that is hypoxanthine, xanthine and uric acid, were assayed (Hellsten Westing et al 1989). The concentrations of amino acids and of the above-mentioned variables were followed for 72 hours after exercise. Materials and methods
Animals Two litters of weaned pigs (Swedish Landrace cross Yorkshire cross Hampshire) were split into
331
332
A. R. P6s6, M. Jensen- Waern
two groups containing eight pigs each. On arrival at the department of medicine and surgery the pigs were three months old and their mean weight was 30 kg. One group was to undergo exercise on a treadmill, while the other was to be left unexercised and served as a control group. The exercising group was habituated to the treadmill exercise before the first test which was performed one month after arrival. The animals were housed in single pens at the department and they were fed ad libitum (Slaktfor 290). On the days of the experiment, the pigs were fed after blood sampling at -60 minutes. To facilitate frequent blood sampling, a catheter was inserted in a jugular vein by the method described by Rodriquez and Kunavongkrit (1983) and left in situ. Briefly, the surgical procedure was carried out under general anaesthesia (azaperone combined with metomidate) and under strictly sterile conditions. The catheter was drawn subcutaneously from the point of insertion in the jugular vein to the cervical region, where it was attached to a cannula (Venflon; Viggo, Sweden) and then stitched to the skin. The cannula was protected by a canvas cover, sutured on to the skin. In order to prevent clotting and infections, the catheter was flushed twice daily with heparinised saline (25 iu ml-1) and benzylpenicillin.
Experimental design Each pig in the exercise group performed two exercise tests on a large-animal treadmill (Sgtotreadmill, Uppsala). In the first test (test A) the pigs ran a distance of 930 m at a speed of 1.5 m sec-1 for three minutes and 2.0 m sec-I for 5.5 minutes. The intensity of this test was considered to be maximal for untrained pigs. In the second test (test B), which was carried out one week later, the pigs ran a distance of 450 m at a speed of 1.5 m sec-1 for five minutes. The intensity of this test was considered to be submaximal. The experiments started at 09.00. During and after the tests the state of fatigue of the animals was visually appraised. Blood samples were drawn into 10 ml heparinised vacutainer tubes 60 minutes before the test when the pigs were still in their pens. After both tests samples were taken at 0, 10, 30 and 60 minutes and at seven, 24 and 72 hours. Plasma samples were prepared instantly. The samples
for lactate analysis were kept on ice and analysed immediately. The rest of the plasma was stored at -70°C until analysed. The experimental design was approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden.
Analytical The plasma concentrations of cortisol were determined by radioimmunoassay according to the method validated for the pig by Nyberg et al (1988). The concentration of lactate in plasma was measured enzymatically with a lactate analyser (Analox GM7; Analox Instruments). The concentrations of hypoxanthine, xanthine and uric acid in plasma were assayed by high performance liquid chromatography (HPLC).The samples were deproteinised by centrifugation for 10 minutes in tubes containing a filter with a cutoff limit of 10 kDa (Ultrafree, Millipore). Hypoxanthine, xanthine and uric acid were separated under isocratic conditions (flow 0.5 ml min 1) with 60 mM ammonium phosphate buffer, pH 3.5, and l per cent methanol in a reversed phase, C-18, column (4 x 125 ram, Merck) and detected at 256 nm. For amino acid analysis a plasma sample was first deproteinised with methanol (Griffin et al 1982) and the free amino acids were derivatised with dansyl chloride and assayed by HPLC as described in detail by Ty6pp6nen (1987). The reversed phase column (C-18, 4 x 125 ram) used was obtained from Merck. The concentrations of amino acids are given as gmol litre-1 plasma.
Statistical analysis The Wilcoxon signed-rank test was used to compare pre- and post-exercise values ofcortisol, lactate, purines and amino acids. The significance of day-to-day variations was calculated by analysis of variance. Differences were regarded as significant at the 0.05 probability level. The results are expressed as mean _+ standard error of the mean, unless otherwise indicated. Results
The animals remained healthy throughout the experimental period. However, two pigs (one from the exercise group and one control) were
Plasma amino acids in exercising pigs
333
excluded because of problems with the catheter in situ. The pigs seemed to enjoy running on the treadmill, though variations between individual animals in the signs of fatigue were observed afterwards.
TABLE 1: Diurnal changes in the concentrations of plasma amino acids in the control group
Exercise-induced changes in plasma concentrations of cortisol, lactate and purines
Glutamine
Except for the diurnal variation in the cortisol level, no day-to-day variation was observed in the concentrations of cortisol, lactate, hypoxanthine, xanthine or uric acid in the control group. As shown in Fig 1, both the first and the second bout of exercise resulted in hormonal and metabolic changes as reflected in plasma values. The cortisol and lactate levels increased significantly up to 10 or 30 minutes after exercise. A similar tendency was found for the plasma concentration of hypoxanthine. The xanthine levels were below the detection limit and the concentration of uric acid remained unchanged throughout the experiment (results not shown).
Lysine Phenylalanine proline Tryptophan Tyrosine valine
Diurnal changes in the concentrations of amino acids Diurnal changes in amino acids were studied by comparing the concentrations in the samples taken from the control pigs at 09.00 and 17.00. The concentrations of glutamate, glutamine, lysine and tyrosine were significantly higher, and the trypt0phan concentration significantlylower, at 17.00 then at 09.00 (Table 1). The concentrations of all other amino acids remained Cortisol
Test A I I I I I I I Test B I I
150 60 oA
15 Lactate
, ,
rm3r-~
60 t Hypoxanthin[~ 20 -60
0
10
30
60
Minutes FIG 1: Plasma concentrations (mean + SE) of cortisol, lactate and hypoxanthine before and after test A and test B. Significant difference from the corresponding resting value is indicated by *
Amino acid Alanine Asparagine Glutamate Glycine Isoleucine Leucine
09.00 (gmol litre 1)
17.00 (gmol litre 1)
687 _+41 64_+6 214_+15 456 _+23 1355 + 79 240 -+ 12 256 _+15 59+6 117_+10 1006 _+143 71 _+7 64 _+6 571 _+26
553 + 50 69+6 333_+24* 574 -+ 22 * 1337 _+125 208 _+15 228 -+ 16 128_+12 121 -+12 856 _+63 29_+4 * 183 _+15 * 510+_27
Statistically significant differences are indicated by *
unchanged. Similar observations were made in the exercise group (results not shown). The day-to-day variation was examined by comparing the concentrations of amino acids in the control pigs at 09.00 on days 1, 2 and 4 of the experiment. Significant variations were observed in the concentrations of glutamate, asparagine, proline and phenylalanine.
Exercise-induced changes in the concentrations of plasma amino acids After both tests the concentrations of arginine, asparagine, histidine, lysine and serine remained unchanged. After test A no changes were observed in the concentrations of alanine, leucine, phenylalanine, tyrosine and valine. The glutamate and proline concentrations were increased in the samples taken immediately after the exercise, but returned to the basal level within 30 minutes. A significantly elevated concentration of isoleucine was found 24 hours after the exercise (Table 2). The concentration of tryptophan was increased 30 minutes, 60 minutes and 24 hours after exercise. The concentrations of alanine and proline were increased immediately after test B, whereas increases in the concentrations of glutamine, phenylalanine and tyrosine were not observed until 30 minutes later (Fig 2). Glutamine and alanine had returned to the basal level 60 minutes after exercise, whereas the increases in tyrosine and phenylalanine were detectable for up to 24 hours. The concentrations of the branched-chain amino acids, that is isoleucine, leucine and valine,
334
A. R. POs6, M. Jensen-Waern TABLE 2: Changes in amino acid concentrations 24 and 72 hours after exercise. The standard error 24 and 72 hours after exercise ranged from 5.1 to 18.5 per cent
Amino acid
Test A 24h 72h Rest % of resting gmol litre-1 level
Test B 24h 72h Rest % of resting gmol litre-1 level
Alanine Asparagine Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Tryptophan Free trytophan Tyrosine Valine
773 ± 60 90 ± 80 244 ± 26 489 ± 24 1201 ± 69 587 ± 37 248 ± 16 266 ± 19 85 ± 12 136 ± 7 1270 ± 112 926 ± 83 70 ± 4 5±1 68±6 539 ± 27
791 ± 54 91 ± 4 272 ± 14 550 ± 35 1746 ± 150 714 ± 22 267 ± 17 278 ± 10 60 ± 8 116 ± 7 635 ± 28 999 ± 28 75 ± 5 4± 1 61 ± 4 592 ± 22
114 129" 90 110 110 115 120" 110 91 115 120 117 104" 142 91 121
111 121 81 105 102 105 101 107 80 107 119 106 111 98 79 112
103 105 82 92 74* 76 116" 113" 123 130" 217" 99 107 90 115" 115"
114 t12 69 96 82 93 105 104 137 128 248* 104 111 126 113 119
*P
Does a single bout of exercise cause adaptation of amino acid metabolism in pigs? A. R. POSI3*, M. JENSEN-WAERN, Department of Medicine and Surgery, Swedish University of Agricultural Sciences, Box 7018, S-750 07 Uppsala, Sweden
Amino acid responses to exercise stress in welltrained racehorses and human athletes are well characterised, but the knowledge of amino acid metabolism during and after exercise in inactive animal species is limited. To study this, plasma amino acid concentrations were measured in previously unexereised pigs which performed two exercise tests on a treadmill with an interval of one week. In general, the changes in amino acids were more pronounced after the second than after the first exercise bout. Alanine, glutamine, phenylalanine and tyrosine were elevated for one hour only after the latter exercise. Twenty-four hours after the second exercise isoleucine, leucine, phenylalanine, tyrosine and valine were increased, but only isoleucine was increased after the first test. These differences between the two tests might be explained by adaptation of the amino acid metabolism after a single exercise bout and suggest that domestic pigs are well suited to study the early effects of exercise.
IN the animal kingdom physical activity appears to be a major determinant of muscle fibre composition. Muscles of trained athletes, racehorses and wild animals, like reindeer, are highly oxidative and well capillarised, whereas skeletal muscles of untrained domestic animals, like pigs, are not (Ess6n-Gustavsson 1986). Porcine muscle contains a high proportion of glycolytic, type IIB fibres. These fibres have a large transverse area, are poorly capillarised and have a low oxidative capacity. Furthermore, domestic pigs, which live a rather inactive life, are highly selected for rapid growth and a large muscle mass. Metabolic patterns differ among fibre types. Oxidative fibres, type I and type IIA, usually rely on aerobic metabolism of carbohydrates and fats in their energy production, whereas type IIB *Present address: Department of Biochemistry, College of Veterinary Medicine, Box 6, SF-00581 Fielsinki, Finland
fibres are dependent on anaerobic glycolysis. Owing to the fibre composition and husbandry of domestic pigs high lactate production is therefore to be expected during physical activity (Essrn-Gustavsson and Lindholm 1983). During and after both short- and long-term exercise the metabolism of protein and amino acids in skeletal muscle has been reported to be enhanced in many species, including man (Dohm 1986, P6s6 et al 1991), but whether all fibre types respond similarly is not known. During exercise, amino acids do not constitute a major fuel for energy production, but it has been suggested that through their participation in several metabolic pathways, such as gluconeogenesis in liver and kidney, they play an essential role in physical performance. The aim of this study was twofold: first, to study whether the exercise-induced changes in plasma amino acid concentrations are similar in inactive domestic pigs with a high proportion of glycolytic fibres to those previously found in other, more active and more oxidative species, such as trained athletes and racehorses; secondly, to study the effect of the exercise intensity on changes in plasma amino acid concentrations. The concentrations ofcortisol and lactate were measured as indicators of physical stress. To evaluate further the intensity of exercise, the concentrations of adenosine triphosphate breakdown products, that is hypoxanthine, xanthine and uric acid, were assayed (Hellsten Westing et al 1989). The concentrations of amino acids and of the above-mentioned variables were followed for 72 hours after exercise. Materials and methods
Animals Two litters of weaned pigs (Swedish Landrace cross Yorkshire cross Hampshire) were split into
331
332
A. R. P6s6, M. Jensen- Waern
two groups containing eight pigs each. On arrival at the department of medicine and surgery the pigs were three months old and their mean weight was 30 kg. One group was to undergo exercise on a treadmill, while the other was to be left unexercised and served as a control group. The exercising group was habituated to the treadmill exercise before the first test which was performed one month after arrival. The animals were housed in single pens at the department and they were fed ad libitum (Slaktfor 290). On the days of the experiment, the pigs were fed after blood sampling at -60 minutes. To facilitate frequent blood sampling, a catheter was inserted in a jugular vein by the method described by Rodriquez and Kunavongkrit (1983) and left in situ. Briefly, the surgical procedure was carried out under general anaesthesia (azaperone combined with metomidate) and under strictly sterile conditions. The catheter was drawn subcutaneously from the point of insertion in the jugular vein to the cervical region, where it was attached to a cannula (Venflon; Viggo, Sweden) and then stitched to the skin. The cannula was protected by a canvas cover, sutured on to the skin. In order to prevent clotting and infections, the catheter was flushed twice daily with heparinised saline (25 iu ml-1) and benzylpenicillin.
Experimental design Each pig in the exercise group performed two exercise tests on a large-animal treadmill (Sgtotreadmill, Uppsala). In the first test (test A) the pigs ran a distance of 930 m at a speed of 1.5 m sec-1 for three minutes and 2.0 m sec-I for 5.5 minutes. The intensity of this test was considered to be maximal for untrained pigs. In the second test (test B), which was carried out one week later, the pigs ran a distance of 450 m at a speed of 1.5 m sec-1 for five minutes. The intensity of this test was considered to be submaximal. The experiments started at 09.00. During and after the tests the state of fatigue of the animals was visually appraised. Blood samples were drawn into 10 ml heparinised vacutainer tubes 60 minutes before the test when the pigs were still in their pens. After both tests samples were taken at 0, 10, 30 and 60 minutes and at seven, 24 and 72 hours. Plasma samples were prepared instantly. The samples
for lactate analysis were kept on ice and analysed immediately. The rest of the plasma was stored at -70°C until analysed. The experimental design was approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden.
Analytical The plasma concentrations of cortisol were determined by radioimmunoassay according to the method validated for the pig by Nyberg et al (1988). The concentration of lactate in plasma was measured enzymatically with a lactate analyser (Analox GM7; Analox Instruments). The concentrations of hypoxanthine, xanthine and uric acid in plasma were assayed by high performance liquid chromatography (HPLC).The samples were deproteinised by centrifugation for 10 minutes in tubes containing a filter with a cutoff limit of 10 kDa (Ultrafree, Millipore). Hypoxanthine, xanthine and uric acid were separated under isocratic conditions (flow 0.5 ml min 1) with 60 mM ammonium phosphate buffer, pH 3.5, and l per cent methanol in a reversed phase, C-18, column (4 x 125 ram, Merck) and detected at 256 nm. For amino acid analysis a plasma sample was first deproteinised with methanol (Griffin et al 1982) and the free amino acids were derivatised with dansyl chloride and assayed by HPLC as described in detail by Ty6pp6nen (1987). The reversed phase column (C-18, 4 x 125 ram) used was obtained from Merck. The concentrations of amino acids are given as gmol litre-1 plasma.
Statistical analysis The Wilcoxon signed-rank test was used to compare pre- and post-exercise values ofcortisol, lactate, purines and amino acids. The significance of day-to-day variations was calculated by analysis of variance. Differences were regarded as significant at the 0.05 probability level. The results are expressed as mean _+ standard error of the mean, unless otherwise indicated. Results
The animals remained healthy throughout the experimental period. However, two pigs (one from the exercise group and one control) were
Plasma amino acids in exercising pigs
333
excluded because of problems with the catheter in situ. The pigs seemed to enjoy running on the treadmill, though variations between individual animals in the signs of fatigue were observed afterwards.
TABLE 1: Diurnal changes in the concentrations of plasma amino acids in the control group
Exercise-induced changes in plasma concentrations of cortisol, lactate and purines
Glutamine
Except for the diurnal variation in the cortisol level, no day-to-day variation was observed in the concentrations of cortisol, lactate, hypoxanthine, xanthine or uric acid in the control group. As shown in Fig 1, both the first and the second bout of exercise resulted in hormonal and metabolic changes as reflected in plasma values. The cortisol and lactate levels increased significantly up to 10 or 30 minutes after exercise. A similar tendency was found for the plasma concentration of hypoxanthine. The xanthine levels were below the detection limit and the concentration of uric acid remained unchanged throughout the experiment (results not shown).
Lysine Phenylalanine proline Tryptophan Tyrosine valine
Diurnal changes in the concentrations of amino acids Diurnal changes in amino acids were studied by comparing the concentrations in the samples taken from the control pigs at 09.00 and 17.00. The concentrations of glutamate, glutamine, lysine and tyrosine were significantly higher, and the trypt0phan concentration significantlylower, at 17.00 then at 09.00 (Table 1). The concentrations of all other amino acids remained Cortisol
Test A I I I I I I I Test B I I
150 60 oA
15 Lactate
, ,
rm3r-~
60 t Hypoxanthin[~ 20 -60
0
10
30
60
Minutes FIG 1: Plasma concentrations (mean + SE) of cortisol, lactate and hypoxanthine before and after test A and test B. Significant difference from the corresponding resting value is indicated by *
Amino acid Alanine Asparagine Glutamate Glycine Isoleucine Leucine
09.00 (gmol litre 1)
17.00 (gmol litre 1)
687 _+41 64_+6 214_+15 456 _+23 1355 + 79 240 -+ 12 256 _+15 59+6 117_+10 1006 _+143 71 _+7 64 _+6 571 _+26
553 + 50 69+6 333_+24* 574 -+ 22 * 1337 _+125 208 _+15 228 -+ 16 128_+12 121 -+12 856 _+63 29_+4 * 183 _+15 * 510+_27
Statistically significant differences are indicated by *
unchanged. Similar observations were made in the exercise group (results not shown). The day-to-day variation was examined by comparing the concentrations of amino acids in the control pigs at 09.00 on days 1, 2 and 4 of the experiment. Significant variations were observed in the concentrations of glutamate, asparagine, proline and phenylalanine.
Exercise-induced changes in the concentrations of plasma amino acids After both tests the concentrations of arginine, asparagine, histidine, lysine and serine remained unchanged. After test A no changes were observed in the concentrations of alanine, leucine, phenylalanine, tyrosine and valine. The glutamate and proline concentrations were increased in the samples taken immediately after the exercise, but returned to the basal level within 30 minutes. A significantly elevated concentration of isoleucine was found 24 hours after the exercise (Table 2). The concentration of tryptophan was increased 30 minutes, 60 minutes and 24 hours after exercise. The concentrations of alanine and proline were increased immediately after test B, whereas increases in the concentrations of glutamine, phenylalanine and tyrosine were not observed until 30 minutes later (Fig 2). Glutamine and alanine had returned to the basal level 60 minutes after exercise, whereas the increases in tyrosine and phenylalanine were detectable for up to 24 hours. The concentrations of the branched-chain amino acids, that is isoleucine, leucine and valine,
334
A. R. POs6, M. Jensen-Waern TABLE 2: Changes in amino acid concentrations 24 and 72 hours after exercise. The standard error 24 and 72 hours after exercise ranged from 5.1 to 18.5 per cent
Amino acid
Test A 24h 72h Rest % of resting gmol litre-1 level
Test B 24h 72h Rest % of resting gmol litre-1 level
Alanine Asparagine Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Tryptophan Free trytophan Tyrosine Valine
773 ± 60 90 ± 80 244 ± 26 489 ± 24 1201 ± 69 587 ± 37 248 ± 16 266 ± 19 85 ± 12 136 ± 7 1270 ± 112 926 ± 83 70 ± 4 5±1 68±6 539 ± 27
791 ± 54 91 ± 4 272 ± 14 550 ± 35 1746 ± 150 714 ± 22 267 ± 17 278 ± 10 60 ± 8 116 ± 7 635 ± 28 999 ± 28 75 ± 5 4± 1 61 ± 4 592 ± 22
114 129" 90 110 110 115 120" 110 91 115 120 117 104" 142 91 121
111 121 81 105 102 105 101 107 80 107 119 106 111 98 79 112
103 105 82 92 74* 76 116" 113" 123 130" 217" 99 107 90 115" 115"
114 t12 69 96 82 93 105 104 137 128 248* 104 111 126 113 119
*P
