Acta physiol. scand. 1975. 95. 391-399 From the Department of Physiology, University of Kuopio, Finland
Comparison of the Effects of Physical Exercise, Cold Acclimation and Repeated Injections of Isoprenaline on Rat Muscle Enzymes BY
M. N . E. HARRIand J. VALTOLA Received 5 May 1975
Abstract HARRI, M. N. E. and J. VALTOLA. Comparison of the effects of physical exercise, cold acclimation and repeated injections of isoprenaline on rat muscle enzymes. Acta physiol. scand. 1975. 95. 391-399. The metabolic effects on rat cardiac and skeletal muscle of a strenuous program of swimming, of cold acclimation and of isoprenaline treatment (0.3 mg/kg daily for 5 five-day weeks) were compared. Exercised and cold-exposed rats gained less body weight than did controls or isoprenaline-treated rats. In all treated groups the heart and the interscapular brown adipose tissue hypertrophied. The size of the adrenals increased only in isoprenaline-treated animals. Cold-acclimation and physical training increased and isoprenaline treatment reduced or did not affect the activities of succinate dehydrogenase, rnalate dehydrogenase and citrate synthase of cardiac muscle. In skeletal muscle all treatments resulted in increased activities of these enzymes. Of the anaerobic enzymes analysed, only the activity of hexokinase increased in response to the treatments used. This increase was the same in cardiac as in skeletal muscle, but it was significantly greater with isoprenaline-treatment than with training or with cold-acclimation. The activities of lactate dehydrogenase and phosphofructokinase did not differ significantly. All treatments improved cold resistance, but only swimming exercise and cold acclimation significantly increased tolerance to exercise. It is concluded that prolonged stimulation of adrenergic p-receptors by catecholamines is responsible for the metabolic changes observed. Key words: Gastrocnemius muscle, cardiac muscle, enzyme activity, physical exercise, cold acclimation, isoprenaline treatment, cold tolerance, exercise tolerance
It is well known that skeletal and cardiac muscle adapt to physical exercise by increasing the number and size of mitochondria and content of mitochondria1 enzymes (Holloszy 1973). On the other hand, attempts to demonstrate changes in the activities of enzymes involved in glycolysis have led to contradictory results (Holloszy et al. 1971, Gollnick and Hermansen 1973). The changes in muscle enzymes as a result of physical training are very similar to those achieved by cold-acclimation (Hannon 1963, Hamilton and Ferguson 1972). Furthermore, acclimation to cold increases the amount of time a rat can swim before exhaustion (Basbashova 1960, Dawson et al. 1970). A relationship between physical fitness and the ability to tolerate cold has been demonstrated in humans (Adams and Heberling 1958, Heberling and Adams 1961, Keatinge 1961, Lange Andersen 1966) and in rats (Strramme and Hammel 1967). On the other hand, repeated injections of isoprenaline results in improved 391
392
M . N. E. HARRl AND J. VALTOLA
cold tolerance of rats, but does not extend swimming time (LeBlanc, Vallieres and Vachon 1972). The present study was undertaken in order to compare the metabolic changes caused by sternuous physical training in the muscle with those caused by cold-acclimation and by repeated injections of isoprenaline.
Material and methods Animals
Four groups of male albino rats of Wistar/af/Han/Mal/(Han 67) strain weighing approximately 240 g at the beginning of the experiments were used in these studies. Th e first group consisted of control animals, and the second group of cold-acclimated rats (4°C for 5 weeks). The rats of the third group were exercised by swimming for 1/2 h daily in water at 30°C. The swimming program was performed five times per week for 5 weeks. A weight of 5 g was attached at the base of the tail, and the weight was gradually increased during the first week to the final weight of 10 g. The rats of the fourth group received daily subcutaneous injections of isoprenaline (0.3 mg/kg) prepared fresh each day in an olive oil suspension. The injections were given five times per week for 5 weeks. The rats were killed by decapitation and hearts, adrenals and interscapular brown adipose tissues (ISBAT) were removed and weighed. The weights were adjusted to a common body weight by analysis of covariance. Enzyme assuys
Samples of the gastrocnemius muscle and of the left ventricular muscle were immediately frozen in liquid nitrogen. The samples were stored frozen at - 80°C until assayed. The muscle samples were then homogenized in a Potter Elvehjem glass homogenizer in Tris-HC1 buffer (0.1 m, p H 7.6) to a 2 % homogenate, and centrifuged for 10 min at 1 OOOxg at 4°C to remove unbroken cells and particulate debris. The supernatants were used for determination of the enzyme activities. Measurements were made at 37°C with a Cary 118 spectrophotometer equipped with a recorder. Succinate dehydrogenase (SDH) activity was analysed according to Earl and Korner (1 96% malate dehydrogenase (MDH) according to Ochoa (1955) and citrate synthase (CS) activity according to Srere (1962). The activities of hexokinase (HK), phosphofructokinase (PFK) were determined according to Bass c r u l . (1969) and Bostrom rt ul. (1973), respectively. For the determination of lactate dehydrogenase (LDH) activity the standardized method of Biochemical Boehringer was used. All enzyme activities are expressed as pmoles substrate utilized per min per g wet tissue weight. The protein contents of homogenates were estimated by the phenol method (Lcwry et at. 1951). Cold tolerance and swimming rime tests
Cold tolerance and swimming time tests were made after the fourth week of treatment. The same animal was never used for both of the tests. Cold tolerance was measured at - 18°C. Four animals in a cage, each belonging to a different group, were placed at that temperature. Colonic temperatures were obtained with a thermocouple inserted to a depth of 4 cm. The swimming time was measured i n water at 30°C with a load of 5 4: of the body weight attached at the base of the tails. Animals in all four groups were used simultaneously in the test. The end point for the swimming time was calculated when the rats remained more than 15 s under the water (LeBlanc rt a6. 1972).
Results Body and organ weights
At the beginning of the experiments the body weights of the rats were identical in all four experimental groups. At the end of the 5-week experimental period the control and the isoprenaline-treated animals weighed significantly more than the cold-acclimated or the exercised animals. Thus the cold-acclimated and the exercised rats gained less weight than did rats of the other groups (Table I). However, the hearts of the control rats were lighter than those of the other groups, while the hearts of the isoprenaline-treated rats were the
393
RAT MUSCLE ENZYMES
TABLE I. Body weight and weight of hearts, interscapular brown adipose tissues (ISBAT) and adrenals in control, cold-acclimated, trained and isoprenaline (IS0)-treated rats. Weight
Control
Cold-acclimated
Trained
Body, g
305k 9.4l (9Y 822+ 13.3 383k 11.5 61.1 -t 2.4
276k9.9a (9) 956+ 21.9' 823 k 34.6' 69.7 1.7
2 7 5 t 10.9' (7) 911+ 15.1' 752+ 58.0' 6 8 . 6 t 1.9
Heart, mg ISBAT, mg Adrenals, mg
ISO-treated 302k9.3 (6) 1211k22.5' 513+ 16.7' 76.5+ 1.5*
Mean+S.E. Number of rats. Significant difference from the control: p
Comparison of the Effects of Physical Exercise, Cold Acclimation and Repeated Injections of Isoprenaline on Rat Muscle Enzymes BY
M. N . E. HARRIand J. VALTOLA Received 5 May 1975
Abstract HARRI, M. N. E. and J. VALTOLA. Comparison of the effects of physical exercise, cold acclimation and repeated injections of isoprenaline on rat muscle enzymes. Acta physiol. scand. 1975. 95. 391-399. The metabolic effects on rat cardiac and skeletal muscle of a strenuous program of swimming, of cold acclimation and of isoprenaline treatment (0.3 mg/kg daily for 5 five-day weeks) were compared. Exercised and cold-exposed rats gained less body weight than did controls or isoprenaline-treated rats. In all treated groups the heart and the interscapular brown adipose tissue hypertrophied. The size of the adrenals increased only in isoprenaline-treated animals. Cold-acclimation and physical training increased and isoprenaline treatment reduced or did not affect the activities of succinate dehydrogenase, rnalate dehydrogenase and citrate synthase of cardiac muscle. In skeletal muscle all treatments resulted in increased activities of these enzymes. Of the anaerobic enzymes analysed, only the activity of hexokinase increased in response to the treatments used. This increase was the same in cardiac as in skeletal muscle, but it was significantly greater with isoprenaline-treatment than with training or with cold-acclimation. The activities of lactate dehydrogenase and phosphofructokinase did not differ significantly. All treatments improved cold resistance, but only swimming exercise and cold acclimation significantly increased tolerance to exercise. It is concluded that prolonged stimulation of adrenergic p-receptors by catecholamines is responsible for the metabolic changes observed. Key words: Gastrocnemius muscle, cardiac muscle, enzyme activity, physical exercise, cold acclimation, isoprenaline treatment, cold tolerance, exercise tolerance
It is well known that skeletal and cardiac muscle adapt to physical exercise by increasing the number and size of mitochondria and content of mitochondria1 enzymes (Holloszy 1973). On the other hand, attempts to demonstrate changes in the activities of enzymes involved in glycolysis have led to contradictory results (Holloszy et al. 1971, Gollnick and Hermansen 1973). The changes in muscle enzymes as a result of physical training are very similar to those achieved by cold-acclimation (Hannon 1963, Hamilton and Ferguson 1972). Furthermore, acclimation to cold increases the amount of time a rat can swim before exhaustion (Basbashova 1960, Dawson et al. 1970). A relationship between physical fitness and the ability to tolerate cold has been demonstrated in humans (Adams and Heberling 1958, Heberling and Adams 1961, Keatinge 1961, Lange Andersen 1966) and in rats (Strramme and Hammel 1967). On the other hand, repeated injections of isoprenaline results in improved 391
392
M . N. E. HARRl AND J. VALTOLA
cold tolerance of rats, but does not extend swimming time (LeBlanc, Vallieres and Vachon 1972). The present study was undertaken in order to compare the metabolic changes caused by sternuous physical training in the muscle with those caused by cold-acclimation and by repeated injections of isoprenaline.
Material and methods Animals
Four groups of male albino rats of Wistar/af/Han/Mal/(Han 67) strain weighing approximately 240 g at the beginning of the experiments were used in these studies. Th e first group consisted of control animals, and the second group of cold-acclimated rats (4°C for 5 weeks). The rats of the third group were exercised by swimming for 1/2 h daily in water at 30°C. The swimming program was performed five times per week for 5 weeks. A weight of 5 g was attached at the base of the tail, and the weight was gradually increased during the first week to the final weight of 10 g. The rats of the fourth group received daily subcutaneous injections of isoprenaline (0.3 mg/kg) prepared fresh each day in an olive oil suspension. The injections were given five times per week for 5 weeks. The rats were killed by decapitation and hearts, adrenals and interscapular brown adipose tissues (ISBAT) were removed and weighed. The weights were adjusted to a common body weight by analysis of covariance. Enzyme assuys
Samples of the gastrocnemius muscle and of the left ventricular muscle were immediately frozen in liquid nitrogen. The samples were stored frozen at - 80°C until assayed. The muscle samples were then homogenized in a Potter Elvehjem glass homogenizer in Tris-HC1 buffer (0.1 m, p H 7.6) to a 2 % homogenate, and centrifuged for 10 min at 1 OOOxg at 4°C to remove unbroken cells and particulate debris. The supernatants were used for determination of the enzyme activities. Measurements were made at 37°C with a Cary 118 spectrophotometer equipped with a recorder. Succinate dehydrogenase (SDH) activity was analysed according to Earl and Korner (1 96% malate dehydrogenase (MDH) according to Ochoa (1955) and citrate synthase (CS) activity according to Srere (1962). The activities of hexokinase (HK), phosphofructokinase (PFK) were determined according to Bass c r u l . (1969) and Bostrom rt ul. (1973), respectively. For the determination of lactate dehydrogenase (LDH) activity the standardized method of Biochemical Boehringer was used. All enzyme activities are expressed as pmoles substrate utilized per min per g wet tissue weight. The protein contents of homogenates were estimated by the phenol method (Lcwry et at. 1951). Cold tolerance and swimming rime tests
Cold tolerance and swimming time tests were made after the fourth week of treatment. The same animal was never used for both of the tests. Cold tolerance was measured at - 18°C. Four animals in a cage, each belonging to a different group, were placed at that temperature. Colonic temperatures were obtained with a thermocouple inserted to a depth of 4 cm. The swimming time was measured i n water at 30°C with a load of 5 4: of the body weight attached at the base of the tails. Animals in all four groups were used simultaneously in the test. The end point for the swimming time was calculated when the rats remained more than 15 s under the water (LeBlanc rt a6. 1972).
Results Body and organ weights
At the beginning of the experiments the body weights of the rats were identical in all four experimental groups. At the end of the 5-week experimental period the control and the isoprenaline-treated animals weighed significantly more than the cold-acclimated or the exercised animals. Thus the cold-acclimated and the exercised rats gained less weight than did rats of the other groups (Table I). However, the hearts of the control rats were lighter than those of the other groups, while the hearts of the isoprenaline-treated rats were the
393
RAT MUSCLE ENZYMES
TABLE I. Body weight and weight of hearts, interscapular brown adipose tissues (ISBAT) and adrenals in control, cold-acclimated, trained and isoprenaline (IS0)-treated rats. Weight
Control
Cold-acclimated
Trained
Body, g
305k 9.4l (9Y 822+ 13.3 383k 11.5 61.1 -t 2.4
276k9.9a (9) 956+ 21.9' 823 k 34.6' 69.7 1.7
2 7 5 t 10.9' (7) 911+ 15.1' 752+ 58.0' 6 8 . 6 t 1.9
Heart, mg ISBAT, mg Adrenals, mg
ISO-treated 302k9.3 (6) 1211k22.5' 513+ 16.7' 76.5+ 1.5*
Mean+S.E. Number of rats. Significant difference from the control: p
