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Effects of training on skeletal muscle in protein-deprived rats
Journal of the Neurological Sciences, 1985, 69:1-8
1
Elsevier
Effects of Training on Skeletal Muscle in Protein-Deprived Rats Anders Oldfors and Patrick Sourander Division of Neuropathology, Department of PathologyL Sahlgren Hospital, S-413 45 Gdteborg(Sweden) (Received 26 November, 1984) (Revised, received 16 January, 1985) (Accepted 18 January, 1985)
SUMMARY
The effects of exercise on atrophy of muscle fibres and loss of mitochondria in the extensor digitorum longus (EDL) and soleus muscles were studied in protein deprived rats. They had smaller muscle fibres than aged-matched control rats, the difference being more evident in type 2 than type 1 fibres both in the fast EDL and slow soleus muscles. The loss of weight was more pronounced in the EDL muscle which is composed mainly of type 2 fibres than in the soleus muscle which is composed mainly of type 1 fibres. Protein deprived rats subjected to a programme of periodic running on a treadmill for 12 weeks showed less muscle atrophy than sedentary, protein deprived rats. This effect of exercise in diminishing the degree of atrophy was more pronounced in the type 2 than type 1 fibres. The protein deprived rats which had been sedentary showed a marked loss of subsarcolemmal mitochondria, which was not seen in protein deprived rats undergoing exercise.
Key words:
Exercise
- Histochemistry
- Protein deprivation - Rat - Skeletal
muscle
INTRODUCTION
The increased knowledge of the importance of skeletal muscle for protein and energy metabolism has promoted interest in how various conditions such as nutritional change influence the structure, function and biochemistry of skeletal muscle. This study was supported by grants from the Swedish Medical Research Council, Proj. Nos. 03488 and 07122. 0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
In a recent study, Oldfors et al. (1983) showed in young rats subjected to severe protein deprivation that atrophy of the fast extensor digitorum longus (EDL) muscle was due primarily to atrophy of type 2 fibres, while the type 1 fibres simply failed to grow. Previous studies showed that the slow soleus muscle which is composed mainly of type 1 fibres is less affected by protein-calorie malnutrition (PCM) than the fast EDL muscle which is composed mainly of type 2 fibres (Rowe 1968; Li and Goldberg 1976; Lammi-Keefe et al. 1981; Layman et al. 1981). It thus seems possible that the relative number of different fibre types in a muscle determines the degree of reduction in size of the whole muscle, since the various fibre types differ in their susceptibility to PCM. A possible explanation for this discrepancy between fast and slow muscles is that the tonic soleus muscle by its greater activity is protected from atrophy during PCM (Goldspink 1978). Protein deprived rats show a marked loss of subsarcolemmal mitochondria (Oldfors et al. 1983), which loss may explain the previous finding that the activity of the respiratory enzymes involved in aerobic metabolism of muscle was reduced in rats subjected to PCM (Wainio et al. 1959; Taskar and Tulpule 1964; Fuge et al. 1968; Hansen-Smith et al. 1977; Layman et al. 1981). However, Fuge et al. (1968) reported that exercise prevented the decrease in the level of activity of some respiratory enzymes of muscle in rats subjected to PCM. This study was undertaken to answer the following: Is the difference in the atrophy of slow and fast muscles in protein deprivation due to a difference in the vulnerability of slow and fast muscle fibres? Does activity of a muscle, during protein deprivation, protect it from atrophy? Does activity of the muscle protect it from loss of subsarcolemmal mitochondria? MATERIALS AND METHODS
Animals and protein deprivation Male Sprague-Dawley rats reared on a standard diet were at the age of 6 weeks divided into two groups. One continued to be fed on a well balanced diet containing 14~o protein by weight. The rats of this group are referred to as NP rats. The other group was fed on a diet containing a very small amount of protein, 1.5 ~o by weight but was supplemented by additional starch so that the diets of both groups were approximately iso-caloric. The group fed on the low protein diet are referred to as protein deprived or LP rats. Details of the composition of the respective diets are given in a previous article (Oldfors et al. 1983). The body weight and food consumption of the rats were assessed weekly. The rats were kept in plastic cages at an ambient temperature of 22 ° C and a relative humidity of 60 Yo. The rats had free access to food and drinking water. Exercise programme Each group included 2 lots each consisting of 5 rats. One lot from each group was trained to run at intervals on a motor driven treadmill consisting of a wide, endless belt running on metal rollers. Ten boxes 50 cm long by 10 cm wide placed on the belt provided a limited area in which each animal could run. Motivation to run was effected
by an electric shock grid at the rear of the boxes. The animals learned within a week to avoid the electric shock by keeping pace with the moving belt. The treadmill was set at a moderate speed of 18 m/min and inclined initially at 5 °. During the 12 weeks of training, the incline was gradually increased to 16 ° and remained so for the last 2 weeks. The animals ran once a day for 5 days per week. The daily exercise period was gradually increased from 15 min the first 2 weeks to 1 h during the last 2 weeks. One lot of each group remained sedentary.
Morphometry and histochemistry Aged 18 weeks, all the rats were anaesthetized by sodium pentobarbital, and the EDL and soleus muscles were removed. The muscles were weighed and the mid part of the belly of the muscles was frozen in a mixture of liquid propane and propylene (Primus Gasol M 11 Esso) chilled with liquid nitrogen. Transverse sections, 10#m thick, were cut on a cryostat mierotome and stained for myofibfiUar ATPase after preincubation at pH 4.6 according to the method recommended by Dubowitz and Brooke (1973) for morphometric analysis of type 1, 2A and 2B fibres. The central parts of the sections were photographed and the size of some 200 adjacent fibres was measured using a particle size analyzer (Zeiss TGZ3) by assessing the diameter of the inscribed circle of each fibre. The mean size of each fibre type of the EDL and soleus muscles of the different groups was compared statistically using the Wilcoxon-Mann-Whitney rank sum test. To study the distribution of mitochondria cryostat 10-pm thick sections were stained by the modified Gomori trichrome stain and for NADH-tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH). RESULTS
The body weight of the rats are illustrated in Fig. la. The NP rats gained weight from the age of 6-18 weeks while the LP rats lost weight. The sedentary NP rats gained more weight than the exercising NP rats while the weight of the sedentary and exercising LP rats did not differ significantly. The daily food consumption in relation to body weight is illustrated in Fig. lb. While the NP rats consumed more food than the LP rats during the In'st 4 weeks of the study the relative food consumption of the groups did not differ significantly from 10 weeks of age. Tablel shows the weights of the EDL and soleus muscles. While the EDL muscle of NP rats aged 18 weeks had a greater weight than the soleus muscle, there was no significant difference between the weights of the EDL and soleus muscles of the LP rats aged 18 weeks, indicating more severe atrophy of the EDL muscle in the LP rats. The weights of the EDL and soleus muscles in relation to body weight are given in Table 2. The relative weights of both the EDL and soleus muscles were significantly higher in exercising NP and LP rats compared with the sedentary NP and LP rats, respectively. The mean size of the different fibre types of the EDL and soleus muscles in exercising and sedentary LP and NP rats is illustrated in Table 3. The type 2B fibres of the EDL muscles of the sedentary LP rats were much smaller (54~o) than the type I
4 Body weight (g)
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Age (weeks)
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17 18 Age (weeks)
Fig. 1. Body weight (a) and daily food consumption(b). A = sedentary NP rats; • = exercised NP rats; (3 = sedentary LP rats; • = exercised LP rats, (64 ~o ) and type 2A (64 %) fibres in relation to the fibres of sedentary NP rats, indicating that the type 2B fibres were more affected by protein deprivation than the type 1 and 2A fibres. The type 1 and type 2A fibres of the soleus muscle were considerably larger than the corresponding fibres of the EDL muscle both in NP and LP rats. There were no type 2B fibres in the soleus muscle. The type 2A fibres were smaller (56~/o) than the type 1 fibres ( 6 9 ~ ) i n the soleus muscle of sedentary LP rats when compared with the respective fibre types in the soleus muscle of sedentary NP rats. This indicates that in the soleus muscle type 2A fibres were more affected by protein deprivation than type 1 fibres. Exercise resulted in statistically significant larger fibre size in the soleus muscle
TABLE 1 WEIGHT (mg) OF THE EDL AND SOLEUS MUSCLES OF SEDENTARY AND EXERCISING NP AND LP RATS AT 6 AND 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. NS, not significant. EDL Sedentary NP 6 weeks Sedentary NP 18 weeks
Soleus
84 ( _+4) 215 ( -+26) NS 203 ( _+8) 61 ( _+3) P < 0.05 67 ( _+4)
Exercised NP 18 weeks Sedentary LP 18 weeks Exercised LP 18 weeks
P < 0.02 P < 0.02 P < 0.02 NS NS
79 ( _+5) 186 ( + 4) NS 186 ( + 4) 59 ( _+5) P < 0.05 66 ( _+3)
TABLE 2 RELATIVE WEIGHTS OF THE EDL AND SOLEUS MUSCLES, PER MILLE OF BODY WEIGHT, IN SEDENTARY NP AND LP RATS AT 6 AND 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. NS, not significant. EDL Sedentary NP 6 weeks Sedentary NP 18 weeks
Soleus
0.43 ( _+0.02) 0.46 ( _+0.03) P < 0.05 0.50 ( + 0.01) 0.50 ( _+0.01) P < 0.01 0.55 ( _+0.02)
Exercised NP 18 weeks Sedentary LP 18 weeks Exercised LP 18 weeks
P < 0.02 P < 0.01
0.39 ( _+0.02) 0.40 ( _+0.02) P < 0.01 0.45 ( _+0.02) 0.48 ( + 0.02) P < 0.01 0.54 ( _+0.02)
P < 0.01 NS NS
TABLE 3 MEAN FIBRE DIAMETER (ttm) OF THE FIBRE TYPES IN SEDENTARY AND EXERCISING NP AND LP RATS AT 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. EDL
Soleus
1
2A
2B
1
2A
Sedentary NP 18 weeks Exercised NP 18 weeks Sedentary LP 18 weeks
33(+1.2) 32(_+0.07) 21 (_+ 1.5)
36(+0.05) 35 (_+ 1.8) 23 (_+ 1.5)
48(+3.7) 46(_+2.1) 26 (+2.9)
Exercised LP 18 weeks
23(_+1.4)
24(_+0.06)
27(+1.4)
52(_+3.4) 48(_+2.4) 36(_+2.1) P < 0.01 40(_+2.6)
50(_+3.9) 50(_+4.5) 28(_+3.6) P < 0.01 36(_+2.6)
of the LP rats. The type 2A fibres were 30~o larger and the type 1 fibres 10~o larger in the exercising LP rats when compared with the sedentary LP rats. The modified Gomori trichrome stain and the stains for N A D H - T R and SDH revealed very different patterns in the sedentary NP and LP rats. The accumulation of subsarcolemmal material corresponding to mitochondria (Oldfors et al. 1983) seen especially in type 2A fibres in the EDL and soleus muscles of sedentary NP rats was almost completely lacking in sedentary LP rats (Fig. 2). The exercising LP rats showed, however, a pattern similar to that of the NP rats (Fig. 2) indicating that the exercising LP rats did not lose subsarcolemmal mitochondria. DISCUSSION In a previous study (Oldfors et al. 1983) it was shown that the type 2 fibres, especially type 2B fibres of the fast EDL muscle in rats, are more affected by protein deprivation with regard to size than the type I fibres. Results from previous studies have shown that the slow soleus muscle, which is composed mainly of type 1 fibres, is less affected by malnutrition than the fast EDL muscle which is composed mainly of type 2 fibres (Rowe 1968: Li and Goldberg 1976; Ogata etal. 1978; Millward 1979; Rosochacki and Millward 1979). The present study, which has shown that the type 2 fibres both of the soleus and EDL muscles are more affected with regard to size by protein deprivation, clearly indicates that it is the relative number of the different fibre types in a muscle that determines the degree of reduction in size of the whole muscle in PCM. Goldspink (1978) suggested that the greater activity of the tonic soleus muscle may protect it from atrophy during PCM. The present study has shown that activity induced by exercise protects the EDL and soleus muscles from atrophy in protein deprivation. Both type 1 and type 2 fibres of the soleus muscle were less atrophic in exercising than in sedentary, protein deprived rats. This effect of exercise was more pronounced in type 2 than type I fibres. The effect of exercise in minimising the degree of atrophy in protein deprived rats was more pronounced in the soleus muscle than the EDL muscle. This may be explained by the greater load on the soleus muscle than the EDL muscle when the animals run upwards. Previous studies have shown that the activity of some respiratory enzymes involved in aerobic metabolism and oxygen uptake in muscle are reduced in rats subjected to PCM (Wainio et al. 1959; Taskar and Tulpule 1964; Fuge et al. 1968; Hansen-Smith et al. 1977; Layman et al. 1981). These findings might well be explained by the loss of muscle mitochondria noted in malnourished rats (Hansen-Smith et al. 1977; Oldfors et al. 1983). Daily exercise of rats subjected to PCM protects the muscles from decreased levels of respiratory enzymes (Fuge et al. 1968). The present study has shown that the large subsarcolemmal accumulations of mitochondria seen preferentially in type 2A and type 1 fibres disappeared in sedentary, protein deprived rats but persisted in protein deprived rats undergoing exercise. Thus exercise not only protects the muscle fibres from atrophy but also from the loss of mitochondria which would otherwise occur in protein deprivation. Loss of mitochondria and atrophy of fast (type 2) muscle fibres is also noted in
Fig. 2. Soleus muscles incubated for N A D H - T R (left) and SDH (fight) in sedentary LP rats (a, b), exercised LP rats (c, d) and sedentary N P rats (e,f). The muscle fibres of the NP rats and exercised LP rats show a large accumulation of subsarcolemmai staining which is not seen in sedentary LP rats. x 375.
fish during seasonal starvation and is considered to be both reversible and nonpathological (Johnston 1981; Beardall and Johnson 1983). The present study supports the concept that skeletal muscle to a great extent adapts to its nutritional supply and that this adaptation may be altered by demands made on the muscle, for example increased activity. ACKNOWLEDGEMENTS
The technical assistance of Miss Hermengild Schucht and the advice of Dr. William Mair are gratefully acknowledged. REFERENCES Beardall, C.H. and I.A. Johnston (1983) Muscle atrophy during starvation in a marine teleost, Europ. d. Cell Biol., 29: 209-217. Dubowitz, V. and M.H. Brooke (1973)Muscle Biopsy--A Modem Approach, W.B. Saunders Co., London, Philadelphia, Toronto. Fuge, K.W., E.L. Crews III, P.K. Pattengale, J.O. Holloszy and R.E. Shank (1968) Effects of protein deficiency on certain adaptive responses to exercise, Amer. J. Physiol., 215: 660-663. Goldspink, D. F. (1978 ) The effects of food deprivation on protein turnover and nucleic acid concentrations of active and immobilized extensor digitorum longus muscles of the rat, Biochem. J., 176: 603-606. Hansen-Smith, F. M., M. G, Maksud and D.L. Van Horn (1977) Effect of dietary protein restriction or food restriction on oxygen consumption and mitochondrial distribution in cardiac and red and white skeletal muscle of rats, J. Nutr., 107: 525-533. Johnston, I.A. (1981) Quantitative analysis of muscle breakdown during starvation in the marine flatfish Pleuronectes platessa, Cell Tiss. Res., 214: 369-386. Lammi-Keefe, C.J., P.V.J. Hegarty and P.B. Swan (1981) Effect of starvation and refeeding on catalase and superoxide dismutase activities in skeletal and cardiac muscles from 12-month-old rats, Experientia (Basel), 37: 25-27. Layman, D.K., M. Merdian-Bender, P.V.J. Hegarty and P.B. Swan (1981) Changes in aerobic and anaerobic metabolism in rat cardiac and skeletal muscles after total or partial dietary restrictions, J. Nutr., 111: 994-1000. Li, J.B. and A.L. Goldberg (1976) Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles, Amer. J. Physiol., 231: 441-448. Millward, D.J. (1979) Protein deficiency, starvation and protein metabolism, Proc. Nutr. Soc., 38: 77-88. Ogata, E.S., S. K. H. Foung and M.A. Holliday (1978) The effects of starvation and refeeding on muscle protein synthesis and catabolism in the young rat, J. Nutr., 108: 759-765. Oldfors, A., W. G. P. Mair and P. Sourander (1983) Muscle changes in protein-deprived young rats - - A morphometricai, histochemical and ultrastructural study, J, NeuroL Sci., 59: 291-302. Rosochacki, S. and D.J. Millward (1979) Cathepsin D and acid autolytic activity in skeletal muscle of protein deficient, severely protein-energy restricted and refed rats, Proc. Nutr. Soc., 38: 137A. Rowe, R.W.D. (1968) Effect of low nutrition on size of striated muscle fibres in the mouse, J. Exp. Zool., 167: 353-358. Taskar, K. and P.G. Tulpule (1964) Influence of protein and calorie deficiencies in the rat on the energy-transfer reactions of the striated muscle, Biochem. J., 92:391-398. Wainio, W.W., J.B. Allison, L.T. Kremzner, E. Bernstein and M. Aronoff (1959) Enzymes in protein depletion, Part 3 (Enzymes of brain, kidney, skeletal muscle and spleen), J. Nutr., 67: 197-204.
1
Elsevier
Effects of Training on Skeletal Muscle in Protein-Deprived Rats Anders Oldfors and Patrick Sourander Division of Neuropathology, Department of PathologyL Sahlgren Hospital, S-413 45 Gdteborg(Sweden) (Received 26 November, 1984) (Revised, received 16 January, 1985) (Accepted 18 January, 1985)
SUMMARY
The effects of exercise on atrophy of muscle fibres and loss of mitochondria in the extensor digitorum longus (EDL) and soleus muscles were studied in protein deprived rats. They had smaller muscle fibres than aged-matched control rats, the difference being more evident in type 2 than type 1 fibres both in the fast EDL and slow soleus muscles. The loss of weight was more pronounced in the EDL muscle which is composed mainly of type 2 fibres than in the soleus muscle which is composed mainly of type 1 fibres. Protein deprived rats subjected to a programme of periodic running on a treadmill for 12 weeks showed less muscle atrophy than sedentary, protein deprived rats. This effect of exercise in diminishing the degree of atrophy was more pronounced in the type 2 than type 1 fibres. The protein deprived rats which had been sedentary showed a marked loss of subsarcolemmal mitochondria, which was not seen in protein deprived rats undergoing exercise.
Key words:
Exercise
- Histochemistry
- Protein deprivation - Rat - Skeletal
muscle
INTRODUCTION
The increased knowledge of the importance of skeletal muscle for protein and energy metabolism has promoted interest in how various conditions such as nutritional change influence the structure, function and biochemistry of skeletal muscle. This study was supported by grants from the Swedish Medical Research Council, Proj. Nos. 03488 and 07122. 0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
In a recent study, Oldfors et al. (1983) showed in young rats subjected to severe protein deprivation that atrophy of the fast extensor digitorum longus (EDL) muscle was due primarily to atrophy of type 2 fibres, while the type 1 fibres simply failed to grow. Previous studies showed that the slow soleus muscle which is composed mainly of type 1 fibres is less affected by protein-calorie malnutrition (PCM) than the fast EDL muscle which is composed mainly of type 2 fibres (Rowe 1968; Li and Goldberg 1976; Lammi-Keefe et al. 1981; Layman et al. 1981). It thus seems possible that the relative number of different fibre types in a muscle determines the degree of reduction in size of the whole muscle, since the various fibre types differ in their susceptibility to PCM. A possible explanation for this discrepancy between fast and slow muscles is that the tonic soleus muscle by its greater activity is protected from atrophy during PCM (Goldspink 1978). Protein deprived rats show a marked loss of subsarcolemmal mitochondria (Oldfors et al. 1983), which loss may explain the previous finding that the activity of the respiratory enzymes involved in aerobic metabolism of muscle was reduced in rats subjected to PCM (Wainio et al. 1959; Taskar and Tulpule 1964; Fuge et al. 1968; Hansen-Smith et al. 1977; Layman et al. 1981). However, Fuge et al. (1968) reported that exercise prevented the decrease in the level of activity of some respiratory enzymes of muscle in rats subjected to PCM. This study was undertaken to answer the following: Is the difference in the atrophy of slow and fast muscles in protein deprivation due to a difference in the vulnerability of slow and fast muscle fibres? Does activity of a muscle, during protein deprivation, protect it from atrophy? Does activity of the muscle protect it from loss of subsarcolemmal mitochondria? MATERIALS AND METHODS
Animals and protein deprivation Male Sprague-Dawley rats reared on a standard diet were at the age of 6 weeks divided into two groups. One continued to be fed on a well balanced diet containing 14~o protein by weight. The rats of this group are referred to as NP rats. The other group was fed on a diet containing a very small amount of protein, 1.5 ~o by weight but was supplemented by additional starch so that the diets of both groups were approximately iso-caloric. The group fed on the low protein diet are referred to as protein deprived or LP rats. Details of the composition of the respective diets are given in a previous article (Oldfors et al. 1983). The body weight and food consumption of the rats were assessed weekly. The rats were kept in plastic cages at an ambient temperature of 22 ° C and a relative humidity of 60 Yo. The rats had free access to food and drinking water. Exercise programme Each group included 2 lots each consisting of 5 rats. One lot from each group was trained to run at intervals on a motor driven treadmill consisting of a wide, endless belt running on metal rollers. Ten boxes 50 cm long by 10 cm wide placed on the belt provided a limited area in which each animal could run. Motivation to run was effected
by an electric shock grid at the rear of the boxes. The animals learned within a week to avoid the electric shock by keeping pace with the moving belt. The treadmill was set at a moderate speed of 18 m/min and inclined initially at 5 °. During the 12 weeks of training, the incline was gradually increased to 16 ° and remained so for the last 2 weeks. The animals ran once a day for 5 days per week. The daily exercise period was gradually increased from 15 min the first 2 weeks to 1 h during the last 2 weeks. One lot of each group remained sedentary.
Morphometry and histochemistry Aged 18 weeks, all the rats were anaesthetized by sodium pentobarbital, and the EDL and soleus muscles were removed. The muscles were weighed and the mid part of the belly of the muscles was frozen in a mixture of liquid propane and propylene (Primus Gasol M 11 Esso) chilled with liquid nitrogen. Transverse sections, 10#m thick, were cut on a cryostat mierotome and stained for myofibfiUar ATPase after preincubation at pH 4.6 according to the method recommended by Dubowitz and Brooke (1973) for morphometric analysis of type 1, 2A and 2B fibres. The central parts of the sections were photographed and the size of some 200 adjacent fibres was measured using a particle size analyzer (Zeiss TGZ3) by assessing the diameter of the inscribed circle of each fibre. The mean size of each fibre type of the EDL and soleus muscles of the different groups was compared statistically using the Wilcoxon-Mann-Whitney rank sum test. To study the distribution of mitochondria cryostat 10-pm thick sections were stained by the modified Gomori trichrome stain and for NADH-tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH). RESULTS
The body weight of the rats are illustrated in Fig. la. The NP rats gained weight from the age of 6-18 weeks while the LP rats lost weight. The sedentary NP rats gained more weight than the exercising NP rats while the weight of the sedentary and exercising LP rats did not differ significantly. The daily food consumption in relation to body weight is illustrated in Fig. lb. While the NP rats consumed more food than the LP rats during the In'st 4 weeks of the study the relative food consumption of the groups did not differ significantly from 10 weeks of age. Tablel shows the weights of the EDL and soleus muscles. While the EDL muscle of NP rats aged 18 weeks had a greater weight than the soleus muscle, there was no significant difference between the weights of the EDL and soleus muscles of the LP rats aged 18 weeks, indicating more severe atrophy of the EDL muscle in the LP rats. The weights of the EDL and soleus muscles in relation to body weight are given in Table 2. The relative weights of both the EDL and soleus muscles were significantly higher in exercising NP and LP rats compared with the sedentary NP and LP rats, respectively. The mean size of the different fibre types of the EDL and soleus muscles in exercising and sedentary LP and NP rats is illustrated in Table 3. The type 2B fibres of the EDL muscles of the sedentary LP rats were much smaller (54~o) than the type I
4 Body weight (g)
a 500-t~ ~x
400--
A
A
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300 --
200--
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%
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18
Age (weeks)
Dail
food consumption body weight
g/100g
b
8~
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17 18 Age (weeks)
Fig. 1. Body weight (a) and daily food consumption(b). A = sedentary NP rats; • = exercised NP rats; (3 = sedentary LP rats; • = exercised LP rats, (64 ~o ) and type 2A (64 %) fibres in relation to the fibres of sedentary NP rats, indicating that the type 2B fibres were more affected by protein deprivation than the type 1 and 2A fibres. The type 1 and type 2A fibres of the soleus muscle were considerably larger than the corresponding fibres of the EDL muscle both in NP and LP rats. There were no type 2B fibres in the soleus muscle. The type 2A fibres were smaller (56~/o) than the type 1 fibres ( 6 9 ~ ) i n the soleus muscle of sedentary LP rats when compared with the respective fibre types in the soleus muscle of sedentary NP rats. This indicates that in the soleus muscle type 2A fibres were more affected by protein deprivation than type 1 fibres. Exercise resulted in statistically significant larger fibre size in the soleus muscle
TABLE 1 WEIGHT (mg) OF THE EDL AND SOLEUS MUSCLES OF SEDENTARY AND EXERCISING NP AND LP RATS AT 6 AND 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. NS, not significant. EDL Sedentary NP 6 weeks Sedentary NP 18 weeks
Soleus
84 ( _+4) 215 ( -+26) NS 203 ( _+8) 61 ( _+3) P < 0.05 67 ( _+4)
Exercised NP 18 weeks Sedentary LP 18 weeks Exercised LP 18 weeks
P < 0.02 P < 0.02 P < 0.02 NS NS
79 ( _+5) 186 ( + 4) NS 186 ( + 4) 59 ( _+5) P < 0.05 66 ( _+3)
TABLE 2 RELATIVE WEIGHTS OF THE EDL AND SOLEUS MUSCLES, PER MILLE OF BODY WEIGHT, IN SEDENTARY NP AND LP RATS AT 6 AND 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. NS, not significant. EDL Sedentary NP 6 weeks Sedentary NP 18 weeks
Soleus
0.43 ( _+0.02) 0.46 ( _+0.03) P < 0.05 0.50 ( + 0.01) 0.50 ( _+0.01) P < 0.01 0.55 ( _+0.02)
Exercised NP 18 weeks Sedentary LP 18 weeks Exercised LP 18 weeks
P < 0.02 P < 0.01
0.39 ( _+0.02) 0.40 ( _+0.02) P < 0.01 0.45 ( _+0.02) 0.48 ( + 0.02) P < 0.01 0.54 ( _+0.02)
P < 0.01 NS NS
TABLE 3 MEAN FIBRE DIAMETER (ttm) OF THE FIBRE TYPES IN SEDENTARY AND EXERCISING NP AND LP RATS AT 18 WEEKS OF AGE Standard deviation in parentheses. P values are indicated between the compared groups. EDL
Soleus
1
2A
2B
1
2A
Sedentary NP 18 weeks Exercised NP 18 weeks Sedentary LP 18 weeks
33(+1.2) 32(_+0.07) 21 (_+ 1.5)
36(+0.05) 35 (_+ 1.8) 23 (_+ 1.5)
48(+3.7) 46(_+2.1) 26 (+2.9)
Exercised LP 18 weeks
23(_+1.4)
24(_+0.06)
27(+1.4)
52(_+3.4) 48(_+2.4) 36(_+2.1) P < 0.01 40(_+2.6)
50(_+3.9) 50(_+4.5) 28(_+3.6) P < 0.01 36(_+2.6)
of the LP rats. The type 2A fibres were 30~o larger and the type 1 fibres 10~o larger in the exercising LP rats when compared with the sedentary LP rats. The modified Gomori trichrome stain and the stains for N A D H - T R and SDH revealed very different patterns in the sedentary NP and LP rats. The accumulation of subsarcolemmal material corresponding to mitochondria (Oldfors et al. 1983) seen especially in type 2A fibres in the EDL and soleus muscles of sedentary NP rats was almost completely lacking in sedentary LP rats (Fig. 2). The exercising LP rats showed, however, a pattern similar to that of the NP rats (Fig. 2) indicating that the exercising LP rats did not lose subsarcolemmal mitochondria. DISCUSSION In a previous study (Oldfors et al. 1983) it was shown that the type 2 fibres, especially type 2B fibres of the fast EDL muscle in rats, are more affected by protein deprivation with regard to size than the type I fibres. Results from previous studies have shown that the slow soleus muscle, which is composed mainly of type 1 fibres, is less affected by malnutrition than the fast EDL muscle which is composed mainly of type 2 fibres (Rowe 1968: Li and Goldberg 1976; Ogata etal. 1978; Millward 1979; Rosochacki and Millward 1979). The present study, which has shown that the type 2 fibres both of the soleus and EDL muscles are more affected with regard to size by protein deprivation, clearly indicates that it is the relative number of the different fibre types in a muscle that determines the degree of reduction in size of the whole muscle in PCM. Goldspink (1978) suggested that the greater activity of the tonic soleus muscle may protect it from atrophy during PCM. The present study has shown that activity induced by exercise protects the EDL and soleus muscles from atrophy in protein deprivation. Both type 1 and type 2 fibres of the soleus muscle were less atrophic in exercising than in sedentary, protein deprived rats. This effect of exercise was more pronounced in type 2 than type I fibres. The effect of exercise in minimising the degree of atrophy in protein deprived rats was more pronounced in the soleus muscle than the EDL muscle. This may be explained by the greater load on the soleus muscle than the EDL muscle when the animals run upwards. Previous studies have shown that the activity of some respiratory enzymes involved in aerobic metabolism and oxygen uptake in muscle are reduced in rats subjected to PCM (Wainio et al. 1959; Taskar and Tulpule 1964; Fuge et al. 1968; Hansen-Smith et al. 1977; Layman et al. 1981). These findings might well be explained by the loss of muscle mitochondria noted in malnourished rats (Hansen-Smith et al. 1977; Oldfors et al. 1983). Daily exercise of rats subjected to PCM protects the muscles from decreased levels of respiratory enzymes (Fuge et al. 1968). The present study has shown that the large subsarcolemmal accumulations of mitochondria seen preferentially in type 2A and type 1 fibres disappeared in sedentary, protein deprived rats but persisted in protein deprived rats undergoing exercise. Thus exercise not only protects the muscle fibres from atrophy but also from the loss of mitochondria which would otherwise occur in protein deprivation. Loss of mitochondria and atrophy of fast (type 2) muscle fibres is also noted in
Fig. 2. Soleus muscles incubated for N A D H - T R (left) and SDH (fight) in sedentary LP rats (a, b), exercised LP rats (c, d) and sedentary N P rats (e,f). The muscle fibres of the NP rats and exercised LP rats show a large accumulation of subsarcolemmai staining which is not seen in sedentary LP rats. x 375.
fish during seasonal starvation and is considered to be both reversible and nonpathological (Johnston 1981; Beardall and Johnson 1983). The present study supports the concept that skeletal muscle to a great extent adapts to its nutritional supply and that this adaptation may be altered by demands made on the muscle, for example increased activity. ACKNOWLEDGEMENTS
The technical assistance of Miss Hermengild Schucht and the advice of Dr. William Mair are gratefully acknowledged. REFERENCES Beardall, C.H. and I.A. Johnston (1983) Muscle atrophy during starvation in a marine teleost, Europ. d. Cell Biol., 29: 209-217. Dubowitz, V. and M.H. Brooke (1973)Muscle Biopsy--A Modem Approach, W.B. Saunders Co., London, Philadelphia, Toronto. Fuge, K.W., E.L. Crews III, P.K. Pattengale, J.O. Holloszy and R.E. Shank (1968) Effects of protein deficiency on certain adaptive responses to exercise, Amer. J. Physiol., 215: 660-663. Goldspink, D. F. (1978 ) The effects of food deprivation on protein turnover and nucleic acid concentrations of active and immobilized extensor digitorum longus muscles of the rat, Biochem. J., 176: 603-606. Hansen-Smith, F. M., M. G, Maksud and D.L. Van Horn (1977) Effect of dietary protein restriction or food restriction on oxygen consumption and mitochondrial distribution in cardiac and red and white skeletal muscle of rats, J. Nutr., 107: 525-533. Johnston, I.A. (1981) Quantitative analysis of muscle breakdown during starvation in the marine flatfish Pleuronectes platessa, Cell Tiss. Res., 214: 369-386. Lammi-Keefe, C.J., P.V.J. Hegarty and P.B. Swan (1981) Effect of starvation and refeeding on catalase and superoxide dismutase activities in skeletal and cardiac muscles from 12-month-old rats, Experientia (Basel), 37: 25-27. Layman, D.K., M. Merdian-Bender, P.V.J. Hegarty and P.B. Swan (1981) Changes in aerobic and anaerobic metabolism in rat cardiac and skeletal muscles after total or partial dietary restrictions, J. Nutr., 111: 994-1000. Li, J.B. and A.L. Goldberg (1976) Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles, Amer. J. Physiol., 231: 441-448. Millward, D.J. (1979) Protein deficiency, starvation and protein metabolism, Proc. Nutr. Soc., 38: 77-88. Ogata, E.S., S. K. H. Foung and M.A. Holliday (1978) The effects of starvation and refeeding on muscle protein synthesis and catabolism in the young rat, J. Nutr., 108: 759-765. Oldfors, A., W. G. P. Mair and P. Sourander (1983) Muscle changes in protein-deprived young rats - - A morphometricai, histochemical and ultrastructural study, J, NeuroL Sci., 59: 291-302. Rosochacki, S. and D.J. Millward (1979) Cathepsin D and acid autolytic activity in skeletal muscle of protein deficient, severely protein-energy restricted and refed rats, Proc. Nutr. Soc., 38: 137A. Rowe, R.W.D. (1968) Effect of low nutrition on size of striated muscle fibres in the mouse, J. Exp. Zool., 167: 353-358. Taskar, K. and P.G. Tulpule (1964) Influence of protein and calorie deficiencies in the rat on the energy-transfer reactions of the striated muscle, Biochem. J., 92:391-398. Wainio, W.W., J.B. Allison, L.T. Kremzner, E. Bernstein and M. Aronoff (1959) Enzymes in protein depletion, Part 3 (Enzymes of brain, kidney, skeletal muscle and spleen), J. Nutr., 67: 197-204.