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Effect of chronic ethanol ingestion and exercise training on skeletal muscle in rat
Drug and Alcohol Dependence 64 (2001) 27 – 33 www.elsevier.com/locate/drugalcdep
Effect of chronic ethanol ingestion and exercise training on skeletal muscle in rat L. Vila, A. Ferrando, J Voces, C. Cabral de Oliveira, J.G. Prieto, A.I. Alvarez * Department of Physiology, The Uni6ersity of Leo´n, Leo´n, 24071, Spain Received 16 June 2000; received in revised form 27 October 2000; accepted 14 November 2000
Abstract The aim of this study was to investigate the interactive effects of exercise training and chronic ethanol consumption on metabolism, capillarity, and myofibrillar composition in rat limb muscles. Male Wistar rats were treated in separate groups as follows: non exercised-control; ethanol (15%) in animals’ drinking water for 12 weeks; exercise training in treadmill and ethanol administration plus exercise for 12 weeks. Ethanol administration decreased capillarity and increased piruvate kinase and lactate dehydrogenase activities in white gastrocnemius; in plantaris muscle, ethanol increased citrate synthase activity and decreased cross-sectional area of type I, IIa, and IIb fibres. Exercise increased capillarity in all four limb muscles and decreased type I fibre area in plantaris. The decreased capillarity effect induced by ethanol in some muscles, was ameliorated when alcohol was combined with exercise. While alcoholic myopathy affects predominantly type IIb fibres, ethanol administration and aerobic exercise in some cases can affect type I and type IIa fibre areas. The exercise can decrease some harmful effects produced by ethanol in the muscle, including the decrease in the fibre area and capillary density. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Exercise; Muscle metabolism; Fiber area; Fiber type; Capillarity; Rats
1. Introduction The action of alcohol on body systems is complex and the problems associated with its excessive and frequent consumption are numerous. Alcohol affects several organs, such as the brain, nerves, heart, digestive system, liver, skeletal muscle and blood, and produces metabolic alterations either directly or indirectly through the products formed in its biotransformation (Lieber, 1992). The study of alcohol consumption and exercise performance is normally restricted to the area of ‘drugs and exercise’, including substances that interfere with the normal development of exercise. The vitamin deficit elicited by alcohol and the accumulation of lactate to block uric acid secretion are the best studied aspects affecting physical activity (Lowenthal and Kendrick, 1985; Kendrick and Lowenthal, 1988).
* Corresponding author. Tel.: + 34-87-291263; fax: +34-87291267. E-mail address: [email protected] (A.I. Alvarez).
Alcohol consumption decreases the use of glucose and amino-acids by muscles. The hypoglycaemia thus generated can promote a failure in energy supply and an impairment in skeletal muscle metabolism during physical exercise. In fact, alcoholic myopathy is usually described in glycolytic muscles, in which protein turnover and glucose metabolism deficiencies associated with chronic alcohol consumption have been reported (Preedy et al., 1994). Glucose transport and uptake induced by physical exercise have been found to correlate with muscle GLUT-4 contents (Neufer et al., 1992). Under certain conditions exercise increases muscular protein synthesis, leading to muscle hypertrophy (Antonio and Gonyea, 1993; McComas, 1994). Siciliano et al. (2000) have shown that the accumulation of lactate during exercise is decreased after aerobic training in mitochondrial myopathies. With respect to the ethanol –exercise interaction, Ardies et al. (1987, 1989) reported that exercise may attenuate the ethanol-induced decline in hepatic mitochondria and that exercise performance accelerates ethanol metabolism by hepatic microsomes.
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Trudell et al. (1995) were the first to document a potential mechanism able to explain how repeated exercise prevents ethanol-induced fatty liver. Husain and Somani (1997a,b, 1998) showed that exercise training seems to reduce the extent of the oxidative damage caused by ethanol on the liver, myocardium, and testes of rats. Myopathy is related to impaired glycolytic metabolism and to restricted capillary density. The aim of this work was to study how the enhanced oxidative metabolism, the increase in capillarity and the increase in alcohol removal from blood can contribute to ameliorating alcohol myopathy. The metabolic responses of the organism to exercise and the generation of alcoholic myopathy have the same physiological consequences in rat and humans, although it is clear that the information obtained is specific to rat muscles and is not always applicable to human muscle. With this background, we addressed the effect of aerobic exercise on the skeletal muscle of ethanoltreated rats. The experimental design included study of muscle metabolism, capillary distribution, myofibrillar distribution and fibre morphometry.
2. Materials and methods
2.2. Training program The trained rats were run on a motor-driven treadmill 4 days per week for 12 weeks. The training session started out as a 30 min run at 20 m min − 1, 0% grade. Duration was increased by 5 min each 2 week until the animals were running for 1 h. Thereafter, running speed was increased by 1 m min − 1 every 2 wk and the grade was increased up to a final elevation of 15%. The final running speed obtained for the 60 min training period was 24 m min − 1. The ethanol treated-trained rats performed the program when they had started to drink 15% ethanol. To eliminate effects of the last bout of exercise, tissue samplings were performed 36–40 h after the final exercise session.
2.3. Tissue sampling Muscles were obtained after the animals had been anaesthetized intraperitoneally with urethane (30% w/v) at a dose of 1 ml/250 g body weight. The red, mixed and white gastrocnemius (predominantly fibre type I, IIa; IIa, IIb; and IIb, respectively), plantaris (predominantly fibre type IIa, IIb) and soleus (fibre type I) were surgically removed from the left and right hind limbs and the connective tissue trimmed away. All muscles were frozen in isopentane, cooled in liquid N2 and stored at − 80°C.
2.1. Animals and treatments 2.4. Determination of enzyme acti6ity Male Wistar rats obtained from IFFA Credo (Madrid, Spain), with a mean body weight of 1009 10 g were used for this study. The animals were housed three per cage, except the ethanol treated animals, which were housed individually to control daily ethanol consumption. The rats were housed in a temperaturecontrolled environment (2291°C) with a 12:12 h light –dark cycle The study was performed with four experimental groups of ten animals each. The animals were divided into control (Cont), ethanol-treated (Eth), exercised (Exer) and ethanol-treated+ exercise (Eth-Exer) groups. Alcohol administration was performed on one group of sedentary and one group of exercised rats. Ethanol was given to the rats for 12 weeks in their drinking water. The concentration of ethanol was gradually increased from 5 to 15% (v/v). Control rats had free access to water. All groups received commercial rat chow ad libitum (Panlab, Barcelona Spain). The animals were housed according to the Principles of Council Directive 86/609/EEC: ‘‘On the Approximation of Laws, Regulation and Administrative Provision of the Member States regarding the Protections of Animal used for Experimental and other Scientific Purposes’’.
Muscle from right hind limb was weighed and homogenized in a buffer (2 mM MgCl2, 2 mM EDTA, 50 mM Tris, pH 7.4) at a dilution of 1:40 (w/v). The homogenizing tube was immersed in ice-cold water and the tissue was subjected to a 1 min burst with a Potter–Elvehjem instrument. The medium was frozen at − 80°C until analysis. Spectrophotometric assays were performed on a HITACHI U-2000 spectrophotometer in 1 ml cuvettes, of 1 cm light path. Reaction rates were proportional to enzyme concentrations. One unit of enzymatic activity is defined as the amount of enzyme that catalyses the uptake of 1 mmole of substrate per minute. Citrate synthase (CS, EC 4.1.3.7) activity, a marker of the tricarboxylic acid cycle, was measured at 37°C by the method of Srere et al. (1963). The activity of hexokinase (HK, EC 2.7.1.1), an enzyme required for glucose uptake, was measured by the method of Bergmeyer (1974a). Pyruvate kinase (PK, EC 2.7.1.40) and lactate dehydrogenase (LDH, 1.1.1.27) activities, markers of the glycolysis, were measured at 37°C by the methods of Bergmeyer (1974b) and Kornberg (1955), respectively. The determination of b-hydroxyacetyl-CoA-dehydrogenase (HAD, EC 1.1.1.35) activity, a marker of b-oxidation, was performed at 30°C according the pro-
L. Vila et al. / Drug and Alcohol Dependence 64 (2001) 27–33
cedures described by Bradshaw and Noyes (1975). All values are expressed in mmol g − 1 wet muscle min − 1.
Table 1 Mean 9 SD values of body weight and liver (g)
2.5. Histochemistry The soleus, plantaris and gastrocnemius from left hind limb were cut in 12 mm sections in a cryostat maintained at −20°C and mounted on glass coverslips. In order to determine the correct sample orientation, a rapid stain with hematoxilin-eosin was performed. Serial sections were stained for histochemical demonstration of myosin ATPase after acid preincubation, based on the method originally described by Padykula and Herman (1955) and Guth and Samaha (1970). This stain identifies three fibre types according to assessment of the contractile properties of the muscle. Capillaries were determined from ATPase stains preincubated at pH 4 (Fouces et al., 1993). Fibre typing and field measurements were obtained using a light microscope equipped with a camera (Optiphot-2 Nikon, Tokyo, Japan). Photomicrographs were taken at a magnification of 200× and fibre measurements were carried out by means of a digitising tablet (Genitizer-GT-1212B) connected to a PC, using suitable software (Sigma-Scan, Jandel Scientific, Erkrath, Germany). Photographs of a stage micrometer were taken at the beginning and at the end of each film as a calibration control (Fouces et al., 1993). A minimum of 200 fibres per muscle for each animal was studied, following the techniques of Blomstrang and Ekblom, (1982) and Shorey and Cleland, (1983). The number of capillaries per square millimetre (cap mm − 2) and the capillary/fibre ratio (capillaries per fibre) were determined from an area of 0.16 mm2, four fields per sample being measured.
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Control Ethanol Exercise Ethanol–Exercise
Total
Liver
416 9 37 395 9 35 383 9 55 336 9 32a
32.3 93.9 33.3 91.5 30.4 9 3.1 32.99 1.8
a
Significantly different from control rats (PB0.05). n =10 animals per group.
(body weight) in the trained animals. Body and liver weights are shown in Table 1, with no significant differences among the groups; only the final weight of the ethanol-trained rats was statistically different from that of the controls (3369 22 vs. 4169 37 g).
3.1. Muscle enzymes Fig. 1 and Fig. 2 show the muscle enzyme activities in the control, trained, alcohol-treated and trained alcohol-treated rats. The data obtained for CS activity as a marker of oxidative metabolism revealed a greater oxidative trend in red gastrocnemius, followed by plantaris muscle (both red gastrocnemius and plantaris muscles having
2.6. Statistical analysis One-way analysis of variance was performed on all variables. The significance of the difference between the means obtained was determined using the Newman– Keuls test. A level of P B0.05 was set for significance for all tests, and all values are expressed as means9 S.D.
3. Results Because of a possible failure in inducing ethanol toxicity by chronic administration and its consequences on the nutritional status of the animals, the animals’ weights and daily ethanol consumptions were recorded during the experimental period. During week 4 of treatment and onwards, the average daily consumption of ethanol was 10.69 1.8 g (ethanol) kg − 1 (body weight) in the untrained rats, and 10.191.6 g (ethanol) kg − 1
Fig. 1. The activities of the tricarboxylic acid cycle enzyme citrate synthase and the b– hydroxyacetyl-CoA dehydrogenase determined from hindlimb muscles of control (Cont), ethanol (Eth), exercise (Exer) and ethanol-exercise (Eth +Exer) groups. * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (PB 0.05).
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red gastrocnemius and soleus than in white gastrocnemius (3.009 0.13 vs. 1.8990.25 mmol g − 1 min − 1).
3.2. Capillarity and fibre area
Fig. 2. The activities of the hexokinase, piruvate kinase and lactate dehydrogenase determined from hindlimb muscles of control (Cont), ethanol (Eth), exercise (Exer) and ethanol-exercise (Eth +Exer) groups. * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (PB 0.05). ‡ Significantly different from exercise group (PB 0.05).
the highest percentages of IIa fibres), and soleus, the lowest activity being found in white gastrocnemius. HAD activity was highest in soleus and red gastrocnemius, including mainly type I fibers (Fig. 1). As regards glycolytic enzymes, HK was slightly increased after exercise; PK and LDH showed the highest values in muscles with type IIb fibers, such as white gastrocnemius and plantaris (Fig. 2). The present results showed that oxidative enzyme activities were unaffected by ethanol except in plantaris muscle, where CS activity increased significantly from 27.591.8 in the controls to 36.792.7 mmol g wet muscle − 1 min − 1 in the alcohol-treated rats. Alcohol administration modified the glycolytic metabolism in muscles with a higher percentage in type IIb fibers, such as white gastrocnemius, where PK and LDH activities were significantly increased. LDH activity was reduced in red gastrocnemius and plantaris, although this effect was largely a consequence of the exercise performed. HK activity, which is related to exogenous glucose utilization, was significantly higher (P B 0.05) in the ethanol-trained group in oxidative muscles such as
The results concerning capillary distribution are shown in Table 2. The most relevant finding after ethanol administration was a significant reduction in white gastrocnemius muscle of both the number of capillaries per fibre (1.269 0.1 to 0.979 0.06) and capillary density (2459 25 to 2129 26 capillaries mm − 2). The remaining muscles showed no changes following ethanol administration. Exercise significantly increased capillarity in all muscles. The effects on capillarity of the action of both ethanol and exercise treatments were that exercise reversed the effect of alcohol in muscles with greater percentages of type IIb fibres. Thus, in white gastrocnemius the number of capillaries per fibre and capillary density were increased with respect to the group that received alcohol (1.109 0.08 vs. 0.979 0.06 capillaries per fibre). In red gastrocnemius and soleus muscles, the most important effect was elicited by exercise and both muscles showed increased capillary distribution, showing no effects associated with alcohol administration. In plantaris muscle, also affected by alcohol but to a lesser extent, the effect of exercise on the increase in capillarity was less pronounced than in white gastrocnemius. The fibrillar area was decreased by alcohol in type I and type II fibbers as compared to the control group (data not shown) but the decrease was not statistically significant except in plantaris muscle, for which alcohol administration decreased the area in all
Table 2 Capillarity of skeletal muscle of ethanol treated, training and ethanol treated-exercised rats Control
Ethanol
Exercise
Ethanol – Exercise
Plantaris Capillaries per fiber 1.72 9 0.17 Capillaries per mm2 477 9 35
1.58 9 0.12 504 9 19
1.95 9 0.07*a 1.59 90.08 575 9 46* 536 9 22
G. White Capillaries per fiber 1.26 9 0.1 Capillaries per mm2 245 9 25
0.97 9 0.06* 1.45 9 0.14* 212 9 26* 273 9 21
1.10 9 0.08† 219 921
Soleus Capillaries per fiber 1.67 9 0.16 Capillaries per mm2 380 9 21
1.97 9 0.23 431 9 34
2.24 9 0.17* 553 9 30*
2.21 90.26* 583 9 47*
G. Red Capillaries per fiber 2.08 9 0.15 1.92 9 0.12 Capillaries per mm2 650 9 68 581 9 30
2.38 9 0.11* 673 9 70
2.40 9 0.13*† 727 9 59
a *Significantly different from control rats (PB0.05). †Significantly different from ethanol group (PB0.05).
L. Vila et al. / Drug and Alcohol Dependence 64 (2001) 27–33
Fig. 3. Effect of alcohol administration, exercise and alcohol administration plus exercise in the area of type I, type IIa and type IIb fibres of plantaris (a) and in type I fibres of soleus, red and mixed gastrocnemius (b). * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (P B0.05). ‡ Significantly different from exercise group (P B0.05).
three fibre types. This decrease persisted in the exercised group (Fig. 3a). Type I fibre area was decreased in the Eth + Exer group only in mixed gastrocnemius muscle (Fig. 3b).
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to the glycogen synthesis. Spolarics et al. (1994) showed that acute ethanol administration inhibits glucose uptake by 50% in rat skeletal muscles (soleus, gastrocnemius and red quadriceps). This inhibition even appeared in hyperinsulinaemic animals, in which the basal glucose level is doubled. Chronic alcohol consumption reduces enzyme activities such as LDH, HK and PK in the rat vastus lateralis (Trounce et al., 1990). From our results no inhibition of glucose uptake can be inferred. One plausible explanation could be the low level of alcoholization in our experiment, since the values for rat serum transaminases (ALT and AST) remained unmodified. Alcohol intake coupled with physical exercise lowers muscle glucose (Juhlin-Dannfelt et al., 1977). By contrast, Shelmet et al. (1988) reported the existence of an acute peripheral insulin resistance after alcohol ingestion, which would reduce glucose oxidation. However, these variations occur in the short term and are attenuated and disappear with prolonged treatments (Cook et al., 1992). It has previously been described that alcohol administration markedly inhibits glucose use by skeletal muscles (Spolarics et al., 1994) but hexokinase activity is induced in ethanol-exercised animals in all muscles studied except plantaris. Exercise increases muscle glucose uptake as a result of an increase in muscle transporter concentration (GLUT-4) (Hargreaves, 1995), this effect being mediated by hormones and by muscular contraction (Yeh et al., 1995). Increasing calcium levels during muscle contraction induces the activation of glucose uptake (Youn et al., 1991).
4. Discussion
4.2. Capillarity distribution
4.1. Enzyme acti6ities
It is generally assumed that capillarity is related to muscle oxidative capacity. The present results also show a correlation between capillarity and citrate synthase activity, the highest values being seen for red gastrocnemius muscle, followed by plantaris, soleus, and white gastrocnemius. This correlation has also been indicated by Tesch and Karlsson (1985). Training increases capillary distribution in exercised muscles. The outcome of capillary development is to allow a greater surface for distribution and to increase the efficiency of respiratory gases, metabolites and substrate exchange (Saltin et al., 1986). Although studies on the effect of ethanol in skeletal muscle capillarity are scarce, it has been established that acute alcohol ingestion produces peripheral tissue vasodilation (Friedman et al., 1982). However, chronic ingestion of alcohol induces a reduction in the vascular lumen, which may be related to the development of alcoholic myopathy. This effect is not mediated by
The results concerning rat oxidative and glycolytic metabolism are in agreement with those described by Armstrong (1988) as regards the greater oxidative capacity of rat type IIa muscle fibres. With respect to glycolytic capacity, white gastrocnemius was the muscle with the highest PK and LDH activities, the soleus muscle showing the lowest values. The exercise protocol was chosen based on that described by Ardies et al. (1989), who reported increased rates of ethanol clearance despite the low intensity of training. Alcoholic myopathy mainly affects type IIb fibres. Several authors have attempted to correlate this selective atrophy with alterations in glycolytic metabolism. In this regard, Cook et al. (1992) indicated that the extensor digitorum longus (EDL) and soleus muscles of chronic alcohol-treated rats underwent serious alterations in glucose metabolism, especially linked
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metabolites but is rather a result of metabolite oxidation (Altura et al., 1990). According to the present results, alcohol affects muscles with the highest percentages of type IIb fibres since a reduction in the number of capillaries per fibre and also in capillary density in white gastrocnemius muscle was more prominent. The rest of the muscles did not show any changes due to ethanol administration. Altura et al. (1990) obtained similar results, relating the decrease in capillarity to alcoholic myopathy. Decreased capillary density seems to be attenuated by the effect of exercise, and is consistent with increased blood alcohol removal due to exercise (Ardies et al. 1989). Exercise reversed the ethanol-due damage in muscles with a higher percentage of type IIb fibers, such as white gastrocnemius muscle.
4.3. Fiber area The effects of alcohol on type I and type II fibre muscle protein content has been studied by several authors. Preedy et al. (1988) reported that ethanol ingestion by rats reduced muscle weight and protein DNA and RNA contents, mainly in muscles with type II fibres. When muscles with different type I and type II fibre contents were compared, the decrease in protein synthesis was more relevant in plantaris than soleus muscle and both ethanol and its metabolite — acetaldehyde — were seen to be responsible, although via independent mechanisms (Conde et al., 1992; Preedy et al., 1994). Our results are in agreement with those described above since alcohol administration decreased type II fibre area, except in plantaris muscle where approximately 30% each fibre subtype was reduced. The variation in fibre area due to the effect of alcohol has been studied by Langohr et al. (1983), who in vastus lateralis muscle biopsies obtained from alcoholics described a decrease in the diameter of type II fibres and an increase in the diameter of type I fibres. The authors related the muscular atrophy to alterations of glycolytic metabolism and neurogenic damage. Conde et al. (1992) associated the fibre atrophy produced in ethanol-administered rats with nutritional deficiencies, although malnutrition induces a decrease in the area of type I fibres that does not occur with alcohol. Amaladevi et al. (1995) observed that in muscles with mainly type I fibres, the repeated muscle contractions associated with physical exercise enhanced the effects of alcohol. Since the results obtained in the present study showed that the plantaris type IIb fibre area was reduced by 13% in the ethanol-trained group while a reduction of 25% was found in the ethanoltreated group, exercise can be said to decrease some of the harmful effects produced by ethanol in the muscle, including the decrease in capillary density.
Acknowledgements The authors thank the Anatomy Department of the School of Veterinary Sciences, University of Leon, for its invaluable technical assistance.
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Effect of chronic ethanol ingestion and exercise training on skeletal muscle in rat L. Vila, A. Ferrando, J Voces, C. Cabral de Oliveira, J.G. Prieto, A.I. Alvarez * Department of Physiology, The Uni6ersity of Leo´n, Leo´n, 24071, Spain Received 16 June 2000; received in revised form 27 October 2000; accepted 14 November 2000
Abstract The aim of this study was to investigate the interactive effects of exercise training and chronic ethanol consumption on metabolism, capillarity, and myofibrillar composition in rat limb muscles. Male Wistar rats were treated in separate groups as follows: non exercised-control; ethanol (15%) in animals’ drinking water for 12 weeks; exercise training in treadmill and ethanol administration plus exercise for 12 weeks. Ethanol administration decreased capillarity and increased piruvate kinase and lactate dehydrogenase activities in white gastrocnemius; in plantaris muscle, ethanol increased citrate synthase activity and decreased cross-sectional area of type I, IIa, and IIb fibres. Exercise increased capillarity in all four limb muscles and decreased type I fibre area in plantaris. The decreased capillarity effect induced by ethanol in some muscles, was ameliorated when alcohol was combined with exercise. While alcoholic myopathy affects predominantly type IIb fibres, ethanol administration and aerobic exercise in some cases can affect type I and type IIa fibre areas. The exercise can decrease some harmful effects produced by ethanol in the muscle, including the decrease in the fibre area and capillary density. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Exercise; Muscle metabolism; Fiber area; Fiber type; Capillarity; Rats
1. Introduction The action of alcohol on body systems is complex and the problems associated with its excessive and frequent consumption are numerous. Alcohol affects several organs, such as the brain, nerves, heart, digestive system, liver, skeletal muscle and blood, and produces metabolic alterations either directly or indirectly through the products formed in its biotransformation (Lieber, 1992). The study of alcohol consumption and exercise performance is normally restricted to the area of ‘drugs and exercise’, including substances that interfere with the normal development of exercise. The vitamin deficit elicited by alcohol and the accumulation of lactate to block uric acid secretion are the best studied aspects affecting physical activity (Lowenthal and Kendrick, 1985; Kendrick and Lowenthal, 1988).
* Corresponding author. Tel.: + 34-87-291263; fax: +34-87291267. E-mail address: [email protected] (A.I. Alvarez).
Alcohol consumption decreases the use of glucose and amino-acids by muscles. The hypoglycaemia thus generated can promote a failure in energy supply and an impairment in skeletal muscle metabolism during physical exercise. In fact, alcoholic myopathy is usually described in glycolytic muscles, in which protein turnover and glucose metabolism deficiencies associated with chronic alcohol consumption have been reported (Preedy et al., 1994). Glucose transport and uptake induced by physical exercise have been found to correlate with muscle GLUT-4 contents (Neufer et al., 1992). Under certain conditions exercise increases muscular protein synthesis, leading to muscle hypertrophy (Antonio and Gonyea, 1993; McComas, 1994). Siciliano et al. (2000) have shown that the accumulation of lactate during exercise is decreased after aerobic training in mitochondrial myopathies. With respect to the ethanol –exercise interaction, Ardies et al. (1987, 1989) reported that exercise may attenuate the ethanol-induced decline in hepatic mitochondria and that exercise performance accelerates ethanol metabolism by hepatic microsomes.
0376-8716/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 6 - 8 7 1 6 ( 0 0 ) 0 0 2 2 3 - 4
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Trudell et al. (1995) were the first to document a potential mechanism able to explain how repeated exercise prevents ethanol-induced fatty liver. Husain and Somani (1997a,b, 1998) showed that exercise training seems to reduce the extent of the oxidative damage caused by ethanol on the liver, myocardium, and testes of rats. Myopathy is related to impaired glycolytic metabolism and to restricted capillary density. The aim of this work was to study how the enhanced oxidative metabolism, the increase in capillarity and the increase in alcohol removal from blood can contribute to ameliorating alcohol myopathy. The metabolic responses of the organism to exercise and the generation of alcoholic myopathy have the same physiological consequences in rat and humans, although it is clear that the information obtained is specific to rat muscles and is not always applicable to human muscle. With this background, we addressed the effect of aerobic exercise on the skeletal muscle of ethanoltreated rats. The experimental design included study of muscle metabolism, capillary distribution, myofibrillar distribution and fibre morphometry.
2. Materials and methods
2.2. Training program The trained rats were run on a motor-driven treadmill 4 days per week for 12 weeks. The training session started out as a 30 min run at 20 m min − 1, 0% grade. Duration was increased by 5 min each 2 week until the animals were running for 1 h. Thereafter, running speed was increased by 1 m min − 1 every 2 wk and the grade was increased up to a final elevation of 15%. The final running speed obtained for the 60 min training period was 24 m min − 1. The ethanol treated-trained rats performed the program when they had started to drink 15% ethanol. To eliminate effects of the last bout of exercise, tissue samplings were performed 36–40 h after the final exercise session.
2.3. Tissue sampling Muscles were obtained after the animals had been anaesthetized intraperitoneally with urethane (30% w/v) at a dose of 1 ml/250 g body weight. The red, mixed and white gastrocnemius (predominantly fibre type I, IIa; IIa, IIb; and IIb, respectively), plantaris (predominantly fibre type IIa, IIb) and soleus (fibre type I) were surgically removed from the left and right hind limbs and the connective tissue trimmed away. All muscles were frozen in isopentane, cooled in liquid N2 and stored at − 80°C.
2.1. Animals and treatments 2.4. Determination of enzyme acti6ity Male Wistar rats obtained from IFFA Credo (Madrid, Spain), with a mean body weight of 1009 10 g were used for this study. The animals were housed three per cage, except the ethanol treated animals, which were housed individually to control daily ethanol consumption. The rats were housed in a temperaturecontrolled environment (2291°C) with a 12:12 h light –dark cycle The study was performed with four experimental groups of ten animals each. The animals were divided into control (Cont), ethanol-treated (Eth), exercised (Exer) and ethanol-treated+ exercise (Eth-Exer) groups. Alcohol administration was performed on one group of sedentary and one group of exercised rats. Ethanol was given to the rats for 12 weeks in their drinking water. The concentration of ethanol was gradually increased from 5 to 15% (v/v). Control rats had free access to water. All groups received commercial rat chow ad libitum (Panlab, Barcelona Spain). The animals were housed according to the Principles of Council Directive 86/609/EEC: ‘‘On the Approximation of Laws, Regulation and Administrative Provision of the Member States regarding the Protections of Animal used for Experimental and other Scientific Purposes’’.
Muscle from right hind limb was weighed and homogenized in a buffer (2 mM MgCl2, 2 mM EDTA, 50 mM Tris, pH 7.4) at a dilution of 1:40 (w/v). The homogenizing tube was immersed in ice-cold water and the tissue was subjected to a 1 min burst with a Potter–Elvehjem instrument. The medium was frozen at − 80°C until analysis. Spectrophotometric assays were performed on a HITACHI U-2000 spectrophotometer in 1 ml cuvettes, of 1 cm light path. Reaction rates were proportional to enzyme concentrations. One unit of enzymatic activity is defined as the amount of enzyme that catalyses the uptake of 1 mmole of substrate per minute. Citrate synthase (CS, EC 4.1.3.7) activity, a marker of the tricarboxylic acid cycle, was measured at 37°C by the method of Srere et al. (1963). The activity of hexokinase (HK, EC 2.7.1.1), an enzyme required for glucose uptake, was measured by the method of Bergmeyer (1974a). Pyruvate kinase (PK, EC 2.7.1.40) and lactate dehydrogenase (LDH, 1.1.1.27) activities, markers of the glycolysis, were measured at 37°C by the methods of Bergmeyer (1974b) and Kornberg (1955), respectively. The determination of b-hydroxyacetyl-CoA-dehydrogenase (HAD, EC 1.1.1.35) activity, a marker of b-oxidation, was performed at 30°C according the pro-
L. Vila et al. / Drug and Alcohol Dependence 64 (2001) 27–33
cedures described by Bradshaw and Noyes (1975). All values are expressed in mmol g − 1 wet muscle min − 1.
Table 1 Mean 9 SD values of body weight and liver (g)
2.5. Histochemistry The soleus, plantaris and gastrocnemius from left hind limb were cut in 12 mm sections in a cryostat maintained at −20°C and mounted on glass coverslips. In order to determine the correct sample orientation, a rapid stain with hematoxilin-eosin was performed. Serial sections were stained for histochemical demonstration of myosin ATPase after acid preincubation, based on the method originally described by Padykula and Herman (1955) and Guth and Samaha (1970). This stain identifies three fibre types according to assessment of the contractile properties of the muscle. Capillaries were determined from ATPase stains preincubated at pH 4 (Fouces et al., 1993). Fibre typing and field measurements were obtained using a light microscope equipped with a camera (Optiphot-2 Nikon, Tokyo, Japan). Photomicrographs were taken at a magnification of 200× and fibre measurements were carried out by means of a digitising tablet (Genitizer-GT-1212B) connected to a PC, using suitable software (Sigma-Scan, Jandel Scientific, Erkrath, Germany). Photographs of a stage micrometer were taken at the beginning and at the end of each film as a calibration control (Fouces et al., 1993). A minimum of 200 fibres per muscle for each animal was studied, following the techniques of Blomstrang and Ekblom, (1982) and Shorey and Cleland, (1983). The number of capillaries per square millimetre (cap mm − 2) and the capillary/fibre ratio (capillaries per fibre) were determined from an area of 0.16 mm2, four fields per sample being measured.
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Control Ethanol Exercise Ethanol–Exercise
Total
Liver
416 9 37 395 9 35 383 9 55 336 9 32a
32.3 93.9 33.3 91.5 30.4 9 3.1 32.99 1.8
a
Significantly different from control rats (PB0.05). n =10 animals per group.
(body weight) in the trained animals. Body and liver weights are shown in Table 1, with no significant differences among the groups; only the final weight of the ethanol-trained rats was statistically different from that of the controls (3369 22 vs. 4169 37 g).
3.1. Muscle enzymes Fig. 1 and Fig. 2 show the muscle enzyme activities in the control, trained, alcohol-treated and trained alcohol-treated rats. The data obtained for CS activity as a marker of oxidative metabolism revealed a greater oxidative trend in red gastrocnemius, followed by plantaris muscle (both red gastrocnemius and plantaris muscles having
2.6. Statistical analysis One-way analysis of variance was performed on all variables. The significance of the difference between the means obtained was determined using the Newman– Keuls test. A level of P B0.05 was set for significance for all tests, and all values are expressed as means9 S.D.
3. Results Because of a possible failure in inducing ethanol toxicity by chronic administration and its consequences on the nutritional status of the animals, the animals’ weights and daily ethanol consumptions were recorded during the experimental period. During week 4 of treatment and onwards, the average daily consumption of ethanol was 10.69 1.8 g (ethanol) kg − 1 (body weight) in the untrained rats, and 10.191.6 g (ethanol) kg − 1
Fig. 1. The activities of the tricarboxylic acid cycle enzyme citrate synthase and the b– hydroxyacetyl-CoA dehydrogenase determined from hindlimb muscles of control (Cont), ethanol (Eth), exercise (Exer) and ethanol-exercise (Eth +Exer) groups. * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (PB 0.05).
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red gastrocnemius and soleus than in white gastrocnemius (3.009 0.13 vs. 1.8990.25 mmol g − 1 min − 1).
3.2. Capillarity and fibre area
Fig. 2. The activities of the hexokinase, piruvate kinase and lactate dehydrogenase determined from hindlimb muscles of control (Cont), ethanol (Eth), exercise (Exer) and ethanol-exercise (Eth +Exer) groups. * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (PB 0.05). ‡ Significantly different from exercise group (PB 0.05).
the highest percentages of IIa fibres), and soleus, the lowest activity being found in white gastrocnemius. HAD activity was highest in soleus and red gastrocnemius, including mainly type I fibers (Fig. 1). As regards glycolytic enzymes, HK was slightly increased after exercise; PK and LDH showed the highest values in muscles with type IIb fibers, such as white gastrocnemius and plantaris (Fig. 2). The present results showed that oxidative enzyme activities were unaffected by ethanol except in plantaris muscle, where CS activity increased significantly from 27.591.8 in the controls to 36.792.7 mmol g wet muscle − 1 min − 1 in the alcohol-treated rats. Alcohol administration modified the glycolytic metabolism in muscles with a higher percentage in type IIb fibers, such as white gastrocnemius, where PK and LDH activities were significantly increased. LDH activity was reduced in red gastrocnemius and plantaris, although this effect was largely a consequence of the exercise performed. HK activity, which is related to exogenous glucose utilization, was significantly higher (P B 0.05) in the ethanol-trained group in oxidative muscles such as
The results concerning capillary distribution are shown in Table 2. The most relevant finding after ethanol administration was a significant reduction in white gastrocnemius muscle of both the number of capillaries per fibre (1.269 0.1 to 0.979 0.06) and capillary density (2459 25 to 2129 26 capillaries mm − 2). The remaining muscles showed no changes following ethanol administration. Exercise significantly increased capillarity in all muscles. The effects on capillarity of the action of both ethanol and exercise treatments were that exercise reversed the effect of alcohol in muscles with greater percentages of type IIb fibres. Thus, in white gastrocnemius the number of capillaries per fibre and capillary density were increased with respect to the group that received alcohol (1.109 0.08 vs. 0.979 0.06 capillaries per fibre). In red gastrocnemius and soleus muscles, the most important effect was elicited by exercise and both muscles showed increased capillary distribution, showing no effects associated with alcohol administration. In plantaris muscle, also affected by alcohol but to a lesser extent, the effect of exercise on the increase in capillarity was less pronounced than in white gastrocnemius. The fibrillar area was decreased by alcohol in type I and type II fibbers as compared to the control group (data not shown) but the decrease was not statistically significant except in plantaris muscle, for which alcohol administration decreased the area in all
Table 2 Capillarity of skeletal muscle of ethanol treated, training and ethanol treated-exercised rats Control
Ethanol
Exercise
Ethanol – Exercise
Plantaris Capillaries per fiber 1.72 9 0.17 Capillaries per mm2 477 9 35
1.58 9 0.12 504 9 19
1.95 9 0.07*a 1.59 90.08 575 9 46* 536 9 22
G. White Capillaries per fiber 1.26 9 0.1 Capillaries per mm2 245 9 25
0.97 9 0.06* 1.45 9 0.14* 212 9 26* 273 9 21
1.10 9 0.08† 219 921
Soleus Capillaries per fiber 1.67 9 0.16 Capillaries per mm2 380 9 21
1.97 9 0.23 431 9 34
2.24 9 0.17* 553 9 30*
2.21 90.26* 583 9 47*
G. Red Capillaries per fiber 2.08 9 0.15 1.92 9 0.12 Capillaries per mm2 650 9 68 581 9 30
2.38 9 0.11* 673 9 70
2.40 9 0.13*† 727 9 59
a *Significantly different from control rats (PB0.05). †Significantly different from ethanol group (PB0.05).
L. Vila et al. / Drug and Alcohol Dependence 64 (2001) 27–33
Fig. 3. Effect of alcohol administration, exercise and alcohol administration plus exercise in the area of type I, type IIa and type IIb fibres of plantaris (a) and in type I fibres of soleus, red and mixed gastrocnemius (b). * Significantly different from control rats (PB 0.05). † Significantly different from ethanol group (P B0.05). ‡ Significantly different from exercise group (P B0.05).
three fibre types. This decrease persisted in the exercised group (Fig. 3a). Type I fibre area was decreased in the Eth + Exer group only in mixed gastrocnemius muscle (Fig. 3b).
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to the glycogen synthesis. Spolarics et al. (1994) showed that acute ethanol administration inhibits glucose uptake by 50% in rat skeletal muscles (soleus, gastrocnemius and red quadriceps). This inhibition even appeared in hyperinsulinaemic animals, in which the basal glucose level is doubled. Chronic alcohol consumption reduces enzyme activities such as LDH, HK and PK in the rat vastus lateralis (Trounce et al., 1990). From our results no inhibition of glucose uptake can be inferred. One plausible explanation could be the low level of alcoholization in our experiment, since the values for rat serum transaminases (ALT and AST) remained unmodified. Alcohol intake coupled with physical exercise lowers muscle glucose (Juhlin-Dannfelt et al., 1977). By contrast, Shelmet et al. (1988) reported the existence of an acute peripheral insulin resistance after alcohol ingestion, which would reduce glucose oxidation. However, these variations occur in the short term and are attenuated and disappear with prolonged treatments (Cook et al., 1992). It has previously been described that alcohol administration markedly inhibits glucose use by skeletal muscles (Spolarics et al., 1994) but hexokinase activity is induced in ethanol-exercised animals in all muscles studied except plantaris. Exercise increases muscle glucose uptake as a result of an increase in muscle transporter concentration (GLUT-4) (Hargreaves, 1995), this effect being mediated by hormones and by muscular contraction (Yeh et al., 1995). Increasing calcium levels during muscle contraction induces the activation of glucose uptake (Youn et al., 1991).
4. Discussion
4.2. Capillarity distribution
4.1. Enzyme acti6ities
It is generally assumed that capillarity is related to muscle oxidative capacity. The present results also show a correlation between capillarity and citrate synthase activity, the highest values being seen for red gastrocnemius muscle, followed by plantaris, soleus, and white gastrocnemius. This correlation has also been indicated by Tesch and Karlsson (1985). Training increases capillary distribution in exercised muscles. The outcome of capillary development is to allow a greater surface for distribution and to increase the efficiency of respiratory gases, metabolites and substrate exchange (Saltin et al., 1986). Although studies on the effect of ethanol in skeletal muscle capillarity are scarce, it has been established that acute alcohol ingestion produces peripheral tissue vasodilation (Friedman et al., 1982). However, chronic ingestion of alcohol induces a reduction in the vascular lumen, which may be related to the development of alcoholic myopathy. This effect is not mediated by
The results concerning rat oxidative and glycolytic metabolism are in agreement with those described by Armstrong (1988) as regards the greater oxidative capacity of rat type IIa muscle fibres. With respect to glycolytic capacity, white gastrocnemius was the muscle with the highest PK and LDH activities, the soleus muscle showing the lowest values. The exercise protocol was chosen based on that described by Ardies et al. (1989), who reported increased rates of ethanol clearance despite the low intensity of training. Alcoholic myopathy mainly affects type IIb fibres. Several authors have attempted to correlate this selective atrophy with alterations in glycolytic metabolism. In this regard, Cook et al. (1992) indicated that the extensor digitorum longus (EDL) and soleus muscles of chronic alcohol-treated rats underwent serious alterations in glucose metabolism, especially linked
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metabolites but is rather a result of metabolite oxidation (Altura et al., 1990). According to the present results, alcohol affects muscles with the highest percentages of type IIb fibres since a reduction in the number of capillaries per fibre and also in capillary density in white gastrocnemius muscle was more prominent. The rest of the muscles did not show any changes due to ethanol administration. Altura et al. (1990) obtained similar results, relating the decrease in capillarity to alcoholic myopathy. Decreased capillary density seems to be attenuated by the effect of exercise, and is consistent with increased blood alcohol removal due to exercise (Ardies et al. 1989). Exercise reversed the ethanol-due damage in muscles with a higher percentage of type IIb fibers, such as white gastrocnemius muscle.
4.3. Fiber area The effects of alcohol on type I and type II fibre muscle protein content has been studied by several authors. Preedy et al. (1988) reported that ethanol ingestion by rats reduced muscle weight and protein DNA and RNA contents, mainly in muscles with type II fibres. When muscles with different type I and type II fibre contents were compared, the decrease in protein synthesis was more relevant in plantaris than soleus muscle and both ethanol and its metabolite — acetaldehyde — were seen to be responsible, although via independent mechanisms (Conde et al., 1992; Preedy et al., 1994). Our results are in agreement with those described above since alcohol administration decreased type II fibre area, except in plantaris muscle where approximately 30% each fibre subtype was reduced. The variation in fibre area due to the effect of alcohol has been studied by Langohr et al. (1983), who in vastus lateralis muscle biopsies obtained from alcoholics described a decrease in the diameter of type II fibres and an increase in the diameter of type I fibres. The authors related the muscular atrophy to alterations of glycolytic metabolism and neurogenic damage. Conde et al. (1992) associated the fibre atrophy produced in ethanol-administered rats with nutritional deficiencies, although malnutrition induces a decrease in the area of type I fibres that does not occur with alcohol. Amaladevi et al. (1995) observed that in muscles with mainly type I fibres, the repeated muscle contractions associated with physical exercise enhanced the effects of alcohol. Since the results obtained in the present study showed that the plantaris type IIb fibre area was reduced by 13% in the ethanol-trained group while a reduction of 25% was found in the ethanoltreated group, exercise can be said to decrease some of the harmful effects produced by ethanol in the muscle, including the decrease in capillary density.
Acknowledgements The authors thank the Anatomy Department of the School of Veterinary Sciences, University of Leon, for its invaluable technical assistance.
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