Malate-aspartate and alpha-glycerophosphate shuttle enzyme levels in human skeletal muscle: methodological considerations and effect of endurance training

The aim of the present study was to investigate whether the levels of the malate-aspartate and alpha-glycerophosphate shuttle enzymes in human skeletal muscle are affected by endurance training. The approach used was to compare six untrained and six endurance-trained subjects as well as through performing a longitudinal study of endurance training on eight untrained subjects. Biopsy samples were obtained from the lateral part of the quadriceps femoris muscle. The trained muscles were characterized by higher levels of oxidative enzymes (55%) as well as enhanced capillary supply (30%). In both the cross-sectional and longitudinal studies, the malate-aspartate shuttle enzyme levels were about 50% higher in the trained state (cytoplasmic malate dehydrogenase 36%, mitochondrial malate dehydrogenase 46%, cytoplasmic aspartate aminotransferase 52% and mitochondrial aspartate aminotransferase 48%). Contrary to this, the alpha-glycerophosphate shuttle enzyme levels did not differ significantly (cytoplasmic and mitochondrial glycerol-3-phosphate dehydrogenase: 10 and -4%, respectively). The activity ratios of the enzymes involved in respective shuttle did not differ significantly between the untrained and endurance-trained states. It is concluded that endurance training may induce increased levels of malate-aspartate shuttle enzymes in human skeletal muscle while the levels of the alpha-glycerophosphate shuttle enzymes are not affected. The study also includes results from several methodological experiments.     

Skeletal muscle of trained and untrained paraplegics and tetraplegics

The effect of physical conditioning on skeletal muscle of individuals with spinal cord injuries (SCI) has been investigated. The anterior portion of the deltoid muscle (active in wheel-chair propulsion) of untrained and endurance-trained paraplegics and tetraplegics, as well as that of untrained able-bodied subjects, was studied. The characterization involved fibre type distribution, capillarization, fibre areas and also oxidative and glycolytic enzyme levels. A general trend towards a successively higher proportion of type I fibres and lower proportion of type IIB fibres was noted in the order of able-bodied subjects (type I, 42%; type IIB, 41%, n=8), paraplegics (type I, 57%; type IIB, 13%, n=13) and tetraplegics (type I, 74%; type IIB, 4.5%, n=11). The trained SCI groups had significantly higher levels of the citric acid cycle marker enzyme citrate synthase (34% and 63%) than the untrained SCI groups and able-bodied subjects, respectively. The glycolytic marker enzyme 6-phosphofructokinase was 32% lower in the tetraplegic groups than in the other groups. In contrast, the fatty acid oxidation marker enzyme 3-hydroxyacyl-CoA dehydrogenase was markedly higher in the tetraplegic group than in the able-bodied subjects (58%) and tended to be higher (21%, P<0.1) than in the paraplegic group. The trained SCI groups displayed significantly higher (28%) levels of capillaries per fibre than the untrained SCI groups, which had about the same levels as the untrained able-bodied subjects. It is concluded that several of the findings are in line with normal muscular adaptation, whereas others are unexpected and support a hypothesis that some of the findings might be due to differences between the groups in, for instance, hormone levels or in types of muscular load.     

Enzyme levels in pools of microdissected human muscle fibres of identified type. Adaptive response to exercise

Enzyme activities were determined in pools of type I (slow twitch) and II A and II B (fast twitch) fibres of the thigh muscle from individuals engaged to a high degree in physical training of an endurance character and from non-endurance-trained controls. The endurance-trained (ET) group had significantly higher activity levels of the mitochondrial enzymes citrate synthase, malate dehydrogenase, and 3-OH-acylCoA dehydrogenase both in type I (2.1X, 1.7X, 1.4X) and in type II A (2.3X, 1.8X, 1.4X) and II B fibres (2.0X, 1.5X, 1.5X) than the non-endurance-trained (NET) group. Of the glycolytic enzymes, phosphofructokinase (PFK) in type I fibres was significantly higher (1.8X) in the ET than in the NET group whereas glyceraldehydephosphate dehydrogenase (GAPDH) in type I fibres was similar in the two groups. In type II fibres both PFK and GAPDH levels tended to be higher in the ET group. Lactate dehydrogenase (LDH) of both fibre types were not different in the two groups. Type I fibres differed significantly from type II fibres for all the six enzymes measured in both groups. However, no significant difference between fibres of types II A and II B was found. The results indicate that fibres of types I, II A and II B in human skeletal muscle all possess great adaptability with regard to their oxidative capacity. Furthermore, the data suggest that extensive endurance training may enhance the glycolytic capacity in both type I and type II fibres although the glycolytic capacity of the muscle as a whole generally is low in endurance trained subjects owing to a predominance of type I fibres. It is concluded that further studies are needed to determine whether there is a metabolic distinction between fibres of types II A and II B.     

Enzyme levels in pools of microdissected human muscle fibres of identified type: Adaptive response to exercise

Enzyme activities were determined in pools of type I (slow twitch) and IIA and II B (fast twitch) fibres of the thigh muscle from individuals engaged to a high degree in physical training of an endurance character and from non-endurance-trained controls. The endurance-trained (ET) group had significantly higher activity levels of the mitochondrial enzymes citrate synthase, malate dehydrogenase, and 3-OH-acylCoA dehydrogenase both in type I (2.1×, 1.7×, 1.4×) and in type IIA (2.3×, 1.8×, 1.4×) and IIB fibres (2.0×, 1.5 ×, 1.5×) than the non-endurance-trained (NET) group. Of the glycolytic enzymes, phosphofructokinase (PFK) in type I fibres was significantly higher (I.8×) in the ET than in the NET group whereas glyceraldehydephosphate dehydrogenase (GAPDH) in type I fibres was similar in the two groups. In type II fibres both PFK and GAPDH levels tended to be higher in the ET group. Lactate dehydrogenase (LDH) of both fibre types were not different in the two groups. Type 1 fibres differed significantly from type II fibres for all the six enzymes measured in both groups. However, no significant difference between fibres of types IIA and IIB was found. The results indicate that fibres of types I, IIA and IIB in human skeletal muscle all possess great adaptability with regard to their oxidative capacity. Furthermore, the data suggest that extensive endurance training may enhance the glycolytic capacity in both type I and type II fibres although the glycolytic capacity of the muscle as a whole generally is low in endurance trained subjects owing to a predominance of type I fibres. It is concluded that further studies are needed to determine whether there is a metabolic distinction between fibres of types IIA and IIB.     

Enzyme activities in type I and II muscle fibres of human skeletal muscle in relation to age and torque development

The quadriceps muscles from 20- 30- and 70-year-old clinically healthy men and women were studied regarding maximal isometric and isokinetic muscle torque in Newton metres (Nm), morphology and enzyme activity. Biopsy specimens were taken from the vastus lateralis muscle and freeze-dried, and individual fibres were dissected out and identified as type I or type II. The activities of citrate synthase (CS), 3-OHacyl-coA dehydrogenase (HAD), lactate dehydrogenase (LDH), myokinase (MK) and creatine phosphokinase (CPK) were determined in pools of type I and type II fibres. In both age groups a higher oxidative (CS, HAD, 1.3-1.5 x) and a lower glycolytic (LDH, 0.7 x) capacity was found in type I than in type II fibres. The myokinase activity was higher in type II (2 x) than in type I, whereas CPK activity was similar. The young men showed higher CS activity in both type I and type II fibres (1.5 x) and higher CPK activity in type I fibres (1.4 x) than the young women. There were only minor changes in oxidative or glycolytic capacities in relation to age. Myokinase was the only enzyme that decreased markedly with age in both pools of fibre types. Type II fibre area and mean fibre area correlated significantly to muscle torque in both sexes. In men, myokinase activity in type II fibres was significantly correlated to type II fibre area and to maximal muscle torque.     

Muscle Adaptation to Extreme Endurance Training in Man

To evaluate the effect of extreme endurance training on muscle fibre composition and activities of oxidative enzymes in different fibre types biopsies were taken from vastus lateralis, gastrocnemius and deltoideus of elite orienteers. Comparisons were made between the (trained) leg muscles and the (relatively untrained) arm muscles, and with leg muscles of 16–18 years old boys. The orienteers had the same percentage type I fibres in vastus lateralis and gastrocnemius as in deltoideus, but higher percentage type I fibres in vastus lateralis compared with the controls. The similarity between trained and untrained muscle in the orienteers suggests that training had not caused the high percentage type I fibres which rather might be the result of selection of individuals with the best prerequisites for high oxidative capacity. However, the distribution of type II subgroups in the leg muscles of the orienteers differed from both their own deltoideus and leg muscles of the controls, the relationship IIA/IIB being altered in favour of the more oxidative IIA. The leg muscles of the orienteers also showed an increased occurrence of the normally rare IIC fibre. These latter findings point at the possibility of a training induced alteration in the subgroup pattern. Unlike in the controls there was no significant difference in succinate dehydrogenase activity, measured in single fibres, between type I and II fibres in gastrocnemius of the orienteers. Thus, type II fibres have the ability metabolically to adapt to high oxidative demands. This might to some extent be mediated by a conversion from IIB to IIA form.     

Muscle adaptation to extreme endurance training in man.

To evaluate the effect of extreme endurance training on muscle fibre composition and activities of oxidative enzymes in different fibre types biopsies were taken from vastus lateralis, gastrocnemius and deltoideus of elite orienteers. Comparisons were made between the (trained) leg muscles and the (relatively untrained) arm muscles, and with leg muscles of 16--18 years old boys. The orienteers had the same percentage type I fibres and vastus lateralis and gastrocnemius as in deltoideus, but higher percentage type I fibres in vastus lateralis compared with the controls. The similarity between trained and untrained muscle in the orienteers suggests that training had not caused the high percentage type I fibres which rather might be the result of selection of individuals with the best prerequisites for high oxidative capacity. However, the distribution of type II subgroups in the leg muscles of the orienteers differed from both their own deltoideus and leg muscles of the controls, the relationship IIA/IIB being altered in favour of the more oxidative IIA. The leg muscles of the orienteers also showed an increased occurrence of the normally IIC fibre. These latter findings point at the possibility of a training induced alteration in the subgroup pattern. Unlike in the controls there was no significant difference in succinate dehydrogenase activity, measured in single fibres, between type I and II fibres in gastrocnemius of the orienteers. Thus, type II fibres have the ability metabolically to adapt to high oxidative demands. This might to some extent be mediated by a conversion from IIB to IIA form.     

Age and sex affect human muscle fibre adaptations to heavy-resistance strength training

This study assessed age and sex effects on muscle fibre adaptations to heavy-resistance strength training (ST). Twenty-two young men and women (20-30 years old) and 18 older men and women (65-75 years old) completed 9 weeks of heavy-resistance knee extension exercises with the dominant leg 3 days week(-1); the non-dominant leg served as a within-subject, untrained control. Bilateral vastus lateralis muscle biopsies were obtained before and after ST for analysis of type I, IIa and IIx muscle fibre cross-sectional area (CSA) and fibre type distribution. One-repetition maximum (1-RM) strength was also assessed before and after ST. ST resulted in increased CSA of type I, IIa and IIx muscle fibres in the trained leg of young men, type I and IIa fibres in young women, type IIa fibres in older men, and type IIx fibres in older women (all P<0.05). Analysis of fibre type distribution revealed a significant increase in the percentage of type I fibres (P<0.05) along with a decrease in type IIx fibres (P=0.054) after ST only in young women. There were no significant changes in muscle fibre CSA or fibre type distribution in the untrained leg for any group. All groups displayed significant increases in 1-RM (27-39%; all P<0.01). In summary, ST led to significant increases in 1-RM and type II fibre CSA in all groups; however, age and sex influence specific muscle fibre subtype responses to ST.     

Histochemical and biochemical changes in human skeletal muscle with age in sedentary males, age 22--65 years.

Biopsies for histochemical and biochemical analyses were taken from the vastus lateralis muscle of 55 untrained, healthy male subjects from 22 to 65 years of age. Fibre type distribution changed towards a decrease in the percentage of type II fibres, both in type IIA and type IIB fibres, whereas type IIB/IIA fibre ratio and type IIC percentage did not change with increasing age. It was found that the type IIB/IIA fibre ratio was inversely related to type I fibres, i.e. subjects rich in type I fibres had a relatively smaller proportion of type IIB fibres. Fibre area determinations revealed a selective decrease in type II fibre area. Consequently, the type II/I fibre area ratio and relative type II fibre area decreased. No changes in the specific activities of Mg2+ stimulated ATPase and myokinase were observed, while the activity of lactate dehydrogenase (LDH) was higher in the youngest groups than in the oldest. LDH isozyme pattern shifted towards a decrease in percentage distribution of the muscle specific isozymes and a corresponding decrease in muscle specific activity while the activity of the heart specific isozymes did not change.     

Biochemical characterization of stage-specific isoforms of aspartate aminotransferases from Trypanosoma cruzi and Trypanosoma brucei

Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Trypanosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T. cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T. brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T. brucei ASATs were very similar to those determined for T. cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T. cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T. cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T. cruzi, and suggest that the succinate excreted by amastigotes more likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism.     

Histochemical and biochemical changes in human skeletal muscle with age in sedentary males,age 22–65 years

Biopsies for histochemical and biochemical analyses were taken from the vastus lateralis muscle of 55 untrained, healthy male subjects from 22 to 65 years of age. Fibre type distribution changed towards a decrease in the percentage of type II fibres, both in type IIA and type IIB fibres, whereas type IIB/IIA fibre ratio and type IIC percentage did not change with increasing age. It was found that the type IIB/flA fibre ratio was inversely related to type I fibres, i.e. subjects rich in type I fibres had a relatively smaller proportion of type IIB fibres. Fibre area determinations revealed a selective decrease in type II fibre area. Consequently, the type II/I fibre area ratio and relative type II fibre area decreased. No changes in the specific activities of Mg²⁺ stimulated ATPase and myokinase were observed, while the activity of lactate dehydrogenase (LDH) was higher in the youngest groups than in the oldest. LDH isozyme pattern shifted towards a decrease in percentage distribution of the muscle specific isozymes and a corresponding decrease in muscle specific activity while the activity of the heart specific isozymes did not change.     

Creatine kinase MB and citrate synthase in type I and type II muscle fibres in trained and untrained men

Summary Total creatine kinase (CK), creatine kinase MB (CK-MB) and citrate synthase (CS) were determined in isolated and pooled type I and type II skeletal muscle fibres. Determinations were made on biopsies from 3 sedentary men, 3 junior cyclists and 2 elite cyclists. CS and CK-MB activities were higher in the trained groups in both fibre types. The total CK activity was not related to training status, although it was lower in type I fibres than in type II fibres (p<0.05). The reverse relation was observed for CS and CK-MB activities (p<0.01). The ratio of type I/type II for CS was not related to training status, while the corresponding ratio for CK-MB increased with a greater degree of endurance training. For a given increase in CS activity, the increase in CK-MB activity was greater in type I fibres than in type II fibres (p<0.01). Thus, with endurance training there seems to be a specific adaptation for CK-MB, particularly in type I fibres.     

Effect of exercise on concentrations of free amino acids in pools of type I and type II fibres in human muscle with reduced glycogen stores

A few animal studies have shown that some amino acid concentrations vary between different muscle fibre types. In the present study, amino acid concentrations were measured in separate pools of different fibre types in human skeletal muscle, with reduced glycogen stores, before and after sustained exercise. Five subjects exercised at a submaximal work rate for 60 min and then at a maximal rate for 20 min. Biopsy samples were taken from the vastus lateralis muscle before and after exercise; they were freeze-dried and individual fibres were dissected out. Fragments of these fibres were stained for myosin-adenosine triphosphatase (ATPase) and identified as type I or type II fibres. The concentrations of free amino acids were measured by high performance liquid chromatography (HPLC) in perchloric acid (PCA) extracts containing pools of either type of fibre. After exercise, glycogen was decreased in type I fibres (53%) and in four subjects also in type II fibres. The concentrations of most amino acids were similar in the two fibre types before exercise, but the glutamate, aspartate and arginine levels were 10% higher in type II than in type I fibres. After exercise, the glutamate concentration was decreased by 45% in both fibre types and the branched-chain amino acids (BCAA) were decreased in type II fibres (14%). Exercise caused an increase by 25-30% in tyrosine concentration in both type I and type II fibres. The results show that amino acids can be measured in pools of fibre fragments and suggest that amino acid metabolism play an important role in both type I and type II fibres during exercise.     

UCP3 Protein Regulation in Human Skeletal Muscle Fibre Types I, IIa and IIx is Dependent on Exercise Intensity

It has been proposed that mitochondrial uncoupling protein 3 (UCP3) behaves as an uncoupler of oxidative phosphorylation. In a cross-sectional study, UCP3 protein levels were found to be lower in all fibre types of endurance-trained cyclists as compared to healthy controls. This decrease was greatest in the type I oxidative fibres, and it was hypothesised that this may be due to the preferential recruitment of these fibres during endurance training. To test this hypothesis, we compared the effects of 6 weeks of endurance (ETr) and sprint (STr) running training on UCP3 mRNA expression and fibre-type protein content using real-time PCR and immunofluorescence techniques, respectively. UCP3 mRNA and protein levels were downregulated similarly in ETr and STr (UCP3 mRNA: by 65 and 50 %, respectively; protein: by 30 and 27 %, respectively). ETr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 54, 29 and 16 %, respectively. STr significantly reduced UCP3 protein content in type I, IIa and IIx muscle fibres by 24, 31 and 26 %, respectively. The fibre-type reductions in UCP3 due to ETr, but not STr, were significantly different from each other, with the effect being greater in type I than in type IIa, and in type IIa than in type IIx fibres. As a result, compared to STr, ETr reduced UCP3 expression significantly more in fibre type I and significantly less in fibre types IIx. This suggests that the more a fibre is recruited, the more it adapts to training by a decrease in its UCP3 expression. In addition, the more a fibre type depends on fatty acid β oxidation and oxidative phosphorylation, the more it responds to ETr by a decrease in its UCP3 content.     

Cycling efficiency in humans is related to low UCP3 content and to type I fibres but not to mitochondrial efficiency

The purpose of this study was to investigate the hypothesis that cycling efficiency in vivo is related to mitochondrial efficiency measured in vitro and to investigate the effect of training status on these parameters. Nine endurance trained and nine untrained male subjects (     , respectively) completed an incremental submaximal efficiency test for determination of cycling efficiency (gross efficiency, work efficiency (WE) and delta efficiency). Muscle biopsies were taken from m. vastus lateralis and analysed for mitochondrial respiration, mitochondrial efficiency (MEff; i.e. P/O ratio), UCP3 protein content and fibre type composition (% MHC I). MEff was determined in isolated mitochondria during maximal (state 3) and submaximal (constant rate of ADP infusion) rates of respiration with pyruvate. The rates of mitochondrial respiration and oxidative phosphorylation per muscle mass were about 40% higher in trained subjects but were not different when expressed per unit citrate synthase (CS) activity (a marker of mitochondrial density). Training status had no influence on WE (trained 28.0 ± 0.5, untrained 27.7 ± 0.8%, N.S.). Muscle UCP3 was 52% higher in untrained subjects, when expressed per muscle mass ( P < 0.05 versus trained). WE was inversely correlated to UCP3 ( r =−0.57, P < 0.05) and positively correlated to percentage MHC I ( r = 0.58, P < 0.05). MEff was lower ( P < 0.05) at submaximal respiration rates (2.39 ± 0.01 at 50%     ) than at state 3 (2.48 ± 0.01) but was neither influenced by training status nor correlated to cycling efficiency. In conclusion cycling efficiency was not influenced by training status and not correlated to MEff, but was related to type I fibres and inversely related to UCP3 . The inverse correlation between WE and UCP3 indicates that extrinsic factors may influence UCP3 activity and thus MEff in vivo .     

Differences of morphometrical parameters in hind limb muscle fibres between ovarectomized and sexually intact female dogs.

Slow and fast twitch fibres of the Mm. tibialis cranialis, semitendinosus and sartorius of seven sexually intact and seven ovarectomized female beagles were histochemically and morphometrically analysed. Along with type I and type IIA fibres, another main type II fibre (IIS), which seems to be peculiar to the dog, was found in the Mm. semitendinosus and tibialis cranialis. Type I fibers comprised 26% and type II fibres 74% of all recorded muscle fibres in the M. tibialis cranialis, 29% (type I) and 71% (type II) in the M. semitendinosus and 51% (type I) and 49% (type II) in the M. sartorius, respectively. The average single profile area and the corresponding mean diameter of fibre types I and II in the investigated hind limb muscles were generally larger in ovarectomized than in sexually intact animals. This was more evident in type II than in type I fibres. However only the type II fibres of the M. tibialis cranialis and sartorius exhibited a statistically significant increase in diameter (p < 0.01 and p < 0.05, respectively). Accordingly, the mean density (number of fibres/mm2) of both fibre types in the hind limb muscles of spayed dogs was generally reduced. Again, this reduction attained statistical relevance in the type I and II fibres of the tibialis cranialis. In addition, the fibre densities of type I in the semitendinosus and type II in the sartorius muscles were also significantly reduced in ovarectomized dogs. In conclusion, ovarectomized beagles showed a generally increased mean diameter of the investigated type I and II hind limb muscle fibres and a concomitant decreased average fibre density of the respective types when compared to sexually intact animals.     

The relationship between anaerobic performance and muscle metabolic capacity and fibre distribution

Summary Relationships between functional anaerobic indicators and the character of cellular muscle energy metabolism were studied. Twelve untrained male students were tested by a specific anaerobic test on the treadmill. The mean values of the anaerobic test were as follows: blood lactate 10.69 mmol · 1⁻¹, running speed 16.08 km · h⁻¹ and duration 92.67 s. The average distribution of muscle fibres (m. vastus lateralis) was: type I 52.2%, type II A 29.0% and type II B 18.8%. The mean enzyme activity values were: triosephosphate dehydrogenase (TPDH) 4.67 Μkat · g⁻¹ w.w., lactate dehydrogenase (LDH) 5.76 Μkat · g⁻¹ w.w, citrate synthase (CS) 0.21 Μkat · g⁻¹ w.w. and hydroxyacyl-CoA dehydrogenase (HAD) 0.12 Μkat · g⁻¹ w.w. Significant negative correlations were found between δLA and CS (r=0.64) and % of fibre type II B and CS (r=0.78) and positive correlations between % of fibre type I and CS and/or HAD (r=0.60 andr=0.62, respectively).     

Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of ‘pure’ I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n=1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in numerous previous publications based on the use of histochemistry alone. This has important implications in attempts to study the physiological properties of fast fibre types adequately in horses.     

The GLUT4 density in slow fibres is not increased in athletes. How does training increase the GLUT4 pool originating from slow fibres?

The influence of training on GLUT4 expression in slow- and fast-twitch skeletal muscle fibres was studied in male endurance-trained athletes and control subjects. The trained state was ensured by elevated maximal oxygen uptake (29%), as well as citrate synthase (60%) and 3-hydroxy-acyl-CoA dehydrogenase (38%) activities in muscle biopsy samples of the vastus lateralis. GLUT4 densities in slow- and fast-twitch fibres were measured by the use of a newly developed, sensitive method combining immunohistochemistry with morphometry, and no effect of training was found. GLUT4 density was higher in slow-twitch fibres compared to fast-twitch fibres (P<0.05) when biopsy samples from untrained subjects were examined. In athletes GLUT4 density was identical in slow- and fast-twitch fibres. Slow-twitch fibre diameters were 10% larger in the athletes (P<0.01), and slow-twitch fibre fractions were 140% of the fraction in the control group. Thus, GLUT4 originating from slow-twitch fibres was increased by 30% (P<0.02) in athletes. We conclude that long-lasting endurance training increases slow-twitch fibre GLUT4 expression by means of an elevated slow-twitch fibre mass in human skeletal muscle.