dehydrogenase increased with increased training. Values of maximum rate of 02

Transcription

1 J. Physiol. (1986), 381, pp. l With 1 text-figure Printed in Great Britain MAXIMUM ACTIVITIES OF KEY GLYCOLYTIC AND OXIDATIVE ENZYMES IN HUMAN MUSCLE FROM DIFFERENTLY TRAINED INDIVIDUALS BY E. BLOMSTRAND*, B. EKBLOM* AND E. A. NEWSHOLMEt From the * Department of Physiology III, Karolinska Institute, Liding6vdgen 1, Stockholm, Seden and the t Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU (Received 28 August 1985) SUMMARY 1. The maximum activities ofhexokinase, 6-phosphofructokinase and oxoglutarate dehydrogenase, together ith capillary density and fibre composition, have been measured in muscle from male and female untrained, medium-trained and ell trained individuals. 2. The activity of hexokinase as almost identical in muscle from the three groups, hereas that of 6-phosphofructokinase decreased and that of oxoglutarate dehydrogenase increased ith increased training. Values of maximum rate of 2 uptake (Vo, max) ere also measured and ere 21 % higher in medium-trained and 49 % higher in ell trained compared to untrained individuals, hereas oxoglutarate dehydrogenase activities ere 39 % and 9% higher respectively. 3. There as a good positive correlation beteen the activity of oxoglutarate dehydrogenase and the percentage of type I fibres but the correlation beteen 2, max and oxoglutarate dehydrogenase activity as less good. Changes in the values of V2 max represent the effects on the circulatory and respiratory system hereas those in oxoglutarate dehydrogenase represent local effects of endurance training. INTRODUCTION In a number of studies, the effects of endurance training on physiological parameters such as the maximum rate of 2 uptake (2, max) and capillary density have been compared ith changes in the activities of glycolytic and oxidative enzymes (Holloszy & Booth, 1976; Gollnick & Saltin, 1983). Evidence based on animal studies indicates that only a very limited number of enzymes can provide quantitative information concerning the maximum flux through metabolic pathays. To the authors' knoledge, comparison of changes in the activities of those enzymes that provide quantitative information ith physiological data has not been previously reported. It has been shon that the activities of hexokinase, 6-phosphofructokinase and oxoglutarate dehydrogenase provide quantitative information on the maximum Experimental ork as carried out mainly in the Karolinska Institute.

2 112 E. BLOMSTRAND, B. EKBLOM AND E. A. NEWSHOLME capacities of glycolysis-from-glucose, glycolysis-from-glycogen and oxidative metabolism, respectively (Nesholme, Crabtree & Zammit, 198; Cooney, Taegtmeyer & Nesholme, 1981; Nesholme & Leech, 1983). From these activities it is possible to calculate the maximum rate of adenosine triphosphate (ATP) formation in muscle under anaerobic or aerobic conditions and this has been carried out for muscles of birds and mammals (Blomstrand, Challiss, Cooney & Nesholme, 1983). In man, all three activities have not been measured in the same study nor have the effects of endurance training been investigated. Consequently, in the present study the maximum activities of hexokinase, 6-phosphofructokinase and oxoglutarate dehydrogenase, together ith the capillary density and fibre composition, have been measured in biopsy samples from men and omen ho ere either untrained, medium-trained or ell trained: in addition, the values of V2 max ere also measured. METHODS Subjects Sixteen male and seventeen female subjects volunteered to participate in this study after being fully informed about the risks involved. Telve of the subjects ere endurance-trained, most of them ere competing in long-distance running or orienteering. Eleven individuals ere mediumtrained, i.e. they performed regular endurance training to or three times a eek. Ten individuals ere untrained: none of them had been engaged in any regular endurance training during the last 2 years and most of them had never been engaged in physical training. Pertinent physical characteristics for the different groups are given in Table 1. The study as approved by the Ethical Committee of the Karolinska Institute. Maximal rate of oxygen uptake (Po2, max) t 2, max as determined during running on a motor-driven treadmill at > 3 deg uphill inclination according to the procedure of Astrand & Rodahl (1977). Expired air as collected in Douglas bags and the volume as subsequently measured in a balanced Tissot spirometer and gas samples ere analysed for 2 and CO2 content in a mass spectrometer (Centronic 2 MGA). Fibre typing and capillary density Muscle biopsies ere taken from the left vastus lateralis muscle using a forceps technique (Radner, 1962). The biopsies ere taken one-third of the length from the proximal edge of the patella to the spina iliaca anterior superior, and 1-2 cm deep into the muscle. For histochemical analyses, the biopsy as mounted in an embedding medium (Tissue TekR II, O.C.T. Compound) and frozen in isopentane cooled to its freezing point in liquid N2. Serial cross-sections, 1,um thick, ere cut ith a microtome at -2 C and stained for myofibrillar ATPase at ph 9*4 (Gomori, 1941; Padykula & Herman, 1955) after pre-incubation at ph 4*3, 4-6 and 1-3 (Brooke & Kaiser, 1969). The fibres ere classified into type I, IIA, IIB and IIC (Brooke & Kaiser, 197; Duboitz & Brooke, 1973). To biopsies from each individual ere taken to determine fibre-type composition. To visualize capillaries, the cross-sections ere stained by the amylase-pas method (Andersen, 1975). From photographs of amylase-pas sections one or several areas, ithout artifacts and ith no tendency to longitudinal cuts of the fibres, ere determined planimetrically. On average, -42 mm2 (range mm2) cross-sectional area as measured from each individual. pillaries ere identified on the photographs. Enzyme activities For these analyses, 3-7 mg of muscle as rapidly freed from blood and connective tissue, eighed and then homogenized in 1 volumes of ice-cooled extraction medium using a Potter- Elvehjem homogenizer and homogenized by hand. The extraction medium contained 5 mm-tris hydrochloride, 5 mm-mgcl2, 1 mm-edta and 2 mm-mercaptoethanol at ph 8-2 (Blomstrand et al. 1983). Hexokinase, 6-phosphofructokinase and oxoglutarate dehydrogenase ere assayed as described previously (Nesholme et al. 198; Blomstrand et al. 1983).

3 KEY ENZ YMES IN HUMAN MUSCLE 113 Statistics Differences beteen the groups ere evaluated by a one-ay analysis of variance (ANOVA). The least significant difference beteen groups as calculated as described by Snedecor & Cochran (1968) for variables for hich a significant F value as obtained in the ANOVA. RESULTS The mean percentage difference in the value of P2, max beteen untrained and medium-trained individuals as 24 % (P < 1) and beteen medium-trained and ell trained the difference as 21 % (P < 1) (Table 1). TABLE 1. Physical characteristics for the different groups Age Height Weight '1O2, max Group (years) (cm) (kg) (ml kg-' min') Untrained Male (n = 6) P18 Female (n = 4) P15 Medium-trained Male (n = 4) Female (n= 7) Well trained Male (n = 6) Female (n = 6) P16 Data are presented as means + S.E. of means. The proportion of fibres classified as type I in the ell trained as almost tice (P < -1) that in the untrained individuals. Regarding the subgroups of type II fibres, the ratio of type IIA/JIB as 2- for untrained, 2-6 for medium-trained and 7-7 for ell trained individuals. The proportion of type IIC fibres in most subjects as very lo (Table 2). The activity of the key mitochondrial enzyme oxoglutarate dehydrogenase as 39 % higher (P < 5) in the medium-trained than in the untrained group and 36 % higher (P < 1) in the ell trained compared to that in the medium-trained group (Table 2). There as a positive correlation beteen the activity of oxoglutarate dehydrogenase and the percentage of type I fibres (r = 73, P < 1) (Fig. 1) and also a negative correlation in relation to the percentage of II B fibres (r = -7, P < 1). There as no difference in hexokinase activity beteen the different groups, hereas the maximum activity of 6-phosphofructokinase as 33 % loer (P < 1) in the ell trained group compared ith the untrained one (Table 2). DISCUSSION The values of V2, max ere greater according to the amount of training the groups had received, as expected. This as also true of the proportion of fibres classified as type I. Whether these latter differences represent an effect of selection or an effect

4 114 E. BLOMSTRAND, B. EKBLOM AND E. A. NEWSHOLME ~._ C.) - C.) 2 o (.) W... c3 C.).) (L (1) W E r.5 1 Ē E CO ea E P-- C.) 1. r- Z-.) 4.) m r.) 4.) 2,C ^ ;._ N l +l +l I* xm U:d *-L.: 1: N o 66o dn o C;- -_- 1tl 2 co Co 1 C = to _ C Co I-L 1: 1- Co +1 Co cs - - o6oo o > eo ONC> n N m~ N 2 -- N _~ N *c * CO - M,- cai -4 CO CO -o C>1 -_ * 1 -~ - sc LsU 2t C>2 "t. C> CD o- o C o1 +1 +l r.i 2) +l 2 C- 4.) C-. ce o._ W _._ 2 4._ C.) F._ ~c 1.~CJ C> * -- * N 2 9C~4 Ct - - t CO _ Ct _ + _+ k -l +l2 +l +o+ + e o e. 2 _i o +l Al ec X eccc cb cb G. E. Po Co C1 Co +l+1 +1 tbcc2 S; CZ I CO e mp _I W- t_ - -a CO ; s =t o la - ol r l

5 KEY ENZYMES IN HUMAN MUSCLE of training is still an open question. Hoever, the higher ratio of type II A/II B fibres in the ell trained group compared ith those in the other groups probably represents a major effect of training, as indicated in earlier studies (Andersen & Henriksson, 1977; Green, Thomson, Daub, Houston & Ranney, 1979). Aerobic training increases the activity of the mitochondrial enzyme oxoglutarate dehydrogenase. This is the first report of the effects of training on the activity of this ~~~~~~~~~~~~~~~~~~~~~~~ >,4-J o1 4-C E -&~~~~~~~ 1 L * *. o X~~~~~~~~~~~ E Type fibres (%) Fig. 1. Maximum activity of oxoglutarate dehydrogenase in relation to the percentage of type I fibres in the vastus lateralis muscle of differently trained individuals. The correlation coefficient r = 73 (P < -1). key enzyme of the aerobic system in the muscle of man. A positive correlation beteen oxoglutarate dehydrogenase and the percentage of type I fibres as found. Hoever, ithin the different groups there as no correlation beteen enzyme activity and fibre composition, except for the medium-trained group. This indicates that the correlation indicated above for the hole group is an effect of training rather than a true relationship beteen oxoglutarate dehydrogenase activity and the percentage of type I fibres. This is supported by the observation that endurance training has been found to increase the activity of mitochondrial enzymes almost to the same extent in type I and type II fibres (Jansson & Kaijser, 1977; Essen- Gustavsson & Henriksson, 1984). The correlation beteen oxoglutarate dehydrogenase and the values of g2 max is less strong (r = 52 for all groups). This is expected since the V2 max is not equivalent to aerobic endurance. Noadays it is agreed by most exercise physiologists that endurance is determined by to main factors: maximum aerobic poer and local muscular adaptation to exercise. The latter may be improved by endurance training ithout any major changes in 2, max' especially in ell trained individuals. It should be pointed out, hoever, that the subjects in the present study ere classified into the different groups, untrained, medium-trained and ell trained, on the basis of their eekly amount of training, not their o2, max* The three groups, therefore, mainly represent various degrees of endurance capacity. In addition, it should be noted that the average age for the untrained and medium-trained males as higher than for the ell trained males and higher than for all the female groups:

6 116 E. BLOMSTRAND, B. EKBLOM AND E. A. NEWSHOLME it is not clear ho far this age difference contributes to the difference in enzyme activities. The mean percentage differences in the values of 12 max and oxoglutarate dehydrogenase beteen untrained and medium-trained individuals ere 21 and 39 % respectively and those beteen untrained and ell trained ere 49 and 9 % respectively. These differences in IQ2, max and oxoglutarate dehydrogenase are much closer than has been observed for other enzymes (Costill, Daniels, Evans, Fink, TABLE 3. lculated maximum rates of ATP formation in human muscle based on maximal activities of key indicator enzymes under aerobic and anaerobic conditions lculated maximum rate of ATP formation from glucose or glycogen (,jmol min- g'l fresh t. at 25 C) Anaerobic Oxidation via Group Sex glycolysis Krebs cycle Untrained Male Female Medium-trained Male Female Well trained Male Female Maximum rates of ATP formation are calculated as follos: for anaerobic glycolysis 6- phosphofructokinase activity is multiplied by 3; for oxidation by the Krebs cycle, oxoglutarate dehydrogenase activity is multiplied by 18 (Blomstrand et al. 1983). Krahenbuhl & Saltin, 1976; Saltin & Roell, 198). This suggests that the maximum activity of oxoglutarate dehydrogenase is the best biochemical quantitative indicator of aerobic performance and also of the effects of training on aerobic endurance. There as no effect of training on hexokinase activity. This is in agreement ith the findings of Wallberg-Henriksson, Gunnarsson, Henriksson, Ostman & Wahren (1984), but in contrast to those of Morgan, Cobb, Short, Ross & Gunn (1971) and Bylund, Bjur6, Cederblad, Holm, Lundholm, Sj6strom, Xngquist & Scherst6n (1977) ho observed an increased hexokinase activity after aerobic training. Hoever, in the present study the activity of this enzyme as already high in untrained subjects, although in the males there as considerable variability in its activity (range gmol min' ga1 muscle) hereas in the ell trained males the range as It is possible that the amount of carbohydrate in the diet may influence this activity. The maximum activity of 6-phosphofructokinase as loer in the ell trained group than in the untrained: this indicates a decrease in the capacity of the pathay of glycolysis-from-glycogen (Nesholme et al. 198). Previous studies have shon either a decrease or no change in the activity of 6-phosphofructokinase ith aerobic training (Schantz, Billeter, Henriksson & Jansson, 1982; Sj6din, Jacobs & Svedenhag, 1982; Gollnick, Armstrong, Saubert, Piehl & Saltin, 1972; Henriksson & Reitman, 1977; Jansson & Kaijser, 1977). The higher proportion of type I fibres in the ell trained group found in the present study might contribute to the loer enzyme activity.

7 KEY ENZYMES IN HUMAN MUSCLE Knoledge of the activities of 6-phosphofructokinase and oxoglutarate dehydrogenase permit calculations to be performed on the maximum rate of ATP generation from aerobic and anaerobic metabolism (see Table 3). These data indicate that maximum capacity for producing ATP under anaerobic conditions is decreased by training (approximately 3 %). This suggests that aerobic training ould be detrimental to the performance in short sprints (1 and 2 m). Maintenance of capacity for ATP generation for short sprints in untrained subjects probably reflects the evolutionary significance of glycolysis as a system for generating ATP to poer escape mechanisms (Nesholme & Leech, 1983). The data in Table 3 also indicate that the ability to generate ATP from aerobic metabolism is doubled in the muscle of the ell trained subjects. This ill be of the utmost importance in endurance activity since it permits a higher poer output ithout the need to rely on the inefficient anaerobic process hich can rapidly lead to fatigue due to the accumulation of protons (see Nesholme & Leech, 1983). None the less, the maximum anaerobic capacity to generate ATP in the muscles of ell trained individuals is at least tice that of the aerobic system, hich indicates the importance of utilizing both aerobic and anaerobic metabolism in the shorter distance athletic running events, including 8 and 15 m races. We thank Professor P.-O. Astrand for his interest and encouragement. This ork as supported by grants from the Karolinska Institute to B. E. and from the Ministry of Defence, London, to E.A.N. 117 REFERENCES ANDERSEN, P. (1975). pillary density in skeletal muscle of man. Acta phy8iologica 8candinavica 95, ANDERSEN, P. & HENRIKSSON, J. (1977). Training induced changes in the subgroups of human type II skeletal muscle fibres. Acta phy8iologica 8candinavica 99, ASTRAND, P.-O. & RODAHL, K. (1977). Textbook of Work Physiology, 2nd edn., Ne York: McGra-Hill. BLOMSTRAND, E., CHALLISS, R. A. J., COONEY, G. J. & NEWSHOLME, E. A. (1983). Maximal activities of hexokinase, 6-phosphofructokinase, oxoglutarate dehydrogenase and carnitine palmitoyltransferase in rat and avian muscles. Bioscience Reports 3, BROOKE, M. H. & KAISER, K. K. (1969). Some comments on the histochemical characterization of muscle adenosine triphosphatase. Journal of Hi8tochemistry and Cytochemietry 17, BROOKE, M. H. & KAISER, K. K. (197). Three 'myosin adenosine triphosphatase' systems: the nature of their ph lability and sulfhydryl dependence. Journal of Hi8tochemi8try and Cytochemi8try 18, BYLUND, A.-C., BJUR6, T., CEDERBLAD, G., HOLM, J., LUNDHOLM, K., SJ6STR6M, M., ANGQUIST, K. A. & SCHERSTEN, T. (1977). Physical training in man. European Journal of Applied Physiology 36, COONEY, G. J., TAEGTMEYER, H. & NEWSHOLME, E. A. (1981). Tricarboxylic acid cycle flux and enzyme activities in the isolated orking heart. Biochemical Journal 2, COSTILL, D. L., DANIELS, J., EVANS, W., FINK, W., KRAHENBUHL, G. & SALTIN, B. (1976). Skeletal muscle enzymes and fiber composition in male and female track athletes. Journal of Applied Physiology 4, DUBOWITZ, V. & BROOKE, M. H. (1973). Major Problems in Neurology, vol. 2, Muscle Biopsy: A Modern Approach. W. B. Saunders. ESSEN-GUSTAVSSON, B. & HENRIKSSON, J. (1984). Enzyme levels in pools of microdissected human muscle fibres of identified type. Acta phyeiologica 8candinavica 12, GOLLNICK, P. D., ARMSTRONG, R. B., SAUBERT, C. W., PIEHL, K. & SALTIN, B. (1972). Enzyme

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