MITOCHONDRIAL OXIDATIVE ENZYME ACTIVITY IN INDIVIDUAL FIBRE TYPES IN HYPO- AND HYPERTHYROID RAT SKELETAL MUSCLES

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1 Quarterly Journal of Experimental Physiology (1984), 69, Printed in Great Britain MITOCHONDRIAL OXIDATIVE ENZYME ACTIVITY IN INDIVIDUAL FIBRE TYPES IN HYPO- AND HYPERTHYROID RAT SKELETAL MUSCLES Department of Neurology, University of Newcastle upon Tyne, Newcastle upon Tyne NE] 7RU (RECEIVED FOR PUBLICATION 2 JUNE 1983) SUMMARY Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa. INTRODUCTION Differences in the response of slow- and fast-twitch muscles to altered thyroid hormone levels have been observed in experimentally induced dysthyroid states (Winder, Fitts, Holloszy, Kaiser & Brooke, 1980). Studies of mitochondrial 'marker' enzymes in rat muscles have shown that relatively high dosage levels of triiodothyronine (T3) or thyroxine (T4) are necessary in order to produce even a modest rise in oxidative enzyme activity in fast-twitch gastrocnemius (Winder, Baldwin, Terjung & Holloszy, 1975), whereas subsequent studies have shown that the response of the slow-twitch soleus muscle to equivalent hormone levels is much greater (Winder et al. 1980). It has also been demonstrated that alterations in iodothyronine levels result in the interconversion of the main metabolic fibre types (Ianuzzo, Patel, Chen, O'Brien & Williams, 1977). The proportion of slow-twitch fibres is increased in hypothyroid muscles, whereas in hyperthyroidism there is an increase in the proportion of fast-twitch fibres. These changes are associated with alterations in the myosin phenotype expressed in the muscle fibres of the dysthyroid animals, with consequent decrease or increase in myofibrillar ATPase activity (Johnson, Mastaglia, Montgomery, Pope & Weeds, 1980a). The degree of fibre-type interconversion seen in slow-twitch muscles such as soleus is consistently greater than that observed in fast-twitch e.d.l. This is true of the responses to both hypothyroidism and hyperthyroidism (Johnson, Mastaglia, Montgomery, Pope & Weeds, 1980b; Johnson, Mastaglia & Montgomery, 1980). Previous studies of the effects of altered iodothyronine levels on muscle mitochondrial oxidative enzymes have relied largely on data derived from biochemical assay of muscle homogenates (Winder et al. 1980) but the comparison of the effect of the hormone on muscles which are predominantly slow- or fast-twitch has indicated that there may be selective hormonal effects on particular metabolic fibre types (Kubista, Kubistova & Pette, 1971).

2 258 The present study comprises a correlative biochemical and quantitative cytochemical examination of the response to iodothyronine levels of mitochondrial oxidative enzymes in individual fibre types. A primary objective is to examine the possibility that the changes in mitochondrial enzyme activity found in homogenates of dysthyroid muscles could be due primarily or even wholly to changes in fibre-type constitution as defined in terms of the myosin phenotype expressed. Under these circumstances, one would expect the oxidative activity of individual fibre types to approximate that seen in the corresponding euthyroid muscles. If however, the underlying mechanism or mechanisms whereby thyroid hormone exerts an influence on (a) mitochondrial oxidative enzyme activity and (b) myosin phenotype and myofibrillar ATPase activity, are not identical, then no such parallel response would be expected. Available evidence suggests that some of the effects of iodothyronines on the contractile properties of skeletal muscle may be mediated via its motor innervation, either by means of altered neural trophic influences or because of changes in the pattern of electrical activity reaching the muscle (Johnson et al. 1980b). The work of Nwoye & Mommaerts (1981) however, appears to indicate a 'direct' (i.e. non-neurally mediated) effect of the hormone on myofibrillar proteins. These viewpoints are by no means mutually exclusive. In addition, a probable role of muscle hormone receptors in mediating a 'direct' effect of thyroid hormone on mitochondrial oxidative enzymes has been postulated (Janssen, Van Hardeveld & Kassenaar, 1978) and the possible interaction of at least two separate mechanisms of hormone action on skeletal muscle needs to be considered. METHODS A group of six male Wistar rats was made hypothyroid by the administration of propylthiouracil (10 mg/kg.24h) in drinking water from the age of 8 to 16 weeks. At the beginning of treatment these rats weighed 200 g (± 5 g). A similar group of six rats was made hyperthyroid by the addition of 3,5,3'-triiodothyronine (T3) to the drinking water in a concentration of 100 1ag/kg.24h from the age of 8-14 weeks. Both groups of rats were sacrificed by cervical dislocation together with nine normal male animals, also weighing 200 g ± 5 g at the age of 8 weeks and kept under identical conditions. Left and right extensor digitorum longus (e.d.l.) and soleus were removed and weighed. The mid-portion of each complete muscle was sandwiched between two blocks of liver together with a corresponding control muscle and the composite blocks were frozen in dichlorodifluoromethane (Arcton 12) atca 'C. The residue of the muscle samples was frozen and stored in liquid nitrogen before biochemical examination. Quantitative cytochemistry Fresh frozen sections l,m) (10 were cut and used to demonstrate the activities of succinate dehydrogenase (E.C ), the mitochondrial, menadione-linked formof L-glycerol-3-phosphate dehydrogenase (E.C ) and D-3-hydroxybutyrate dehydrogenase (E.C ). MTT (3-[dimethyl-thiazolyl-2-]-2,5-diphenyl tetrazolium bromide) was used as electron acceptor in the methods for the demonstration of succinate dehydrogenase and L-glycerol-3-phosphate dehydrogenase and NBT (2,2' di p-nitrophenyl -5, 5'-diphenyl 3, 3'-[3,3' dimethoxy -4, 4'-biphenylene] ditetrazolium chloride) was used for the demonstration of the activity of D-3-hydroxybutyrate dehydrogenase (Pearse, 1972). The incorporation of dysthyroid muscle and the corresponding control tissue in the same block enabled comparative densitometric measurements to be made on experimental and control material of identical section thickness, incubated under identical conditions. At least 100 fibres from each muscle sample were examined, the fibre type of each fibre being determined previously by reference to photomicrographs of serial sections demonstrating the activity of myofibrillar ATPase at ph 9 5 (Hayashi & Freiman, 1966). Densitometric measurements were made using a Vickers M 17 microscope fitted with a stable d.c. power supply and photometer based on a high-efficiency silicon photodiode

3 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE 259 and integrated amplifier (R. S. Components Ltd). The formazan reaction products in the cytochemical assays were measured at their absorption maximum using a bandpass filter with peak absorption of 574 nm and half-width 50 nm. The 1000% transmission level was set using initial readings taken through slide, cover-slip and mounting medium in areas immediately adjacent to the tissue sections, using a measuring spot of 20,um diameter. Densitometric measurements were expressed as relative absorbances, calculated from the formula (2-log100% T) where % T is the percentage of light transmitted. Biochemistry Five per cent homogenates of whole muscle were prepared by first finely chopping with scissors in ice-cold medium (250 nm sucrose, 10 mm Tris HCI, 1 mm EGTA, ph 7 4) and the rest of the procedure was carried out at 0-4 'C. The muscle tissue was then homogenized in the same medium using a Teflon/glass homogenizer and then assayed immediately. Succinate dehydrogenase was determined spectrophotometrically at nm using 0 5 mm-fe(cn) 63- as oxidant in an isotonic medium containing 130 mm-kcl, 10 mm HEPES, 2-5 mm phosphate, 04 mm ADP, 0 1 mm EDTA, defatted bovine serum albumin 1 5 mg/ml, 1 mm-kcn (to inhibit cytochrome oxidase) and 1,ug rotenone (to inhibit NADH dehydrogenase) (Turnbull, Sherratt, Davies & Sykes, 1982). After measuring the endogenous rate the reaction was started by the addition of 1O mm succinate. L-glycerol-3-phosphate dehydrogenase was measured in the same medium, plus 1 jug/ml antimycin (to block the respiratory chain at cytochrome b) and using 20 mm DL-glycerol-3-phosphate as substrate. The activities of succinate dehydrogenase and of L-glycerol-3-phosphate dehydrogenase were linear over the protein concentrations used. RESULTS Densitometric measurement of mitochondrial enzyme activity in normal e.dl. and soleus muscles The mitochondrial oxidative enzymes studied were chosen as 'markers' of the activity of particular metabolic pathways, namely succinate dehydrogenase (SDH) (citric acid cycle), L-glycerol-3-phosphate dehydrogenase (L-GPDH) (glycerophosphate cycle) and D-3- hydroxybutyrate dehydrogenase (D-HBDH) (ketone utilization). Initial studies were concerned with the determination of relative activities of these enzymes in the individual fibre types of rat e.d.l. and soleus. These results are shown graphically in Fig. 1 A and B. Normal rat e.d.l. contains all three major metabolic fibre types, i.e. Type 1 (slow oxidative), Type 2 A (fast oxidative-glycolytic) and Type 2 B (fast glycolytic) (Fig. 2 A). Rat soleus contains only Type 1 and Type 2A fibres with a small percentage (<5%0) of intermediate '2 C' fibres (Fig. 2 C). It can be seen from Fig. 1 that the relative enzyme activity in each fibre type shows a different pattern for each of the enzymes studied. The gradient of activity for SDH is Type 2A > 1 > 2B; that of L-GPDH, Type 2B > 2A > 1 and that of D-HBDH, Type 1 > 2A > 2B. Statistical analysis of the densitometric measurements is given in Table 1. The differences in the oxidative enzyme activity between individual fibre types in both e.d.l. and soleus are, in general, highly significant (P < 0 001). However, less striking differences were seen when the SDH and D-HBDH activities of Type 1 and 2A fibres in e.d.l. and D-HBDH activity of Type 1 and 2A fibres in soleus were compared. Since '2 C' (intermediate) fibres represent a transitional stage between Type 1 and Type 2 A fibres, and not a stable metabolic fibre type, the oxidative enzyme activities of this class of fibre were, predictably, intermediate in relation to those of Types 1 and 2A and the differences were not always statistically significant. Hypothyroid rat e.d.l. and soleus The changes in relative enzyme activity seen in hypothyroid rat e.d.l. and soleus are shown in Fig. 3 A and B. It has been previously reported that the degree offibre-type interconversion

4 260 A 024 S SDH L-GPDH D-HBDH 2A 2B 1 2A 2B 1 2A 2B B 0F24 SDH L-GPDH D-HBDH C 0-~ CO ~0-12- C I 2A 2C I 2A 2C 1 2A 2C Fig. 1. A, densitometric measurement of mitochondrial oxidative enzyme activity (SDH, L-GPDH, D-HBDH) in individual fibre types in normal rat e.d.l. (mean+s.e.m.). B, densitometric measurement of mitochondrial oxidative enzyme activities (SDH, L-GPDH, D-HBDH) in individual fibre types in normal rat soleus (mean + S.E.M.). from Type 2A to Type 1 in soleus frequently results in a soleus muscle which is composed entirely of Type 1 fibres (Johnson et al a). Because of this there were insufficient Type 2 A or 2C (intermediate) fibres remaining in any of the six samples of hypothyroid soleus to permit a valid statistical analysis (see Fig. 4 C). In the case of all three enzymes studied, namely SDH, L-GPDH and D-HBDH, the relative absorbance values in the hypothyroid muscles showed a significant decrease as compared with normal muscles, in all fibre types in e.d.l. The statistical analysis of these results is shown in Table 2 A. The decrease in L-GPDH is illustrated in Fig. 4B (cf. Fig. 2B). Fig. 2.A, normal rat e.d.l. showing small proportion of Type 1 fibres (pale). The remainder of the muscle is composed of Type 2A (dark) and type 2B (intermediate) fibres. Myofibrillar ATPase (m.atpase); bar = 100 /sm. B, serial section to Fig. 2A showing L-GPDH activity in normal rat e.d.l. fibre types. Type I fibres (low), Type 2A fibres (intermediate), Type 2B fibres (high). C, normal rat soleus showing a predominance of Type 1 fibres (pale). There are approximately 20% Type 2A fibres (dark) and a variable proportion of 2C (intermediate) fibres. M.ATPase; bar = 100,um. D, serial section to Fig. 2 C showing L-GPDH activity in normal rat soleus fibre types. Type 1 fibres (low), Type 2A fibres (high), with intermediate reactivity of 2C fibres.

5 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE I 2or legend s Fig. 2. For legend see ie 0 opposite page. 261

6 262 Table 1. Statistical analysis of relative absorbance values in individualfibre types in rat e.d.l. and soleus Normal rat e.d.l. (n = 9) SDH Type 1 vs.2a P < 005 Type 2A vs. 2B < Type 2Bvs.2 <0001 L-GPDH Type 1 vs. 2A P < Type 2A vs. 2B < Type 2Bvs.1 <0001 D-HBDH Type 1 vs. 2A P < 002 Type 2Avs.2B <0001 Type 2Bvs.l <0001 Normal rat soleus (n = 9) SDH Type 1 vs.2a P < 0001 Type 2A vs. 2C < 0 05 Type 2C vs. 1 <0001 L-GPDH Type 1 vs. 2A P < Type 2A vs. 2C < Type 2C vs. 1 < D-HBDH Type 1 vs. 2A P < 0-05 Type 2A vs. 2C N.s. Type 2Cvs. 1 N.s. Two-tailed variant of Student's t test used throughout. In hypothyroid soleus where all fibres were Type 1 in histochemical profile, there were significant decreases in activity of SDH and L-GPDH as compared with normal Type 1 fibres, but only a small (non-significant) decrease in D-HBDH activity (Table 2B). The difference in L-GPDH activity between normal and hypothyroid soleus is illustrated in Figs. 2D and 4D. In normal soleus the Type 2A fibres, comprising about 20% of the total population, show moderate levels of activity. In hypothyroid soleus the Type 2A fibres have been eliminated by conversion to Type 1, and in addition, the L-GPDH activity of these Type 1 fibres is lower than that of the Type 1 fibres in normal soleus. Hyperthyroid rat e.d.l. and soleus The histograms (Fig. 3 A and B) also illustrate the changes in relative enzyme activity seen in hyperthyroid rat e.d.l. and soleus. It should be noted that, due to conversion of Type 1 fibres to Type 2A, Type 1 fibres were virtually absent from hyperthyroid e.d.l. (Fig. 5A) and hence no assessment of their enzyme activity was possible. In hyperthyroid e.d.l. and soleus the most marked increase was seen in the levels ofl-gpdh activity with a greater increase in soleus than in e.d.l. Hyperthyroid soleus also showed a significant increase in SDH activity whereas that of e.d.l. was unaltered. There were moderate increases in D-HBDH activity in both e.d.l. and soleus. However, the muscles from the hyperthyroid animals showed much more inter-sample variation in oxidative enzyme activity than did either the euthyroid or hypothyroid muscles. For this reason some of the observed differences though quite marked were not statistically significant (Table 2A and B).

7 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE 263 A SDH L-GPDH D-HBDH O Hypothyroid O Euthyroid 0 Hyperthyroid B O Hypothyroid 0 Euthyroid E Hyperthyroid D-HBDH r- CZ ~00 I 2A 2C 1 2A 2C I 2A 2C Fig. 3. A, comparison of densitometric measurements of mitochondrial oxidative enzyme activity (SDH, L-GPDH, D-HBDH) in euthyroid, hypothyroid and hyperthyroid rat e.d.l. *Type 1 virtually absent in hyperthyroid e.d.l. B, comparison of densitometric measurements of mitochondrial oxidative enzyme activity (SDH, L-GPDH, D-HBDH) in euthyroid, hypothyroid and hyperthyroid rat soleus. *Types 2A and 2C virtually absent in hypothyroid soleus. The greater degree of variability of the hyperthyroid animals is also reflected in the details of muscle, heart and body weights obtained at autopsy (Table 3). The increase in L-GPDH activity in hyperthyroid e.d.l. and soleus muscles is illustrated in Fig. 5 B and D (cf. Fig. 2B and D). The increase in the proportion of Type 2A and 2C fibres relative to the total fibre population is also apparent (Fig. SC cf. Fig. 2 C). Biochemical assays The results of assays of whole muscle homogenates are shown in Table 4. In e.d.l., SDH activity was significantly decreased in the hypothyroid muscles but there was no significant change in the hyperthyroid muscles (Fig. 6A). A similar picture emerges in the assay of L-GPDH with a significant decrease in activity in hypothyroid muscles but no change in the hyperthyroid animals.

8 264 "'r-- -h w4 I ;i!.. :- r.e~ W ; - I2 Fig. 4. For legend see opposite page.

9 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE 265 Table 2. Comparison of relative absorbance values in euthyroid, hypothyroid and hyperthyroid rat e.d.l. and soleus (values expressed as mean ±S.D.) Euthyroid Hypothyroid Hyperthyroid A, e.d.l. (n = 9) (n = 6) (n = 6) SDH Type t Type2A N.s. Type2B N.s. L-GPDH Type Type 2A t Type 2 B t D-HBDH Type l Type 2A N.s. Type 2B N.s. Euthyroid Hypothyroid Hyperthyroid B, soieus (n = 9) (n = 6) (n = 6) SDH Type T Type 2A t Type 2C N.s. L-GPDH Type t Type 2A t Type 2C N.s. D-HBDH Type N.s N.s. Type2A N.s. Type2C N.s. t, significant increase (P < 0 01). 1, significant decrease (P < 0 01). N.s., non-significant difference. In soleus (Fig. 6B) the changes were more marked with a significant decrease in activity in hypothyroid animals and a significant increase in activity in hyperthyroid animals. The activity of L-GPDH was low in normal soleus but fell further in the hypothyroid animals. Hyperthyroidism induced a marked increase in L-GPDH activity. Assays of the activity of D-HBDH in whole muscle homogenates of normal rat e.d.l. and soleus gave values near to the limits of detection, in contrast to assays of normal rat diaphragm and cardiac muscle which showed high activity. For this reason no statistical analysis of the results of D-HBDH assays in e.d.l. and soleus was attempted. DISCUSSION The results of previous biochemical studies have shown that significant changes in mitochondrial oxidative enzyme activity take place in response to alterations in iodothyronine levels (Winder et al. 1975, 1980) and have indicated a greater response in slow-twitch muscle. Fig. 4. A, hypothyroid rat e.d.l. showing increased numbers oftype 1 fibres (pale) due to fibre-type interconversion. M.ATPase; bar = 100,m. B, serial section to Fig. 4A showing a decrease in L-GPDH activity in all fibre types as compared with normal rat e.d.l. C, hypothyroid rat soleus showing loss of Type 2A and 2C fibres due to interconversion, resulting in a uniform Type 1 fibre population. M.ATPase; bar= 100,m. D, serial section to Fig. 4C showing very low activity of L-GPDH in the Type 1 fibres.

10 266 F e Fig. 5. For legend e -p pa - see opposite page.

11 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE 267 Table 3. Comparison of muscle and body weights in euthyroid, hyperthyroid rats (mean + S.D.) hypothyroid and Euthyroid Hypothyroid Hyperthyroid (n = 6) n = 6) (n = 6) Body wt. (g) E.d.l.wt.(mg) E.d.l. ( x 103):body wt. ratio T N.s. Soleus wt. (mg) Soleus ( x 103):body wt. ratio i+0023 t N.s. Heartwt.(g) t Heart (x 103):body wt. ratio N.s T N.s., non-significant difference from euthyroid. T, significant increase (P < 0 01). 1, significant decrease (P < 0-01). Table 4. Enzyme activities in muscle homogenates A, e.d. SDH L-GPDH Euthyroid Hypothyroid Hyperthyroid N.s N.s. B, soleus SDH L-GPDH Euthyroid Hypothyroid Hyperthyroid T t Results are expressed as mmol of ferricyanide reduced/min mg wet wt. (mean ±S.D.; n = 6): 4, significant decrease (P < 0 01). t, significant increase (P < 0 01). N.s., non-significant difference. In addition it was found that the changes in activity of some enzymes were much greater than others; L-GPDH, for example, showed a greater increase in hyperthyroid skeletal muscle than did SDH. In a previous qualitative histochemical study of the effects of hypoand hyperthyroidism on mitochondrial activity in rat respiratory muscles, we suggested that many of the differences in hormonal effect noted by Winder et al. (1975, 1980) might be due to the interaction of at least two separate mechanisms of iodothyronine action on muscle (Johnson, Olmo & Mastaglia, 1983). One mechanism involves the interconversion of the fibre types within the hypo- or hyperthyroid muscles, a process which not only involves changes in the myosin phenotype and myofibrillar ATPase activity of individual muscle fibres, but also implies corresponding changes in mitochondrial oxidative enzyme activity (Pette, Smith, Staudte & Vrbovai, 1973). Nevertheless, if changes in mitochondrial oxidative Fig. 5. A, hyperthyroid rat e.d.l. showing loss of Type 1 fibres due to interconversion. M.ATPase; bar = 100gm. B, serial section to Fig. SA showing increased activity of L-GPDH in both Types 2A and 2 B. C, hyperthyroid rat soleus showing increased numbers of Type 2A and 2C fibres due to interconversion. M.ATPase; bar = 100 gm. D, Serial section to Fig. S C showing increased activity of L-GPDH in all fibre types.

12 268 A ~: 1*0 -o SDH E] Hypothyroid 0 Euthyroid E Hyperthyroid L-GPDH 1-1 tv 0 5i 1..-.E a) B 1-o -o) a) 0 :3 2.5r 2O0 1-5F 1*0 L-GPDH T 0 5 I&E'' El Hypothyroid CD Euthyroid E Hyperthyroid Fig. 6. A, activity of SDH and L-GDPH in homogenates of euthyroid, hypothyroid and hyperthyroid e.d.l. muscles (mean + S.E.M.). B, activity of SDH and L-GPDH in homogenates of euthyroid, hypothyroid and hyperthyroid soleus muscles (mean +S.E.M.). enzyme activity were merely linked to changes in myofibrillar ATPase activity, one would expect the oxidative enzyme activity of the converted fibres to approximate that of the corresponding euthyroid fibre types. However, the results of the present study clearly demonstrate that this is not the case. In hypothyroid muscles, mitochondrial oxidative enzyme activity shows a significant decrease when individual euthyroid and hypothyroid fibre types are compared, the converse being true of hyperthyroid muscles. These findings lend support to the suggestion that iodothyronines may exert a direct effect on skeletal muscle mitochondria via hormone receptors located in or on the surface of individual muscle fibres, the greater response of slow-twitch muscles being associated with their higher receptor density (Janssen, Van Hardeveld & Kassenaar, 1979). It has also been demonstrated that denervation only partially abolishes the thyroid-hormone-associated

13 OXIDATIVE ENZYME IN DYSTHYROID MUSCLE changes in mitochondrial enzyme activity (Winder et al. 1980) though the same process inhibits virtually all fibre-type interconversion (Johnson et al. 1980b). Available evidence therefore suggests that whereas changes in myosin phenotype in response to altered thyroid hormone levels require intact motor innervation, mitochondrial enzyme changes are to some extent independent of motor influences and fibre-type interconversion. The densitometric measurements made in this study are end-point assays and therefore do not permit the calculation of absolute rates of enzyme activity (Pette, 1981). However, there is good correlation between the results obtained from quantitative cytochemistry and from biochemical assays, confirming the validity of the technique. The application of this combined biochemical and cytochemical approach has identified one probable cause contributing to the differing response of individual mitochondrial enzymes which may be associated with the existence of different mitochondrial sub-populations in individual fibre types. For exampe in Type I fibres of both soleus and e.d.l., very few mitochondria contain cytochemically demonstrable L-GPDH activity whereas a large number show SDH activity. The net result of such differences is seen in the characteristic gradients of cytochemical activity shown in Fig. 1 A and B. Since the content of individual mitochondrial oxidative enzymes differs widely in individual fibre types, it is obvious that fibre-type interconversion will affect the over-all muscle content of individual enzymes in different ways. For example, in hyperthyroid soleus the activity of L-GPDH in the muscle as a whole increases not only because the enzyme activity is higher in both Type 1 and 2A fibres but also because the percentage of Type 2A fibres is greater, thus increasing the proportion of fibres with high L-GPDH activity. These effects are clearly additive. However, in the case of SDH, the increase in enzyme activity seen in homogenates of hyperthyroid soleus is not so marked. This may reflect the relatively small difference in SDH activity between Type 1 and 2A fibres, with the result that, in this instance, fibre-type interconversion will exert less effect on over-all enzyme activity. The combination of biochemical and quantitative cytochemical techniques is of great potential use in the analysis of situations involving alteration in the metabolic profile of skeletal muscle. D.M.T. is in receipt of an M.R.C. Training Fellowship. 269 REFERENCES HAYASHI, M. & FREIMAN, D. G. (1966). An improved method of fixation for formalin sensitive enzymes with special reference to myosin adenosine triphosphatase. Journal of Histochemistry and Cytochemistry 14, IANUzzo, D., PATEL, P., CHEN, V., O'BRIEN, P. & WILLIAMS, C. (1977). Thyroidal trophic influence on skeletal muscle myosin. Nature 270, JANSSEN, J. W., VAN HARDEVELD, C. & KASSENAAR, A. A. H. (1978). Evidence for a different response of red and white skeletal muscle of the rat in different thyroid states. Acta endocrinologica 87, JANSSEN, J. W., VAN HARDEVELD, C. & KASSENAAR, A. A. H. (1979). A methodological study in measuring T3 and T4 concentration in red and white skeletal muscle and plasma of euthyroid rats. Acta endocrinologica 90, JOHNSON, M. A., MASTAGLIA, F. L. & MONTGOMERY, A. (1980). The histochemical and contractile properties of hyperthyroid mammalian skeletal muscle. I.R.C.S. Journal of Medical Science 8, JOHNSON, M. A., MASTAGLIA, F. L., MONTGOMERY, A., POPE, B. & WEEDS, A. G. (1980a). Changes in myosin light chains in the rat soleus after thyroidectomy. Federation ofthe European Biochemistry Society Letters 110,

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