Levels of the NADH shuttle enzymes and cytochrome b5 reductase in human skeletal muscle: effect of strength training

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NADH shuttle enzymes and cytochrome b5 reductase

in human skeletal muscle: effect of strength training

PETER G.

Department

SCHANTZ, PETER G.,

SCHANTZ AND MI KiiLLMAN

of

Physiology III, Karolinska Institute, Lidingiivtigen 1, S-114 33 Stockholm, Sweden

AND MI KALLMAN. NADH shuttle

enzymes and cytochrome bS reductase in human skeletal muscle: effect of strength training. J. Appl. Physiol. 67(l): 123-127,

1989.-The main aim of this study was to investigate whether enzyme levels of the malate-aspartate and a-glycerophosphate

shuttles and of cytochrome bg reductase in human skeletal muscle are affected by strength training. Muscle biopsy samples from the deltoid muscle of the nondominant arm in untrained

(n = 12) and strength-trained (n = 12) subjects were compared. The strength-trained muscles were characterized by a tendency to a higher percentage of type I fibers (67 vs. 59%), a lower percentage of type IIb fibers (12 vs. 18%), 34% larger mean fiber areas, and 19% more capillaries per fiber (P c 0.1). No difference was noted in levels of enzymes representing the citric acid cycle, fatty acid oxidation, and glycolysis, nor in the number of capillaries per square millimeter. Neither did the levels of malate-aspartate and cu-glycerophosphate shuttle enzymes nor cytochrome b5 reductase differ. Levels of cytochrome b5 reductase correlated (r = 0.59, P < 0.01) with levels of the mitochondrial marker enzyme citrate synthase. It is concluded that strength training does not appear to result in increased levels of NADH shuttle enzymes and cytochrome bs reductase. cu-glycerophosphate shuttle; aspartate aminotransferase; enzyme activity; glycerol-3-phosphate dehydrogenase; isoen- zymes; malate-aspartate shuttle; malate dehydrogenase

DURING GLYCOLYSIS, the coenzyme NAD+ is reduced to NADH. The rate of glycolytic NADH production in human skeletal muscle can increase to about 8 mmol kg wet wt-l emin-’ during bicycle exercise that demands the maximal O2 uptake (29). The level of NAD+ in resting ’ muscle is, however, only -0.45 mmol l kg wet wt-’ (25).

Thus a rapid reoxidation of NADH is necessary if glycolysis is to proceed. This reoxidation can be achieved in two different ways: anaerobically through the reduction of cytosolic pyruvate to lactate or aerobically via the mitochondrial respiratory chain. The transfer of NADH into mitochondria is hindered by the impermeability of the inner mitochondrial membrane to NADH. Therefore, aerobic oxidation of cytosolic NADH is achieved through the transfer of reducing equivalents into mitochondria via the malate-aspartate (MA) and cu-glycerophosphate

(GP) shuttles. Another pathway for aerobic reoxidation of cytosolic NADH, involving cytochrome bg reductase in the outer mitochondrial membrane, has recently been described (9, 35).

It has been shown that endurance training augments levels of the MA shuttle enzymes in human skeletal

muscle, whereas those of the GP shuttle do not change (33). A corresponding adaptation with strength training would be functional because very high levels of muscle lactate (17 mM), indicative of a substantial accumulation of cytosolic NADH, have been reported after a strength training exercise regimen (36). The main aim of this study was, therefore, to investigate whether strength training affects levels of NADH shuttle enzymes and cytochrome & reductase. This has been done by compar- ing the enzyme levels in biopsy samples from a nonpostural muscle (deltoid muscle) of 12 strength-trained and 12 untrained male subjects. Data from four endurance- trained subjects (swimmers) are also included.

SUBJECTS AND METHODS

Subjects. Twelve untrained (UT) and 12 strength- trained (ST) subjects, as well as four endurance-trained

(swimmers, ET) subjects were studied. The average age, height, and weight were 26 t 1 (SE) yr, 1.82 t 0.01 m, and 70 t 2 kg for UT; 25 t 1 yr, 1.77 t 0.02 m, and 88 + 4 kg for ST, and 19 t 1 yr, 1.83 t 0.01 m, and 72 t 2 kg for ET. The average training period for ST was 8.6 yr. During the months preceding the biopsy sampling, their average training program involved the deltoid muscle in -18 sets of repetitions with a load that could be lifted a maximum of 5 times (5 RM), 5 times per week. Five of the strength-trained subjects had never used anabolic steroids, whereas six had made use of these agents for shorter periods >l yr before the biopsy sampling. Only one subject had used anabolic steroids during the year preceding the biopsy sampling. The ET subjects were swimmers who averaged 30 km (-18 miles) of swimming per week. The subjects were informed con- cerning the procedure and risks involved in the experi- ments before they volunteered. The study was approved by the Committee on Ethics of the Karolinska Institute.

Biopsy procedure. Muscle biopsies were taken from the middle portion of the deltoid muscle by means of the needle biopsy technique (8). All samples were from the nondominant arm. No training of the deltoid muscle had been undertaken during a period of 24 h before the biopsy sampling. Samples for enzyme assays were frozen im- mediately in liquid nitrogen and stored at -80°C until analysis. Samples for histochemical analyses were mounted in an embedding medium (Tissue-Tek II, OCT compound, Lab-Tek Products, Naperville, IL) frozen in Freon-12 (Frigen 12, Schiessl, Oberhaching, FRG), which had been cooled to its freezing point (158°C) with liquid

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nitrogen and stored at -80°C for subsequent analyses. Histochemical analysis. Serial transverse sections

(10

pm) were cut with a microtome at -20°C and stained for myofibrillar adenosine triphosphatase (ATPase) to iden- tify fiber types

(11, 12, 14)

and by the amylase-periodic acid-Schiff (PAS) method to visualize capillaries (2). Capillary counts were performed as described by Ander- sen and Henriksson (3). Photos of sections stained for myofibrillar ATPase (pH

4.6)

were used to measure cross-sectional fiber areas on an area measuring tablet (Apple Computer). The determinations of muscle fiber type distribution were based on 240 t 20 fibers. The number of capillaries per square millimeter was counted for a cross-sectional area of 0.41 t 0.06 mm’, containing 70 t 9 muscle fibers. The number of fibers included in the measurements of the fiber areas ranged between 12 t

2

and

20

t

1

for the different fiber types. Mean fiber area was calculated on the basis of the percentages of each fiber type and the corresponding fiber type area.

Enzyme assays. Muscle samples were weighed at

-20°C and homogenized

(1:100

wt/vol) by hand in Pot- ter-Elvehjem homogenizers containing ice-cooled potas- sium phosphate buffer (0.3 M, pH 7.7) with 0.05% (wt/ vol) bovine serum albumin. The homogenate was stored at -80°C until analyzed (the exception was cytochrome bg reductase; see below). Activities of the following enzymes were analyzed spectrophotometrically according to the references cited: 6-phosphofructokinase (PFK, EC

2.7.1.11;

Ref. 23), citrate synthase (CS, EC 4.1.3.7; Ref. l), 3-hydroxyacyl-CoA-dehydrogenase (HAD, EC 1.1.1.35; Ref. 5), mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH, EC

1.1.99.5;

Ref.

20), cyto-

plasmic glycerol-3-phosphate dehydrogenase (cGPDH, EC

1.1.1.8;

Ref. 6), aspartate aminotransferase (ASAT, EC 2.6.1.1; Ref. 7), and malate dehydrogenase (MDH, EC 1.1.1.37; Ref. 13). The cytoplasmic and mitochondrial isozymes of malate dehydrogenase (cMDH, mMDH) were assayed as described in detail by Schantz (29). The activity of cytochrome bg reductase (EC 1.6.2.2) was determined with NADH as substrate, and the reduction of cytochrome c was determined spectrophotometrically in a medium containing rotenone and KCN (34). For these assays, homogenate prepared from frozen muscle samples was used. That this procedure was adequate for measuring the activity of all cytochrome bs reductase molecules was indicated by the fact that freezing and thawing of the homogenate (i.e., disruption of any re- maining intact mitochondria) did not result in a different activity. An addition of succinate (34) to the assay system described above (i.e., NADH + succinate as substrates) increased the activity to the same amount as with succinate as the sole substrate. Thus the assay for cytochrome bs reductase appeared to be specific for this enzyme and did not involve reduction of cytochrome c due to electron flow in the respiratory chain. All enzymes were assayed in duplicate at 25°C (cytochrome bg reductase, 30°C) under conditions in which enzyme concen- trations were proportional to enzyme activities. Levels of mitochondrial and cytoplasmic ASAT isozymes (mASAT, cASAT) were determined through electropho- retie separation, enzymatic staining, and densitometric

scanning, essentially as described by Schantz and Hen- riksson (31). The areas under the absorbance curves were used to calculate the mASAT/cASAT scanning area ratios, which together with the total ASAT activity, were used to calculate absolute isoenzyme activities. Linearity between enzyme concentration and mASAT/cASAT ratios was obtained under the assay conditions imposed.

Statistics. Results are expressed as means t SE. Wil- coxon rank sum test for unpaired data was used for comparisons between the untrained and strength-trained subjects. The values for the endurance-trained group were not compared statistically with those of the other groups.

RESULTS

Values for muscle fiber type distribution, capillary supply, fiber areas, and oxidative as well as glycolytic enzymes are presented in Tables 1 and 2. Only the differences between UT and ST groups were evaluated statistically.

Malate-aspartate shuttle enzyme levels. Although the mean values for the activities of mMDH, cMDH, mASAT, and cASAT were slightly higher (16-35%) in ST than in UT, these differences were not statistically significant (Table 2). Neither did the cMDH/mMDH,

cMDH/cASAT, cMDH/mASAT, mMDH/mASAT, and

mMDH/cASAT activity ratios differ between the groups, whereas a 9% higher (P < 0.05) cASAT/mASAT ratio was noted in ST.

a-Glycerophosphate shuttle enzyme levels. Nearly equal enzyme activities were noted in UT and ST with regard to mGPDH and cGPDH (Table 2). Thus no difference was noted between the cGPDH/mGPDH activity ratios for the groups.

Enzyme levels of cytochrome bb reductase. No significant difference was noted in cytochrome bg reductase activities between UT and ST (Table 2). The activity of cytochrome b5 reductase correlated significantly (r = 0.59, P C 0.01) with the activity of the mitochondrial marker enzyme citrate synthase (Fig. 1).

TABLE

1.

Muscle fiber type distribution, capillary supply, and fiber areas in UT, ST,

and ET human deltoid muscle

UT ST ET

(n = 12) (n = 12) (n = 4)

Fiber type, %

I 59k3 67&2* 72k5

IIa 23zt2 21k3 27k5

IIb M&3 12k2" O.lkO.1 Capillaries/fiber 1.6kO.l 1.9kO.l’ 2.4kO.l Capillaries/mm2 325t24 300*13 405k32 Fiber area, pm2

I 4,430&210 5,520&400 5,720+210 IIa 5,100&270 7,350*560* 6,900&520 IIb 4,330&230 7,270*900t

Mean fiber area 4,600f200 6,150+450* 6,080~180 Values are means & SE. Number of subjects included in the fiber area measurements was 10 for untrained (UT), 9 for strength trained (ST), and 3 for endurance trained (ET). Data on ET were not evaluated statistically. * Tendency toward different (P < 0.1) value compared with UT. t Significantly different value (P < 0.05) compared with UT.

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TABLE

2.

Enzyme activities in UT, ST, has previously been reported for citric acid cycle enzymes

and ET human deltoid muscle

(19, 26, 37, 39).

Enzymes UT ST ET (n = 12) (n = 12) (n = 4) ST/UT ET/UT PFK HAD cs mMDH cMDH mASAT cASAT mGPDH cGPDH Cytochrome bs reductase 30.5t1.6 9.3zko.7 12.9kl.O 69.3k4.5 159tlO 30.75fr1.9 27.5rt1.8 0.49t0.04 17.6k0.8 3.020.2 29.7k1.4 10.3kO.6 15.7t1.3 80.6k6.0 188~14 38.6k4.7 37.2t4.5 0.51t0.08 18.4k1.9 3.3k0.2 33.6t4.9 0.97 1.10 12.8k0.9 1.11 1.38 28.8zk3.8 1.22 2.23 138t13 1.16 1.99 252k13 1.18 1.58 61.1t9.1 1.26 1.99 42.6t6.0 1.35 1.55 0.49kO.07 1.04 1.00 17.8k0.7 1.05 1.01 3.8tO.l 1.10 1.27 Values are means 2 SE. Enzyme activities are expressed as prnol. g wet wt-’ . min-‘. PFK, HAD, and CS are marker enzymes for glycolysis, fatty acid oxidation, and the citric acid cycle, respectively. mMDH, cMDH, mASAT, and cASAT are malate-aspartate shuttle enzymes. mGPDH and cGPDH are ar-glycerophosphate shuttle enzymes. See

DISCUSSION for explanation of the function and location of cytochrome

b5 reductase. There were no significant differences between the values of UT and ST. Number of subjects included in the cytochrome b5

reductase measurements was 10 for both UT and ST. The data on ET were not evaluated statistically. See text for further explanation of abbreviations. x c, Y - .0819 * x t 2.18 .e > s- r- .591 ‘$ + : al 4- z c, Y u al L 2 2- 0 E 0 z : 1 - o = untrained c, + -strength trained A * - endurance trained w 0& I I I 1 1 I 0 5 10 15 20 25 30

Citrate synthase activity

FIG. 1. Citrate synthase and cytochrome bb reductase activities of

untrained, strength-trained, and endurance-trained human deltoid muscles. Enzyme activities are expressed as pmol l g wet wt? l min? DISCUSSION

With the aim of illuminating the effects of strength training per se on muscle adaptation, we chose to study a nonpostural muscle, deltoid muscle, of the nondominant arm. The rationale for this was to diminish con- ceivable effects due to usage of muscles in everyday life.

Enzyme ZeveZs. The main findings of this study were that the enzyme levels of the NADH shuttles and cytochrome bg reductase did not differ significantly between the UT and ST subjects. Increased levels of malate- aspartate shuttle enzymes but unaltered levels of CX- glycerophosphate shuttle enzymes have been reported after endurance training (rat,

17, 18;

man, 33; cf. values for the endurance-trained subjects in the present study). Thus strength and endurance training appears to affect the levels of malate-aspartate shuttle enzymes differ- ently. A corresponding difference in training response

In line with the present results for the marker enzymes for fatty acid oxidation, citric acid cycle and glycolysis, no, or only minor, increases

(40%)

in the levels of these enzymes, as well as of high-energy phosphate transfer enzymes, have previously been reported in conjunction with strength training

(19,

26, 37, 39). On the contrary, decreases in mitochondrial volume density with strength training have been reported (triceps brachii,

25%,

Ref.

22;

vastus lateralis,

lo%,

Ref.

21),

and they were inter- preted as a “dilution effect” due to muscle cell hypertrophy. In fact, it was calculated that the absolute volume of mitochondria was unchanged

(21).

Reichmann et al. (24) reported that increases in oxidative enzyme levels on chronic electric stimulation are matched by increases in volume density of mitochondria of the same relative magnitude. The discrepancy between data on oxidative enzyme levels and mitochondrial volume density in con- nection with strength training evokes the question of whether changes in these variables are not always par- allel. Studies on strength training combining measurements of these variables are therefore warranted.

The great need for aerobic reoxidation of extramito- chondrial NADH during exercise focuses interest on cytochrome bs reductase located in the outer mitochondrial membrane, since reduced cytochrome & can transfer electrons to cytochrome c in the intermembrane space, which in turn can be oxidized through cytochrome oxidase in the inner mitochondrial membrane (9, 35). The question as to whether the level of cytochrome bs reductase in the outer mitochondrial membrane can be altered with physical training is complicated, however, by the fact that this enzyme is also found in the micro- somal fraction of skeletal muscle

(10).

Thus the present data from crude muscle homogenate should be inter- preted with caution. The positive correlation between the levels of cytochrome bg reductase and the mitochondrial marker enzyme citrate synthase (Fig. 1) does indicate, however, the possibility of enhanced levels of the mitochondrial fraction of cytochrome bs reductase in conjunction with physical activity that induces increases in oxidative enzyme levels. To further elucidate this matter, analyses of both the cytochrome bs reductase activity per milligram of outer mitochondrial membrane protein and the total amount of outer mitochondrial membrane are needed.

Capillary supply. A tendency toward a larger number of capillaries per fiber (20%) was noted in ST. But because of their greater fiber areas, the number of capillaries per square millimeter did not differ between the groups. Previous reports have described similar (28, 38) or a larger (27) number of capillaries per fiber in strength-trained subjects than in untrained ones, whereas the number of capillaries per square millimeter has been reported to be lower (38) or the same (27,28).

Increases in the number of capillaries per fiber with endurance training have been taken as an index of capillary neoformation (3). However, a prerequisite for using this index is that the training does not involve alterations in fiber number. Whether the hypertrophy of human

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skeletal muscle reflects hypertrophy of an unaltered number of fibers or partially involves hyperplasia is still the subject of debate (26).

With the assumption that no increase in fiber number has occurred, taken together, the present and previous results indicate that strength training might exert a weak stimulus for neoformation of capillaries, which, however, does not result in an increased number of capillaries per square millimeter

growth.

due to the concomitant fiber area

Fiber type composition. Both endurance training and high-intensity intermittent (ice hockey) training can in- duce transformation of fast-twitch type IIb fibers into fast-twitch type IIa fibers (4, 16). The present findings of a tendency toward a lower type IIb percentage in ST poses the question as to whether strength training might stimulate the same conversion. Support for this can, in fact, be found in the literature. Edstrom (15) noted the same type IIa dominance within the type II population in the quadriceps femoris of ST subjects as described for ET subjects. Our own data on the quadriceps femoris (32) and the triceps brachii muscles (28) demonstrate type IIb percentages in ST subjects (5 and 0.5%, respectively) that are clearly lower than those normally seen in these muscles, i.e., lo-20% (3, 4, 30, 32). It therefore appears likely that fiber type IIb to type IIa conversion might occur with a greater spectrum of modes of activa- tion than it is generally associated with.

The main conclusion from this study is that levels of NADH shuttle enzymes and cytochrome bb reductase did not differ significantly between untrained and strength- trained subjects. Thus, contrary to the effect of endur- ante training, strength training does not seem to stimulate to significantly increased levels of malate-aspartate

shuttle enzymes. -

This study was supported by grants from the Karolinska Institute’s Research Funds, the Research Council of the Swedish Sports Federa- tion, and Swedish Medical Research Council Grant B87-14x-07917.

Address reprint requests to P. G. Schantz.

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