The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle

The effects of testectomy and subsequent administration of testosterone propionate on the activity of the alkaline proteinases in rat skeletal muscle were investigated. Castration of the mature rat was followed by a short-term delay in protein accretion in skeletal muscle tissue as measured by the protein/DNA ratio and was paralleled by a 2–3 fold increase in specific activity of the alkaline proteinase(s). This increase of proteolytic activity was equally significant when expressed relative to μg DNA. Although the gain in body weight was significantly lower in the castrated rats, nevertheless the protein/DNA ratio in muscle after 6 weeks approximated the values of sham-operated control rats without normalization of the proteolytic activity.Treatment of the castrated rats with testosterone propoinate resulted in restoring normal levels of previously elevated levels of alkaline proteolytic activity in muscle tissue. The normalization of enzyme activity as well as protein accretion in muscle was dose-dependent. Treatment of the rats with a low dose (0.1 mg/day) of testosterone propionate failed to restore the proteolytic activity, but led to a small increase of the protein/DNA ratio as well as to a progressive increase in body weight. These data indicate a regulatory role of testosterone in the adaptive behaviour of the alkaline proteolytic system in rat skeletal muscle.     

Exercise alters the profile of phospholipid molecular species in rat skeletal muscle.

We have determined the effect of two exercise-training intensities on the phospholipid profile of both glycolytic and oxidative muscle fibers of female Sprague-Dawley rats using electrospray-ionization mass spectrometry. Animals were randomly divided into three training groups: control, which performed no exercise training; low-intensity (8 m/min) treadmill running; or high-intensity (28 m/min) treadmill running. All exercise-trained rats ran 1,000 m/session for 4 days/wk for 4 wk and were killed 48 h after the last training bout. Exercise training was found to produce no novel phospholipid species but was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in glycolytic (white vastus lateralis) than in oxidative (red vastus lateralis) muscle fibers. The largest observed change was a decrease of approximately 20% in the abundance of 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine [PE(18:0/22:6); P < 0.001] ions in both the low- and high-intensity training regimes in glycolytic fibers. Increases in the abundance of 1-oleoyl-2-linoleoyl phopshatidic acid [PA(18:1/18:2); P < 0.001] and 1-alkenylpalmitoyl-2-linoleoyl phosphatidylethanolamine [plasmenyl PE (16:0/18:2); P < 0.005] ions were also observed for both training regimes in glycolytic fibers. We conclude that exercise training results in a remodeling of phospholipids in rat skeletal muscle. Even though little is known about the physiological or pathophysiological role of specific phospholipid molecular species in skeletal muscle, it is likely that this remodeling will have an impact on a range of cellular functions.     

Greater effect of diet than exercise training on the fatty acid profile of rat skeletal muscle.

We determined the interaction of diet and exercise-training intensity on membrane phospholipid fatty acid (FA) composition in skeletal muscle from 36 female Sprague-Dawley rats. Animals were randomly divided into one of two dietary conditions: high-carbohydrate (64.0% carbohydrate by energy, n = 18) or high fat (78.1% fat by energy, n = 18). Rats in each diet condition were then allocated to one of three subgroups: control, which performed no exercise training; low-intensity (8 m/min) treadmill run training; or high-intensity (28 m/min) run training. All exercise-trained rats ran 1,000 m/session, 4 days/wk for 8 wk and were killed 48 h after the last training bout. Membrane phospholipids were extracted, and FA composition was determined in the red and white vastus lateralis muscles. Diet exerted a major influence on phospholipid FA composition, with the high-fat diet being associated with a significantly (P < 0.01) elevated ratio of n-6/n-3 FA for both red (2.7-3.2 vs. 1.0-1.1) and white vastus lateralis muscle (2.5-2.9 vs. 1.2). In contrast, alterations in FA composition as a result of either exercise-training protocol were only minor in comparison. We conclude that, under the present experimental conditions, a change in the macronutrient content of the diet was a more potent modulator of skeletal muscle membrane phospholipid FA composition compared with either low- or high-intensity treadmill exercise training.     

The influence of ethylenediaminetetraacetate on white skeletal muscle myosin.

Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.     

Effects of paraquat on mitochondria of rat skeletal muscle

The effect of the herbicide paraquat (N,N'-dimethyl 4,4'-bipyridium), known to damage the lipid cellular membrane by peroxidation with superoxide radicals and a singlet oxygen, was investigated on skeletal muscle mitochondria. Minced rat gastrocnemius muscles were incubated in 8 mM paraquat solution. Mitochondrial fractions prepared from the incubated muscles were examined with respect to respiratory function and the enzyme activity of cytochrome c oxidase and succinate-cytochrome c reductase in the electron transport chain. The ADP/O ratio, RCR, and state 3 rates (= oxygen consumption in state 3) decreased gradually. State 4 rates (= oxygen consumption in state 4) increased in the initial stages and decreased after longer incubations. Enzyme activities gradually increased. These results suggested that paraquat damaged the mitochondrial membrane and disrupted oxidative phosphorylation in the early stage of incubation. Also, the electron transport chain was accelerated in the earlier stage and broken following a longer incubation. The inhibitory modality of paraquat on mitochondrial respiration was shown to be different from that of other known inhibitors.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Comparison of the phospholipid and triacylglycerol fatty acid profile of rat serum, skeletal muscle and heart

Although several studies have analyzed the fatty acid profile of phospholipids (PL) and, to a lesser degree, triacylglycerols (TG) in one or more tissues concurrently, a systematic comparison of the fatty acid composition of different tissues and/or lipid classes is lacking. The purpose of the present study was to compare the fatty acid composition of major lipid classes (PL and TG) in the rat serum, soleus muscle, extensor digitorum longus muscle and the heart. Lipids were extracted from these tissues and analyzed by a combination of thin-layer chromatography and gas chromatography. We found many significant differences in various tissues and lipid classes. Serum had the most distinct fatty acid profile in PL but this "uniqueness" was less apparent in TG, where differences among tissues were in general less frequent than in PL. These two skeletal muscles exhibited similar fatty acid composition in both lipid classes despite their different muscle fiber type composition, denoting that fiber type is not a major determinant of the fatty acid composition of rat skeletal muscle. The fatty acid profile of heart PL was the most different from that of the other tissues examined. PL were rich in polyunsaturated fatty acids, whereas TG were rich in monounsaturated fatty acids. Although the reasons for the differences in fatty acid profile among the tissues examined are largely unknown, it is likely that these differences have an impact upon numerous biological functions.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Cytomorphometry of skeletal muscle: The influence of age and testosterone on the rat M. levator ani

Summary The levator ani muscle of the rat was examined by correlated light and electron microscopic morphometry. Corrections were made for shrinkage, compression, and differences in stretching. Age, castration, and subsequent testosterone treatment do not affect the fiber number, the filament lattice, and the size of the filaments and myonuclei. The fibers in intact growing males increase in width and length. The number of myonuclei rises, although relatively slower than the amount of contractile material.Castration, performed at six weeks, partially suppresses fiber growth. The increase of mean fiber width is more strongly inhibited than that of fiber length. Myonuclear multiplication is almost completely arrested in castrates, and the amount of contractile material per myonucleus is lower than in intact males of equal age.Testosterone, administered at about two months following orchidectomy, highly accelerates the transversal fiber growth, but fiber length is not significantly influenced. Between the fourth and seventh day of treatment a marked increase in myonuclear number occurs.Analysis of the frequency distribution of the individual fiber widths, which is logarithmic-normal in intact males, revealed that the hormonal influence on the net result of protein anabolism and catabolism markedly differs in the various fibers of a single muscle.With the technical assistance of Tineke J. Hoogenboezem.     

Isoproterenol ameliorates workstress-induced rat skeletal muscle degeneration

Beta-Agonists though have been widely studied for their protein anabolic effects in skeletal muscles, but the lipid status under work stress and agonist treatment have not been understood well in the skeletal muscles and heart of rat. In the present study, adult male Wistar rats were subjected to work overload stress and beta agonist isoproterenol treatment (2 mg kg(-1) day(-1) intraperitoneally) to examine, whether it attenuates work stress-induced changes or not. Simultaneously, beta2 antagonist butoxamine (2 mg kg(-1) day(-1) intraperitoneally) was administered to another isoproterenol-treated group. Work stress led to myofibrillar degeneration as well as rapid utilization of lipid to meet increased energy demands and for muscle repair, which was reflected through histochemical localization of lipids and biochemical estimation of cholesterol and triglycerides. Significantly decreased cholesterol levels in skeletal muscles and heart muscles were noticed. As expected, isoproterenol reversed the conditions by raising cholesterol and triglyceride levels significantly in the skeletal muscles and also by ameliorating the degenerative changes in muscle fibres as induced by work overload. However, severe accumulation of lipids in heart infers towards deleterious effects of isoproterenol on heart and thus remains a limiting factor for its immediate clinical application. Further research is needed to separate desirable effects of beta agonists on skeletal muscles from any undesirable effects on the heart, so as to optimize their therapeutic potential.     

Cortisol binding in rat skeletal muscle.

Studies of the reversible binding of [3H]cortisol by rat gastrocnemius muscle cytoplasm in vitro reveal specific binding in the 27,000 times g supernatant fraction at 0 degrees. The [3H]cortisol-binding molecule had an apparant Kd value of 1.7 times 10-7 M and the number of binding sites was 0.99 pmol per mg of cytosol protein. Only a single class of [3H]cortisol-binding sites could be detected, whose protein nature was suggested by its susceptibility to nagarse. The [3H]cortisol-protein complex sedimented at similar to 4 S in a 5 to 20% sucrose gradient either in the presence or absence of 0.3 M KCl. Binding increased more than 2-fold in adrenalectomized rats and was markedly reduced in the muscle of rats pretreated with cortisol. In contrast to the binding of [3H]dexamethasone and [3H]triamcinolone acetonide to receptor proteins in muscle, no correlation was found between the ability of various steroids to complete wtth [3H]cortisol binding and their glucocorticoid potency: [3H]cortisol binding was inhibited by a 1000-fold higher concentration of unlabeled cortisol and progesterone but not by dexamethasone or triamcinolone acetonide. It is therefore suggested that the [3H]cortisol-binding reaction is not directly involved in the biological effects of all potent glucocorticoids in skeletal muscle. The [3H]cortisol-binding protein in muscle cytosol could not be unequivocally distinguished from rat plasma corticosteroid-binding globulin, because both had similar steroid specificity and temperature stability, were not markedly affected by--SH reagents, and displayed similar sedimentation properties.     

Effect of contractile activity on rat skeletal muscle beta-adrenoceptor properties

The effect of fiber type and endurance exercise training on skeletal muscle beta-adrenoceptor properties were assessed using a direct radioligand binding technique. Six separate muscles, composed of a variety of different fiber types, were examined in treadmill trained and sedentary rats. In trained animals, sarcolemmal preparations from heart and slow twitch soleus muscle exhibited a significantly greater receptor concentration than membranes from white fast twitch glycolytic fibers of the vastus lateralis. No significant changes were observed between trained and sedentary rat muscle beta-adrenoceptor density (beta max, fmole/mg protein) or affinity (Kd, nM) within each muscle type, despite significantly increased myocardial/body weight ratios and skeletal muscle enzyme adaptations associated with the exercise program. These results suggest that muscle beta-adrenoceptor properties may be influenced in part by the motor nerve innervation to that muscle, and are further discussed with respect to a possible relationship between exercise intensity and receptor regulation.     

Eccentric exercise-induced injury to rat skeletal muscle

These experiments were designed to study skeletal muscle pathology resulting from eccentric-biased exercise in rats. The effects on the muscles of running on a treadmill on a 0 degrees incline (similar amounts of concentric and eccentric contractions), down a 16 degrees incline (primarily eccentric contractions), and up a 16 degrees incline (primarily concentric contractions) at 16 m . min-1 for 90 min were assessed by following postexercise changes in 1) plasma creatine kinase and lactate dehydrogenase activities, 2) glucose-6-phosphate dehydrogenase (G-6-PDase) activity (bio- and histochemically) in the physiological extensor muscles, and 3) histological appearance of the muscles. The data indicate the following. 1) Whereas all exercise protocols resulted in elevations of plasma enzymes immediately after running, only eccentric exercise caused late phase elevations 1.5-2 days postexercise. 2) Significant increases in muscle G-6-PDase activity, which were always associated with accumulations of mononuclear cells, always occurred within some muscles of each extensor group 1-3 days following downhill and uphill running and did not occur following level running; the increases in activity were usually of lower magnitude in the muscles of uphill runners than in those of downhill runners; the deeply located, predominantly slow-twitch muscles were most affected by both down- and uphill running. 3) Muscle histology demonstrated localized disruption of normal banding patterns of some fibers immediately after exercise and accumulations of macrophages in the interstitium and in some (less than 5%) muscle fibers by 24 h postexercise in the deep slow muscles of the antigravity groups. Although the data generally indicated that eccentric exercise causes greater injury to the muscles, questions remain.     

Chronic muscle stimulation increases lactate transport in rat skeletal muscle

The aim of this study was to examine the effects of chronic low frequency stimulation on the lactate transport across the plasma membrane of the tibialis anterior (TA) muscle of the rat. Stimulating electrodes were implanted on either side of the peroneal nerve in one hindlimb. Chronic stimulation (10 Hz, 50 psec bursts, 24 h/day) commenced 7 days after surgery, and were continued for 7 days. Animals were then left for 24 h, and thereafter muscles were obtained. Cytochrome C-oxidase activity was increased 1.9-fold in the stimulated TA compared to the control TA (p < 0.05). Lactate transport (zero-trans) was measured in giant sarcolemmal vesicles obtained from the chronically stimulated TA and the control TA. At each of the concentrations used in these studies a significant increase in lactate transport was observed: 2.8-fold increase at 1 mM lactate p < 0.05); 2-fold increases at both 30 mM and 50 mM lactate p < 0.05). These studies have shown that lactate transport capacity is markedly increased in response to chronic muscle contraction.     

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.