The presence, operation and role of the sphingomyelin-signalling pathway have been investigated in different cell types (1-4). Ceramide is the principal second messenger in this pathway. Ceramide is mostly formed in the plasma membrane, from sphingomyelin located there, by action of the enzyme neutral, Mg++ - dependent sphingomyelinase. It can also be formed in endosomes and lysosomes, by the action of acidic sphingomyelinase. Activation of both enzymes is a receptor- mediated process (1,2). A number of stimuli have been shown to increase formation of ceramide in the cell. They have been classified into four groups: inducers of differentiation, inducers of apoptosis, damaging agents and inflammatory cytokines (5). Ceramide is deacylated in a reaction catalysed by the enzyme ceramidase. Sphingosine, the product of deacylation and its phosphorylated form, sphingosine-1-phosphate are also very biologically active compounds (1,2). We have previously described the sphingomyelin-signalling pathway in different skeletal muscle types in the rat (6). We identified and quantified twelve different ceramides and twelve different sphingomyelins based on their fatty acid residues. The major ceramide-saturated fatty acid residues were palmitiate and stearate whereas oleate was the major unsaturated acid residue in each muscle type. The total content of ceramide-fatty acids in the fast-twitch glycolytic muscle was lower than in the fast-twitch oxidative-glycolytic and slow-twitch oxidative muscles. Prolonged exercise of moderate intensity resulted in a considerable reduction in the content of ceramide in each muscle type. The activity of neutral, Mg++ dependent sphingomyelinase was reduced after exercise in the two high oxidative muscles and remained stable in the high-glycolytic muscle (6). Exercise also increased the content of sphinganine (the key precursor of ceramide on the de novo-synthesis pathway) and sphingosne (7). Endurance training has been shown to induce several changes in lipid metabolism in skeletal muscles. The major one is increased capacity of the muscles to utilize free fatty acids as a source of energy. Also, certain changes in phospholipid content and composition and the content of triacylglycerols have been described (8-12). The aim of the present study was to examine the effect of endurance training on the functioning of the sphingomyelin-singalling pathway in the muscles. Specifically, the content and composition of ceramide-, and sphingomyelin-fatty acids, the content of sphinganine and sphingosine and activity of neutral, Mg++ -dependent sphingomyelinase were investigated in different skeletal muscle types of the rat.


MATERIALS AND METHODS
The experiment were carried out on male Wistar rats, 250-280 grams of body weight, fed ad libitum on a commercial pellet diet for rodents. The experimental protocol was approved by the Ethical Committee for Animal Studies in the Medical University of Bia³ystok. The animals were trained on an electrically driven treadmill according to the following protocol (13):1st week- one hour daily at a speed of 960m/h. The same running time was applied during successive weeks but the running speed was increased as follows: 2nd week- 1200m/h, 3rd week 1440m/h, 4th-6th week 1680m/h. 24h after the last exercise bout in the training programme the rats were anaesthetized along with the controls with pentobarbital sodium administered intarperitoneally at a dose of 80mg/kg. Samples of the soleus, red and white section of the gastrocnemius were taken. These muscles are composed mostly of slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively (14,15). The muscle samples were cleaned of any visible non-muscle tissue and frozen in liquid nitrogen. Lipids were extracted with chloroform/methanol (2:1) (16) as described by (17). The samples were pulverized in an aluminium mortar with a stainless steel pestle precooled in liquid nitrogen and transferred to tubes containing methanol at -21° C. Buthylated hydroxytoluene (Sigma), 30mg/100ml, was added to methanol as an antioxidant. The tubes were warmed to room temperature and chloroform was added. Ceramide and sphingomyelin were isolated by means of thin layer chromatography as described previously (6). They were identified according to appropriate standards (Sigma), which were run along with the samples. The bands containing the examined compounds were scraped off the plates into tubes containing methylpentadecanoic acid (Sigma) as an internal standard. Fatty acids were transmethylated using 14% boron fluoride in methanol (18). The resulting methyl esters were identified and quantified by means of gas-liquid chromatography according to the appropriate internal standards (Sigma). The content of sphinganine and sphingosine present in the chloroform layer was determined by means of high performance liquid chromatography (19, 20).

The activity of neutral Mg++ dependent sphingomyelinase was determined spectophotometrically using trinitrophenylaminolaouroyl (THPAL)-Sphingomyelin (Sigma) as the substrate (21). The protein content was determined according to Lowry et al. (22).

The results obtained were evaluated statistically using the Student t-test for unpaired data. P<0.05 was considered significant. N=10 in each group. The results are presented as means ± standard deviation.


RESULTS
Ceramide-fatty acids (Table 1). Training reduced the total content of ceramide-fatty acids in each muscle. It also induced several changes in the composition of ceramide-fatty acids. The major change was a reduction in the percentage content of ceramide containing stearic acid residue and elevation in the percentage content of ceramide containing oleic acid residue in each muscle.

Table 1. The content of individual ceramide - fatty acids in different skeletal muscle types in untrained and trained rats.

Values (nmol/g of wet weight) are mean ± SD. G - gastrocnemius; The acids are: myrystic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), eicosapentaenoic (20:5), behenic (22:0), docosaheksaenoic (22:6), nervonic (24:1). a-p < 0.05, b-p<0.02, c-p < 0.01, d-p<0.001, vs. the respective value at rest; Total - the sum of individual ceramide-fatty acids;

Sphingomyelin-fatty acids (Table 2). Training reduced the total content of sphingomyelin-fatty acids in each muscle. It also produced several changes in the composition of sphingomyelins. In the solues, the major change was a reduction in the percentage content of sphingomyelin containing stearic acid and elevation in the content of sphingomyelin containing oleic acid residue. In the red gastrocnemius only relatively minor changes in the composition of the sphingomyelins occurred. In the white gastrocnemius, the percentage content of sphingomyelin containing palmitic and nervonic acid residues decreased and the percentage content of sphingomyelin containing stearic and oleic residue increased.

Table 2. The content of individual sphingomyelin - fatty acids in different skeletal muscle types in untrained and trained rats.

Values (nmol/g of wet weight) are mean ± SD. G - gastrocnemius. The acids are: myrystic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidonic (20:4), eicosapentaenoic (20:5), behenic (22:0), docosaheksaenoic (22:6), nervonic (24:1). b-p < 0.02, c-p < 0.01, d-p<0.001, vs. the respective value at rest; Total - the sum of individual sphingomyelin-fatty acids;

Sphinganine and sphingosine (Fig. 1). Training increased the content of sphinganine but it had no effect on the content of sphingosine in each muscle.

Fig. 1. Figure 1. Effect of endurance training on the content of free sphinganine (A) and free sphingosine (B) in skeletal muscles of the rat. z-p<0.001, vs. the respective value at rest;

Activity of neutral, Mg++ dependent sphingomyelinase (Fig. 2). Training increased activity of the enzyme in each muscle.

Fig. 2. Effect of endurance training on the activity of neutral Mg2+-dependent sphingomyelinase in skeletal muscles of the rat. x-p<0.02, y-p<0.01, z-p<0.001 vs. the respective resting value;

DISCUSSION
The results obtained clearly show that training affected metabolism of ceramide in the skeletal muscles. This was manifested in the reduction in the total content of ceramide-fatty acids and changes in the composition of ceramide-fatty acids in each skeletal muscle type. We have shown, in the previous study, that a single bout of prolonged exercise reduces the sphingomyelin - signalling pathway activity. This manifests in a reduction in the content of ceramide in each muscle type and reduction in the activity of neutral, Mg++ dependent sphingomyelinase in the soleus and red gastocnemius (6). The data presented here show that training reduces the content of ceramide, in spite of an elevation in the enzyme activity and reduction in the content of sphingomyelin. In accordance with present knowledge, the two latter events should have resulted in elevation in the content of ceramide. There must, therefore, be other mechanisms responsible for the reduction in the content of ceramide as a result of physical training. Another cause for the reduction in the content of ceramide could be its augmented deacylation to sphingosine. It should be mentioned that after prolonged exercise of moderate intensity and after short-term high-intensity contractile activity induced by stimulation of the sciatic nerve, the content of sphingosine has been was found to increase considerably (7). We, therefore, measured the content of this compound in the muscles, but found that it was not affected significantly by training. This would argue against an increased rate of ceramide deacylation in the trained muscles. Of course, this is not final proof, since sphingosine is further converted to sphingosine-1-phosphate in a reaction catalysed by the enzyme sphingoid base kinase (1,2). If the rate of conversion of sphingosine to sphingosine -1-phosphate was augmented by training, the stable content of sphingosine would not reflect the rate of its production from ceramide, i.e. it would not reflect the rate of ceramide deacylation. However, at present we do not have any proof in hands to support this hypothesis, though it is a very likely one. Ceramide is also synthesized de novo from serine and palmitoyl-CoA. One of the major intermediates along this pathway is sphinganine. Sphinanine is acylated to dihydroceramide, which is the direct precursor of ceramide (1,2). We have previously found that the content of sphinganine in the muscles was markedly elevated both after prolonged exercise of moderate intensity and after short- term high intensity contractile activity (7). The present data show that the content of sphinganine is also elevated in trained muscles, thus suggesting increased de novo synthesis of ceramide. As mentioned in the introduction, ceramide is also formed from sphingomyelin in endosomes and lysosomes by acidic sphingomyelinase, which is present there (1-4). This pathway of ceramide formation has not been studied in skeletal muscles, as yet. However, it maybe the case that it is inhibited by training, which could contribute to a reduction in the content of ceramide. The other routes for ceramide removal are: conversion to ceramide-1-phosphate, glucosyl-ceramide, galactosyl-ceramide and ceramide-phosphoethanolamine (1,2). Contribution of these pathways to regulation in the content of ceramide has not been recognized, so far. It remains an open question, as to whether they contribute to the reduction in the content of ceramide in the muscles after training.

Ceramide exerts numerous effects in the cell. The major ones are: induction of differentiation, inhibition of proliferation, induction of apoptosis and regulation of inflammatory processes (1-4). A role for ceramide in skeletal muscles has not been established, as yet. However, muscular training does not change the number of myocytes and thus a reduction in the content of ceramide cannot have an impact on any of the above listed processes. The content of ceramide in skeletal muscles in insulin-resistant Zucker rats is elevated (23). It has also been shown to be inversely related to 2-deoxy-glucose uptake by the muscles after prolonged exercise (6). This compound has also been shown to inhibit insulin-stimulated glucose uptake by the myocytes (24-27). It probably also mediates the inhibitory action of palmitate on glucose uptake by C2C12 myotubes (27). Training increases glucose uptake by skeletal muscles (28). Therefore, a reduction in the content of ceramide after training could partially contribute to this phenomenon.

In summary, we have shown that endurance training reduces the content of ceramide in each skeletal muscle type. Concomitantly, it elevates activity of neutral, Mg++ dependent sphingomyelinase and the content of sphinganine, a precursor of ceramide. The content of sphingosine, the product of ceramide catabolism remains stable. The reduction in the content of ceramide may contribute to increased glucose uptake in the trained muscles.


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