The sphingomyelin-signaling pathway has been described in many tissues. Ceramide is the main second messenger in this pathway. Ceramide has also been shown to be present in skeletal muscles; however, there are few data on the regulation of the content of ceramide in muscle tissue. Moreover, there are no data on the content of particular ceramides or their composition in the muscles. The aim of the present study was to examine the content and composition of fatty acids (FA) in ceramide and sphingomyelin moieties and the activity of neutral Mg2+-dependent sphingomyelinase in different skeletal muscle types of the rat at rest and after exhausting exercise of moderate intensity. The experiments were carried out on male Wistar rats, divided into two groups: 1) control and 2) exercised until exhaustion on a treadmill. Soleus and red and white gastrocnemius muscles were taken. Ceramide and sphingomyelin were separated by TLC. The content of individual FA in the two compounds was determined by gas-liquid chromatography. Twelve different ceramides and sphingomyelins were identified and quantified in each muscle type according to the FA residues. Saturated FA consisted of 68, 67, and 66% of total ceramide-FA and 75, 77, and 78% of total sphingomyelin-FA in soleus and red and white gastrocnemius, respectively. The total content of ceramide- and sphingomyelin-FA and the activity of sphingomyelinase were highest in the soleus and lowest in the white gastrocnemius. Exercise resulted in a reduction in the total content of ceramide- and sphingomyelin-FA in each muscle. This was accounted for mostly by a reduction in content in the amount of saturated FA. Exercise reduced the activity of neutral Mg2+-dependent sphingomyelinase in the soleus and red gastrocnemius and did not affect its activity in the white gastrocnemius. We conclude that the sphingomyelin-signaling pathway is present in skeletal muscles and that it is influenced by prolonged exercise.
- sphingomyelin-signaling pathway
a novel transmembrane signaling pathway, called the sphingomyelin-signaling pathway, has been described and characterized in recent years. Sphingolipid sphingomyelin, which is located mostly in the outer layer of the plasma membrane, is hydrolyzed by the enzyme neutral Mg2+-dependent sphingomyelinase to phosphorylcholine and ceramide. Ceramide may be further converted to sphingosine and a long-chain fatty acid by the enzyme ceramidase. Ceramide has been shown to have the role of the second major messenger in this pathway (5, 7, 13, 21). Several stimuli increase the production of ceramide. They have been grouped as follows: inducers of apoptosis, inducers of differentiation, damaging agents, and inflammatory cytokines. Examples of the stimuli are tumor necrosis factor-α, interleukin-1, interferon-γ, nerve growth factor, and 1,25-dihydroxyvitamin D3 (19). There are very few reports concerning the content of ceramide in the skeletal muscles. The content of ceramide in fast-twitch red muscle (plantaris) was shown to be higher than in slow-twitch red muscle (soleus). It was elevated in both muscles in obese Zucker rats (29). Treatment with insulin, anoxia, and electrical stimulation did not affect the content of ceramide in the calf muscles of the rat (27). Denervation was reported both not to affect (29) and to increase (27) the content of ceramide in the rat soleus and plantaris muscle. Palmitate, but not oleate or linoleate, increases the content of ceramide in incubated C2C12myotubes (24). Studies on human plasma, human brain, and mouse liver showed that ceramides contain different fatty acids (9, 22, 23). So far, however, no data on the content and composition of ceramide-fatty acids in skeletal muscle are available. Moreover, in the aforementioned study (27) examining the effect of contractile activity on the content of ceramide in the muscles, the muscles were stimulated with twitch pulses for 25 min. This does not exclude the possibility that contractile activity of longer duration might affect the content of ceramide in the muscles.
The aim of the present study was to examine the content and composition of ceramide- and sphingomyelin-fatty acids and the activity of neutral Mg2+-dependent shingomyelinase in different muscle types1) at rest and 2) after exhaustive exercise of moderate intensity.
The experiments were performed on male Wistar rats, 250–280 g body wt fed ad libitum with a rodent pellet diet. The experimental protocol was approved by the Ethical Committee on Human and Animal Studies of the Medical Academy of Białystok. The rats were divided into two groups: 1) control and 2) exercised until exhaustion. The rats in this group were made to run on a treadmill set at +10° and moving with the speed of 1,200 m/h. They were familiarized with running at these conditions for 10 min daily during 1 wk preceding the final experiment. Exhaustion was regarded as the point at which the rats refused any further running and, when placed on a table, did not attempt to escape. The running time until exhaustion was 250 ± 40 min. Immediately after the exercise, the rats were anesthetized with pentobarbital sodium (80 mg/100 g), and the soleus and the red and white sections of the gastrocnemius were excised, cleaned of any visible adipose tissue, nerves, and fascias, and frozen in liquid nitrogen. These muscles are composed predominantly of slow-twitch oxidative, fast-twitch oxidative-glycolytic, and fast-twitch glycolytic fibers, respectively (1,26).
Ceramide- and Sphingomyelin-Fatty Acid Assay
The samples were pulverized in an aluminum mortar with a stainless steel pestle precooled in liquid nitrogen. The powder was then transferred to clean glass tubes containing methanol at a temperature of −20°C (31). Butylated hydroxytoluene (Sigma) was added, as an antioxidant, to methanol in a dose of 30 mg/100 ml. Lipids were extracted by the method of Folch et al. (2). Ceramide was isolated using the modified method of Prevati et al. (20). The samples were spotted on thin-layer chromatography silica plates (Kieselgel 60, 0.22 mm, Merck) and developed to one-third of the total length of the plate in chloroform-methanol-25% NH3 (20:5:0.2, vol/vol/vol). Then, the plates were dried and rechromatographed in heptane-isopropyl ether-acetic acid (60:40:3, vol/vol/vol). In the original method, the second developing solvent was composed of hexane-diethyl ether-acetic acid (5:15:0.2). We found that our solvent separates ceramides better. To isolate sphingomyelin, lipids were fractionated on silica plates (as above) using chloroform-methanol-acetic acid-water (50:37.5:3.5:2, vol/vol/vol/vol) as the developing solvent (12). Standards of ceramide (Sigma) and sphingomyelin (Sigma) were run along with the samples. After this procedure, the plates were allowed to dry at room temperature, and lipid bands were visualized under ultraviolet light after spraying with a 0.5% solution of 3′7′-dichlorofluorescein in absolute methanol. The gel bands corresponding to ceramide and sphingomyelin were scraped off the plate and transferred into screw tubes containing methylpentadecanoic acid (Sigma) as an internal standard. Fatty acids were then transmethylated along with the gel in the presence of 1 ml of 14% boron fluoride in methanol at 100°C for 90 min (15). Next, the samples were cooled to room temperature, 1 ml of pentane and 0.5 ml of water were added, and the two phases were separated by centrifugation. The upper pentane phase was transferred into new tubes and dried under nitrogen. The methyl esters were dissolved in 40 μl of hexane and analyzed by gas-liquid chromatography. A Hewlett-Packard 5890 Series II and a fused Hp-INNOWax (50 m) capillary column were used. The gas chromatograph was equipped with a double flame ionization detector. Injector and detector temperatures were set at 250°C each. The oven temperature was increased linearly from 160 to 230°C at a rate of 5°C/min. Individual fatty acid methyl esters were quantified using the area corresponding to the internal standards. The fatty acid standards were purchased from Sigma.
Neutral Mg2+-dependent sphingomyelinase was assayed as described by Murase et al. (16). The muscle samples were homogenized in cold 0.05 M Tris · HCl buffer, pH 7.4, containing 0.1% Triton X-100 and 0.02 M MgCl2 (5% tissue wet, wt/vol). The homogenate was centrifuged, and the supernatant was used for determination of the activity of neutral Mg2+-dependent sphingomyelinase and the concentration of protein. To assay the activity of sphingomyelinase, 150 μl of the supernatant were added to a tube containing 22.2 μg of trinitrophenylaminolauroyl (TNPAL)-sphingomyelin (Sigma) in 50 μl of 0.05 M Tris · HCl buffer (composed as above), pH 7.4. The tube was shaken for 5 min at room temperature, and then it was incubated for 30 min in a water bath at 37°C with continuous shaking. The reaction was stopped by adding 750 μl of the mixture of isopropanol-heptane-5% M H2SO4(40:10:1, vol/vol/vol). Next, 400 μl of heptane were added, the tube was shaken, 400 μl of water were added, and the tube was shaken again and then centrifuged to separate the two phases. The upper, heptane phase contained TNPAL-ceramide, which was formed from TNPAL-sphingomyelin by the neutral Mg2+-dependent sphingomyelinase present in the sample. The absorbency of the heptane phase was read at the wavelength of 330 nm using a Beckman DU 640 spectrophotometer. The activity of the enzyme was measured using a standard curve that was prepared at the same time using sphingomyelinase from human placenta (Sigma). The activity of sphingomyelinase was expressed in nanomoles of ceramide per hour per milligram of protein. Protein was determined by the method of Lowry et al. (11), with the use of bovine serum albumin (Sigma) as a standard.
Measurement of 2-Deoxyglucose Uptake by Muscles
The uptake of 2-deoxyglucose was measured in a separate experiment according to Turinsky et al. (27), with the exception that [3H]mannitol instead of [14C]sucrose was used to measure the extracellular space. The rats were handled as described in Animals. They were anesthetized at rest or immediately after exercise until exhaustion, and 1 μCi of 2-deoxy-d-[1-14C]glucose (specific activity 56 mCi/mmol, NEN Life Science Products, Boston, MA) and 5 μCi of [1-3H]mannitol (specific activity 26.3 Ci/mmol, NEN) per 100 g body wt were administered into the tail vein. The muscle samples and the blood were taken 25 min later. The muscle and plasma samples were digested with 1 M NaOH followed by neutralization with 1 M HCl. The scintillation cocktail (Ultima Gold, Packard) was added, and radioactivity was counted (in Tri-Carb 1900, Packard counter). The 2-deoxyglucose uptake was calculated as the difference between the total muscle radioactivity and the radioactivity of the muscle extracellular space.
Data are presented as means ± SD. The means were calculated from the results obtained in 10 rats. The results were evaluated statistically using Student's t-test for unpaired data. Linear regression analysis was performed using the ratio of the total content of sphingomyelin-fatty acids, i.e., the total content of ceramide-fatty acids and the activity of sphingomyelinase.
Twelve ceramides and sphingomyelins were identified in each muscle type according to the fatty acid residue. They contained the following fatty acid residues: myristic (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), docosahexaenoic (22:6), and nervonic (24:1) acids.
Ceramide-Fatty Acid Content And Composition
The total content of ceramide-fatty acids (i.e., the sum of individual ceramide-fatty acids) depended on the muscle type; it was highest in the soleus and lowest in the white gastrocnemius, with the content in red gastrocnemius in between (Table 1). Quantitatively, ceramides containing palmitic, stearic, and oleic acids constituted the major fractions (76–77% of the total) in each muscle type. Ceramide containing stearic acid was the most abundant (34% in the soleus, 37.6% in the red gastrocnemius, and 38.4% in the white gastrocnemius). Ceramides containing linolenic and docosahexaenoic acids were least represented (<1%) (Table2). The ratio of the content of ceramides containing saturated fatty acids to the content of ceramides containing unsaturated fatty acids was similar (∼2) in the examined muscles (Fig. 1).
Effect of exercise.
Exercise reduced the total content of ceramides in each muscle type. Quantitatively, a reduction in the content of ceramides containing palmitic and stearic acid residues contributed mostly to this phenomenon. The content of ceramide containing arachidonic acid residue was also reduced in each muscle. There were no changes in the content of ceramides containing myristic, palmitoleic, linolenic, and docosahexaenoic acid residues in either muscle. The effect of exercise on the content of ceramides containing other fatty acid residues depended on both the muscle type and the fatty acid residue. This was reduced in the soleus in the case of ceramide containing behenic acid, in the red gastrocnemius in the case of ceramide containing linoleic acid, and in the white gastrocnemius in the case of ceramides containing oleic, linoleic, eicosapentaenoic, behenic, and nervonic acid. The ratio of total content of ceramide containing saturated acid residues to total content of ceramides containing unsaturated acid residues was 1.42, 1.55, and 1.76 in the soleus and red and white gastrocnemius, respectively. In the case of the soleus and red gastrocnemius, the ratio was significantly (P < 0.001 and P < 0.01, respectively) lower than at rest (Fig.1).
Sphingomyelin-Fatty Acid content and Composition
The total content of sphingomyelin-fatty acids (i.e., the sum of individual sphingomyelin-fatty acids) in the soleus was higher than in either section of the gastrocnemius (Table3). There was no difference in the content of sphingomyelin-fatty acids between the red and white sections of the gastrocnemius. Sphingomyelins containing palmitic and stearic acids constituted together 66, 70, and 74% of the total sphingomyelin-fatty acid content in the soleus and red and white gastrocnemius, respectively. Sphingomyelin containing nervonic acid constituted a considerable percentage of the total sphingomyelins: 10.8, 8.9, and 11.4% in the soleus and red and white gastrocnemius, respectively. Sphingomyelins containing linolenic and docosahexaenoic acids constituted the smallest fractions (<0.5% of the total) (Table4). The ratio of the content of sphingomyelins containing saturated fatty acids to the content of sphingomyelins containing unsaturated fatty acids was 2.7 in the soleus and 3.25 in the red and 3.36 in the white gastrocnemius (P < 0.001 vs. the value in the soleus) (Fig. 1).
Effect of exercise.
Exercise reduced the total content of sphingomyelin-fatty acids in each muscle type. In the soleus, this was accounted for by a reduction in the content of sphingomyelin containing myristic, stearic, linoleic, arachidonic, and eicosapentaenoic acid. Concomitantly, the content of sphingomyelins containing oleic and linoleic acid increased. In the red gastrocnemius, a reduction in the content of sphingomyelin containing myristic, palmitoleic, stearic, arachidonic, and eicosapentaenoic acid residues took place. In the white gastrocnemius, the content of sphingomyelin containing myristic, palmitic, linolenic, arachidonic, eicosapentaenoic, behenic, and nervonic acids was reduced after exercise. The ratio of total content of sphingomyelin containing saturated fatty acids to total content of sphingomyelin containing unsaturated fatty acids increased after exercise in the white gastrocnemius and remained stable in the other muscles (Fig. 1).
Sphingomyelin-Fatty Acid-to-Ceramide-Fatty Acid Molar Ratio
The ratio was calculated for the total content of the two compounds and for individual sphingomyelin and ceramide as well. Only the ratio for the total content is shown in Fig.2. At rest, the ratio for the total content in the soleus and in the red gastrocnemius was lower than in the white gastrocnemius. After exercise, the ratio was higher than at rest in each muscle type.
At rest, the activity of sphingomyelinase in the soleus was higher than in either section of the gastrocnemius (Fig.3). Enzyme activity in the white gastrocnemius was much lower than in the red portion of the same muscle. Exercise reduced the activity of the enzyme in the soleus and in the red gastrocnemius but had no effect on its activity in the white gastrocnemius.
The uptake of 2-deoxyglucose by the soleus and by the red section of the gastrocnemius was higher than by the white section of the gastrocnemius both at rest and after exercise (P < 0.001 in each case) (Table 5). The exercise increased the uptake severalfold in each muscle.
The Data at Rest
As already mentioned in the introductory section, the only data on the content of ceramides in skeletal muscles were published by Turinsky and colleagues (27, 29). Our results differ from those reported by these authors. In our study, the total content of ceramide (expressed as the total content of ceramide-fatty acids) was much higher (∼3 times) than the content reported by Turinsky and colleagues. This difference might be due both to the different analytical method used and to the different treatment of rats. Turinsky and colleagues determined the content of ceramide with the diacylglycerol kinase assay, whereas we determined the content of ceramide-fatty acid residues upon their methylation, using gas-liquid chromatography. We isolated ceramides before methylation so that they did not contain any other fatty acid-containing lipids that could increase the content of ceramide. Turinsky et al. (29) used rats fasted overnight, whereas we used rats fed ad libitum, and this factor might contribute to the difference. Another experimental difference was that Turinsky et al. (29) exsanguinated rats before taking the muscle samples, and we did not. However, the procedure of exsanguination did not remove much blood from the muscles. Moreover, we found (unpublished data) that the concentration of ceramide-fatty acids in the rat plasma is only ∼20 nmol/ml; therefore, the plasma ceramides did not contribute much to the content of ceramide-fatty acids in the muscles. Unfortunately, there are no other data available on the content of ceramide in the skeletal muscles to be compared with our results.
Our study is the first to describe the content and composition of ceramides in skeletal muscles according to their fatty acid residues. We found that most ceramides contained saturated fatty acids (saturated-to-unsaturated acid ratio ∼2), and there were no differences between the examined muscles in this regard. The major saturated acids were palmitic and stearic acids, and the major unsaturated acid was oleic acid. These data are different from the data obtained in mouse liver, where most ceramides contained unsaturated acids (22). In the human brain, the ceramides contain only saturated fatty acid (9). Ceramide can be formed in different ways in the cell. The extracellular stimuli increase production of ceramide from sphingomyelin, which is located mostly in the plasma membrane. The reaction is catalyzed by the enzyme neutral Mg2+-dependent sphingomyelinase (7, 21). Therefore, we also measured the content and composition of sphingomyelin-fatty acids in the muscle samples. In the literature, the only data that exist on the content and composition of sphingomyelin-fatty acids in skeletal muscle were obtained in muscles composed of a mixture of different fiber types. The results obtained in human muscle are not consistent. According to Svennerholm et al. (25), palmitic, stearic, behenic, and lignoceric acid constitute most of the saturated sphingomyelin-fatty acids (25, 25, 10, and 6% of total sphingomyelin-fatty acids, respectively). The only unsaturated fatty acid was nervonic acid, which constituted 21% of total fatty acids. According to Kunze et al. (10), unsaturated fatty acids comprise ∼50% and, according to Pearce et al. (17), only ∼20% of the total sphingomyelin-fatty acids. In mouse muscle, the unsaturated fatty acids contribute in ∼28% to the total sphingomyelin-fatty acid pool (18). In the rat, palmitic and stearic acids constitute ∼40% and arachidonic acid >50% of total sphingomyelin-fatty acids (8). In our study, palmitic and stearic acids constituted 66–74% of total sphingomyelin-fatty acids, depending on the muscle type. The major unsaturated acid was nervonic acid (∼10%). Oleic and arachidonic acids contributed 3–6% to the total pool. Overall, the ratio of saturated to unsaturated acids was ∼3. Therefore, our data are different from those reported by Isaev et al. (8). However, it is difficult to find a convincing explanation for the results of these authors, in particular for the high percentage of arachidonic acid. We have shown that there are differences in the content and composition in sphingomyelin-fatty acids between the soleus (a slow-twitch muscle) and both sections of the gastrocnemius (fast-twitch muscles), whereas the differences between the white and red sections of the gastrocnemius were only minor. The activity of sphingomyelinase was found to be highest in the soleus and lowest in the white section of the gastrocnemius. Enzyme activity correlates to the ratio of total content of sphingomyelin-fatty acids to total content of ceramide-fatty acids in each muscle (Fig.4). This strongly suggests that the sphingomyelin-signaling pathway is the main route in the production of ceramide in the muscles at rest. We also calculated the ratio of the content of individual sphingomyelin-fatty acid to the content of individual ceramide-fatty acid (results not shown). This ratio is mostly dependent on the acid residue and only to a minor degree on the type of muscle. For example, in the soleus, it is ∼0.9 for oleic acid and ∼8 for nervonic acid. This suggests that sphingomyelinase selectively hydrolyzes different sphingomyelins. To the best of our knowledge, selectivity in the production of different ceramides has not yet been studied in detail. Certainly, much more research is needed to clarify this issue.
The total content of phospholipids has repeatedly been shown to be stable during exercise (3, 4, 14, 30). So far, there are no reports on the effect of exercise on the content of individual phospholipids in the muscles. It should be mentioned, however, that contractile activity has been reported to increase the incorporation of blood-borne [14C]palmitic acid into different phospholipid fractions, including sphingomyelin (6). In our study, we have shown that exercise reduced the total content of sphingomyelin-fatty acids in each muscle type. The reduction was mild (12, 11, and 16% in the soleus and red and white gastrocnemius, respectively) but significant. Concomitantly, the activity of sphingomyelinase was reduced after exercise in the soleus and in the red section of the gastrocnemius. A number of stimuli (e.g., tumor necrosis factor-α, interferon-γ, nerve growth factor, vitamin D3) were shown to increase the activity of neutral Mg2+-dependent sphingomyelinase in different cell types (13). Now, we have shown that prolonged exercise reduces the activity of the enzyme in muscles composed of fibers with high oxidative capacity. It indicates that activity of the enzyme may be not only up- but also downregulated with resulting changes in the production of ceramide. Exercise reduced the total content and changed the composition of ceramides in each muscle type. These changes in the content of individual ceramides depended mostly on fatty acid residue. The reduction in the concentration of ceramides after exercise could have been caused by either the reduced formation or increased breakdown of the compounds. The reduction in the activity of sphingomyelinase in the two red muscles would suggest a reduction in the formation of ceramide from sphingomyelin for the main reason of the reduction in the content of ceramide. A positive correlation between the ratio of total content of sphingomyelin-fatty acids to total content of ceramide-fatty acids and neutral Mg2+-dependent sphingomyelinase persisted during exercise in the two muscles. It would appear to suggest that sphingomyelin remained the major source of ceramides. Enzyme activity in the white gastrocnemius was stable. This would indicate that increased breakdown of ceramide could contribute to the reduction in its content after exercise in this muscle. Again, it is clear that this does not concern every ceramide but only those containing certain fatty acid residues.
As mentioned in the introductory section, Turinsky et al. (27) did not find any changes in the content of ceramide in the calf muscles of the rat after electrical stimulation of the muscles. However, they stimulated the muscles for only 20 min with twitch pulses. It is likely that the contractile activity was too mild to produce changes in the content of ceramides in the muscles.
Ceramide is involved in many processes in the cells. The major ones are induction of cell differentiation, inhibition of cell proliferation, induction of apoptosis, and regulation of inflammatory responses. The effects of ceramide depend largely on the cell type (5, 7, 13, 19, 21). Studies in skeletal muscles have shown that the content of ceramides increased in insulin-resistant rats (29). However, plasma membrane-permeable ceramides did not increase glucose uptake in incubated soleus muscle (28). On the other hand, ceramide was proposed as mediating the inhibitory effect of palmitate on glucose uptake by C2C12myotubes (24). If the latter is true also in vivo, the reduction in the content of ceramide in working skeletal muscles would facilitate glucose uptake by the muscles in the presence of elevated long-chain fatty acids in the plasma. To shed light on such a possibility, 2-deoxyglucose uptake by skeletal muscle was measured, and it was related to the total content of ceramide-fatty acids in the same muscle. As it can be seen in Fig. 5, there is an inverse relationship between the 2-deoxyglucose uptake and the content of ceramide in each muscle after exercise. These results support the hypothesis that ceramide may be involved in the regulation of glucose uptake in the muscles.
In summary, we identified and quantified 12 different ceramides and sphingomyelins in three different skeletal muscle types according to their fatty acid residues. The presence of neutral Mg2+-dependent sphingomyelinase was also shown in the muscles. Exhausting exercise of moderate intensity resulted in a reduction in the total content of both ceramide-fatty acids and sphingomyelin-fatty acids, with a higher reduction in the case of ceramide-fatty acids. Exercise reduced the activity of sphingomyelinase in the soleus and red gastrocnemius. It is concluded that the sphingomyelin-signaling pathway is present in skeletal muscles and that it is affected by prolonged contractile activity.
This work was supported by the Polish Research Committee Grant 4/P05B/022/15 and Medical Academy of Białystok Grant 3-18489.
Address for reprint requests and other correspondence: J. Górski, Dept. of Physiology, Medical Academy of Białystok, 15–230 Bialystok, Poland (E-mail:).
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