Lipids (2013) 48:697–704 DOI 10.1007/s11745-013-3769-3 ORIGINAL ARTICLE Fiber Specific Changes in Sphingolipid Metabolism in Skeletal Muscles of Hyperthyroid Rats A. Chabowski • M. Zendzian-Piotrowska_ • A. Mikłosz • B. Łukaszuk • K. Kurek • J. Go´rski Received: 1 September 2012 / Accepted: 22 January 2013 / Published online: 7 March 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Thyroid hormones (T3,T4) are well known SPA Sphinganine modulators of different cellular signals including the SPH Sphingosine sphingomyelin pathway. However, studies regarding S-1-P Sphingosine-1-phosphate downstream effects of T3 on sphingolipid metabolism in CER Ceramide skeletal muscle are scarce. In the present work we sought SM Sphingomyelin to investigate the effects of hyperthyroidism on the activity aSM-ase Acid sphingomyelinase of the key enzymes of ceramide metabolism as well as nSM-ase Neutral sphingomyelinase the content of fundamental sphingolipids. Based on fiber/ alCDase Alkaline ceramidase metabolic differences, we chose three different skeletal nCDase Neutral ceramidase muscles, with diverse fiber compositions: soleus (slow- SPT Serine palmitoyltransferase twitch oxidative), red (fast-twitch oxidative-glycolytic) THR Thyroid hormone receptor and white (fast-twitch glycolytic) section of gastrocne- mius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingo- myelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine Introduction palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, Thyroid hormones (T3,T4) are well known modulators of hyperthyroidism induced fiber specific changes in the whole body energy utilization. However, they also serve as content of sphingolipids that were relatively more related an important molecular signaling transducers [1]. A num- to de novo synthesis of ceramide rather than to its gen- ber of cellular signaling pathways are modified by triio- eration via hydrolysis of sphingomyelin. dothyronine (T3), including the sphingomyelin/ceramide pathway [2]. Skeletal muscles constitute the bulk of the Keywords Ceramide Á Sphingolipids Á Triiodothyronine Á body’s total metabolic activity either by virtue of its mass Skeletal muscle or oxidative capacity, and thus represent an important target for the action of T3 [3]. Furthermore, others indicate Abbreviations high expression of T3 receptor (THR) in muscle fibers [4] T3 Triiodothyronine and interestingly, the thyroid hormone receptors density is FFA Free fatty acids muscle specific as slow-twitch (oxidative) fibers are more sensitive to T3 than fast-twitch (glycolytic) muscles [5]. Recently, diminished content of ceramide with a con- A. Chabowski (&) Á M. Zendzian-Piotrowska_ Á A. Mikłosz Á comitant increase in sphingomyelin concentration, in B. Łukaszuk Á K. Kurek Á J. Go´rski skeletal muscles was reported in hypothyroid rats [6]. Department of Physiology, Medical University of Bialystok, ul. Mickiewicza 2C, 15-222 Bialystok, Poland Ceramide content in myocytes is a net result of myocellular e-mail: [email protected] ceramide generation (by hydrolysis of sphingomyelin or de 123 698 Lipids (2013) 48:697–704 novo synthesis pathway) and its degradation (by ceramid- hyperthyroidism in humans [13, 14]. Control rats were ases) [7]. The formation of ceramide de novo begins with treated with saline accordingly. After 10 days, rats were condensation of serine and palmitoyl-CoA, the reaction fasted overnight and anaesthetized by intraperitoneal catalyzed by serine palmitoyltransferase (SPT). However, injection of pentobarbital with a dose of 80 mg/kg of body hydrolysis of sphingomyelin, can also generate substantial weight. Fasting blood samples were collected sodium- amounts of ceramide, through the increased activity of heparinized tubes and centrifuged at 20,0009g for 20 min specific sphingomyelinases (acidic and/or neutral sphing- at 4 °C. Plasma was removed and stored at -80 °C until omyelinases—aSM-ase/nSM-ase). In contrast, degradation analyzed. Concomitantly, the soleus, red and white section of ceramide occurs mainly by its deacylation and formation of gastrocnemius were excised, cleaned of blood and/or of sphingosine in a reaction catalyzed by specific ceram- connective tissue and immediately frozen in liquid nitrogen idases (CDase, acidic, neutral and alkaline). Sphingosine and then stored at -80 °C until analysis. might also be further phosphorylated to sphingosine-1 phosphate. In addition, it should be noted that most of the Sphingomyelin Content steps involved in sphingolipid metabolism is reversible [8]. Increasing evidence strongly supports involvement of dif- The tissue samples were pulverized in an aluminum mortar ferent sphingolipids in the regulation of myocellular precooled previously in liquid nitrogen. The powder was functions [9, 10], and disturbances in sphingolipid forma- then transferred to a tube which contained methanol and tion have been found essential in the induction of pathol- 0.01 % butylated hydroxytoluene (Sigma) as an antioxidant. ogies [2]. More specifically, elevated concentrations of Lipids were extracted by the method described by Bligh ceramide or other sphingolipid intermediates are impli- and Dyer [15]. Then the lipid samples were spotted on cated in the induction of insulin resistance in skeletal thin-layer chromatography (TLC) silica plates (Kieselgel muscle [8]. However, after exercise (a known insulin- 60, 0.22 mm, Merck) and developed as described by Maha- sensitizing effect), higher ceramide content was also devappa et al. [16]. Standards of sphingomyelin (Sigma) reported in muscles, in animals [11] and humans [12]. were run along with the samples. Lipid bands were visual- At present, it is unclear whether thyroid hormones ized under ultraviolet light after spraying with a 0.5 % influence sphingolipid metabolism in skeletal muscles. solution of 3070-dichlorofluorescein in absolute methanol. Therefore, the activity of key enzymes of ceramide The gel bands corresponding to the sphingomyelin were metabolism (serine palmitoyltransferase, neutral sphingo- scraped off the plate and transferred into screw tubes con- myelinase, acid sphingomyelinase, neutral ceramidase and taining pentadecanoic acid (Sigma–Aldrich, St. Louis, MO) alkaline ceramidase) as well as the content of sphingolipid as an internal standard. Sphingomyelin fatty acids were then metabolism products (sphingosine, sphinganine, sphingo- transmethylated and subsequently analyzed by means of sine-1-phosphate, ceramide and sphingomyelin) were gas–liquid chromatography. A Hewlett-Packard 5890 Series measured. To investigate the effects of hyperthyroidism, II system, equipped with a double flame ionization detector rats were made hyperthyroid (n = 8) over 10 days using T3 and Agilent CP-Sil 88 capillary column (100 m, internal i.p. injections and subsequently three types of skeletal diameter of 0.25 mm), was used. The sphingomyelin content muscles were taken: soleus (slow-twitch oxidative), red is presented as the sum of individual fatty acid residues. (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. Ceramide Content A small (50 ll) volume of the chloroform phase, contain- Materials and Methods ing the lipids extract was transferred to a fresh tube which contained an internal standard (C17-sphingosine, Avanti The experimental protocol was approved by the Ethical Polar Lipids, UK). Ceramide (Cer) present in the organic Committee for Animal Experiments at the Medical Uni- phase was hydrolyzed in 1 M KOH in 90 % methanol at versity of Białystok. Male Wistar rats (200–220 g body 90 °C for 60 min. This digestion procedure does not con- weight) were housed under controlled conditions vert complex sphingolipids, such as SM, galactosylcera- (21 °C ± 2, 12 h light/12 h dark cycle) with unlimited mide, or glucosylceramide, into free sphingoid bases [17]. access to standard chow and water. The animals were The content of free sphingosine, liberated from Cer was divided into two groups, control (n = 8) and treated with next analyzed by means of HPLC (Varian Inc. OmniSpher triiodothyronine (T3)(n = 8). Triiodothyronine (Sigma– 5, 4.6 9 150 mm). The calibration curve was prepared Aldrich, St. Louis, MO) was injected subcutaneously with using N-palmitoylsphingosine (Avanti Polar Lipids, UK) as a dose of 100 lg/100 g of body weight, daily for 10 days a standard. The chloroform extract used for the analysis of [13]. This dose is quite commonly used to mimic Cer level also contains small amounts of free sphingoid 123 Lipids (2013) 48:697–704 699 bases. Therefore, the content of Cer was corrected for the proteins in each sample were separated using 10 % level of free sphingosine determined in the same sample. SDS–polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. Equal protein concentra- Sphingosine, Sphinganine and Sphingosine-1- tions were loaded in each lane as confirmed by Ponceau Phosphate Content staining the blot membrane. In the next step, membranes were immunoblotted with selected primary antibodies The ceramide derivatives were measured according to the (THRa, b-actin (Abcam, EU). Quantification of the selected method, described by Min et al. [18]. Prior to sample protein content was achieved by densitometry (OD-Optical homogenization and ultrasonication, internal standards Density; Biorad, Poland). The