Irradiation with 365 nm and 405 nm wavelength shows differences in DNA damage of swine pancreatic islets.

INTRODUCTION: 3D printing is being used more extensively in modern biomedicine. One of the problems is selecting a proper crosslinking method of bioprinted material. Amongst currently used techniques we can distinguish: physical crosslinking (e.g. Ca2+ and Sr2+) and chemical crosslinking-the UV light crosslinking causing the biggest discussion. UV radiation is selectively absorbed by DNA, mainly in the UV-B region but also (to some extent) in UV-A and UV-C regions. DNA excitement results in typical photoproducts. The amount of strand breaks may vary depending on the period of exposition, it can also differ when cells undergo incubation after radiation. AIM: The aim of this study was to show whether and how the time of irradiation with 405 nm and 365 nm wavelengths affect DNA damage in cell lines and micro-organs (pancreatic islets). MATERIALS AND METHODS: The degree of DNA damage caused by different wavelengths of radiation (405 nm and 365 nm) was evaluated by a comet assay. The test was performed on fibroblasts, alpha cells, beta cells and porcine pancreatic islets after 24 hours incubation period. Samples without radiation treatment were selected as a control group. Results analysis consisted of determining the percent of cells with damaged DNA and the tail intensity evaluation. RESULTS: The degree of DNA damage in pancreatic islets after exposure to 405 nm wavelength oscillated between 2% and 6% depending on the tested time period (10 - 300 seconds). However, treating islets using 365 nm wavelength resulted in damage up to 50%. This clearly shows significantly less damage when using 405 nm wavelength. Similar results were obtained for the tested cell lines. CONCLUSIONS: Crosslinking with 405 nm is better for pancreatic islets than crosslinking with 365 nm UV light.

Stearoyl-CoA desaturase--a new player in skeletal muscle metabolism regulation.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (18:1) and palmitoleate (16:1), which are a major component of tissue lipids. SCD1 deficient mice reveal increased energy expenditure and decreased body adiposity due to the upregulation of genes of fatty acid oxidation and the downregulation of genes of lipid synthesis in liver. In this review, we examine data showing that SCD is an important component in the regulation of skeletal muscle metabolism, which affects insulin sensitivity, mitochondrial fatty acid oxidation and ceramide de novo synthesis in oxidative myofibers. The lack of SCD1 gene increases the rate of fatty acid beta-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and by upregulating genes of fatty acid oxidation in soleus and red gastrocnemius muscles. Consistent with increased beta-oxidation, the contents of free fatty acids and long-chain acyl-CoAs are significantly decreased, which together with reduced mRNA level and activity of serine palmitoyltransferase led to reduced ceramide synthesis in oxidative muscles of SCD1-/- mice. Thus, reduced contents of free fatty acids, acyl-CoAs and ceramides as well as increased AMPK phosphorylation, might contribute to increased insulin sensitivity observed in muscle of SCD1-/- mice. SCD1 deficiency also results in downregulation of the expression of the protein-tyrosine phosphatase 1B, which is responsible for the sustained insulin receptor autophosphorylation despite reduced levels of plasma insulin in the SCD1-/- mice. SCD1 deficiency reduced ceramide synthesis, increased AMPK phosphorylation and carnitine palmitoyltransferase 1 activity also in soleus and red gastrocnemius muscles of leptin deficient ob/ob mice. These findings raise the possibility that SCD1 may be a downstream component of the leptin signaling pathway in skeletal muscle.

Ceramides, sphinganine, sphingosine and acid sphingomyelinases in the human umbilical cord blood.

Ceramides, sphingosine, sphinganine, as well as Zn (++)-dependent and Zn (++)-independent acid sphingomyelinase are present in the plasma of adults. The aim of the present study was to examine the concentrations of these compounds and activities of both enzymes in the umbilical cord blood in humans. Twenty-two women with uncomplicated term pregnancy volunteered for the study. Blood was taken from the umbilical cord artery and from the antecubital vein of the mother immediately after delivery. Free ceramides were isolated by thin layer chromatography, and their fatty acids were identified and quantified by gas-liquid chromatography. Free sphingosine and sphinganine concentrations were determined using high-performance liquid chromatography. Acid Zn (++)-dependent and Zn (++)-independent sphingomyelinase activity was measured using sphingomyelin [choline-methyl-14C] as a substrate. We found that the compounds examined are present in the umbilical cord blood. The total fatty acid-containing ceramide concentrations in fetal blood were lower than in mother's blood. The mean sphingosine and sphinganine concentrations in the fetal and maternal serum were similar. The examined enzymes were present in the fetal serum, and their mean activity did not differ from that in the mother. In conclusion, we have shown the presence of ceramides, sphingosine and sphinganine and both isoforms of acid sphingomyelinase in the human fetal cord blood. They are most likely the product of the fetus itself.

Stearoyl-CoA desaturase as a new drug target for obesity treatment.

Stearoyl-CoA desaturase (SCD), the rate-limiting enzyme in monounsaturated fatty acid synthesis, has recently been shown to be the critical control point regulating hepatic lipogenesis and lipid oxidation. As several manifestations of the metabolic syndrome and type 2 diabetes mellitus are associated with alterations in intracellular lipid partitioning, we propose that SCD1 may be a potential therapeutic target in the treatment of obesity and the metabolic syndrome. In support of this notion, we have shown that SCD1-deficient mice have increased energy expenditure, reduced body adiposity, increased insulin sensitivity and are resistant to diet-induced obesity and liver steatosis. Furthermore, SCD1 was found to be specifically repressed during leptin-mediated weight loss, and leptin-deficient ob/ob mice lacking SCD1 showed marked correction of the hypometabolic phenotype and hepatic steatosis. Much evidence indicates that the direct anti-steatotic effect of SCD1 deficiency stems from increased fatty acid oxidation and decreased lipid synthesis. All of these findings reveal that pharmacological manipulation of SCD activity might be of benefit in the treatment of obesity, diabetes, liver steatosis and other diseases of the metabolic syndrome.

Effect of acute exercise and training on metabolism of ceramide in the heart muscle of the rat.

AIM: The sphingomyelin signalling pathway operates in the heart muscle. There are no data on the effect of exercise on the functioning of this pathway in the myocardium and it was the aim of the present study to examine this question. METHODS: The experiments were carried out on male Wistar rats, 300-320 g of body weight. They were divided into three groups: (1) control, (2) run 3 h on a treadmill moving with a speed of 1200 m h(-1) and set at +10 degrees incline, and (3) trained on a treadmill for 6 weeks. The rats were anaesthetized and samples of the left ventricle were taken. They were immediately frozen in liquid nitrogen. Thereafter, lipids were extracted and ceramide and sphingomyelin were isolated by means of thin layer chromatography. Their fatty acids were identified and quantified by means of gas-liquid chromatography. In separate heart samples the activity of neutral, Mg(2+)-dependent sphingomyelinase and acid sphingomyelinase was determined using labelled sphingomyelin as a substrate. RESULTS: Thirteen different ceramides and sphingomyelins were identified based on their fatty acid residue. Exercise markedly reduced the total content of ceramide-fatty acids and had no effect on the total content of sphingomyelin-fatty acids. Training did not affect the total content either of ceramide-, or sphingomyelin-fatty acids. The activity of both neutral Mg(2+)-sphingomyelinase and acid sphingomyelinase was reduced after exercise. Training did not affect the activity of neutral sphingomyelinase and reduced the activity of acid sphingomyelinase. CONCLUSION: It is concluded that acute, prolonged exercise, but not training, markedly affects the operation of the sphingomyelin-signalling pathway in the heart.

Effect of endurance training on the sphingomyelin-signalling pathway activity in the skeletal muscles of the rat.

The sphingomyelin signalling pathway has been shown to function in different skeletal muscle types. The aim of the present study was to examine the effect of endurance training on the functioning of the pathway in the muscles. The experiments were carried out on two groups of male Wistar rats: sedentary and trained for six weeks. 24h after cessation of the training rats were anaesthetized and samples of the soleus, red and white section of the gastrocnemius were taken. The content and composition of sphingomyelin-fatty acids and ceramide - fatty acids was determined by means of gas-liquid chromatography. The content of sphingosine and sphinganine was determined by means of high-pressure liquid chromatography. The activity of neutral Mg(++)-dependent sphingomyelinase was determined spectophotometrically using trinitrophenylaminolauroyl-sphingomyelin as the substrate. It has been found that training reduces the total content of sphingomyelin- and ceramide-fatty acids, increases the content of sphinganine and does not affect the content of sphingosine in individual muscle types. The activity of the enzyme in the muscles is also elevated. It is concluded that training affects functioning of the sphingomyelin -signalling pathway in skeletal muscles. The reduction in the content of ceramide may contribute to elevation in glucose uptake in skeletal muscles observed after training.

Effect of streptozotocin-diabetes on the functioning of the sphingomyelin-signalling pathway in skeletal muscles of the rat.

AIMS/HYPOTHESIS: Ceramide is the main second messenger in the sphingomyelin-transmembrane signalling pathway. The compound is likely to play a role in the induction of insulin resistance. The aim of the present study was to examine the effect of streptozotocin diabetes on the content and composition of ceramides and sphingomyelins and the activity of neutral Mg (2+)-dependent sphingomyelinase and acid sphingomyelinase in different types of skeletal muscle of the rat. METHODS: The experiments were carried out on two groups of male Wistar rats weighing 250-280 g: controls and those treated with streptozotocin at a dose of 60 mg/kg. Determinations were performed on three types of skeletal muscle: the slow-twitch oxidative (soleus), fast-twitch oxidative-glycolytic (red section of the gastrocnemius) and fast-twitch glycolytic (white section of the same muscle). The content and composition of ceramide- and sphingomyelin-fatty acids were determined using gas-liquid chromatography. The activity of the enzymes was measured using N-[(14)CH (3)]-sphingomyelin as the substrate. RESULTS: Twelve different ceramides and sphingomyelins were identified and quantified in each muscle with regard to the fatty acid residue. The ratio of total content of ceramide-saturated fatty acids to the total content of ceramide-unsaturated fatty acids was more than two. In the case of sphingomyelin, the ratio was similar to ceramide in the soleus and much higher in both sections of the gastrocnemius. Treatment with streptozotocin increased the total content of ceramide-fatty acids by 78% (p < 0.001) in the soleus, 27.5% (p < 0.01) in the red and 36.9% (p < 0.001) in the white section of the gastrocnemius. Concomitantly, the total content of sphingomyelin-fatty acids decreased by 43.8%, 31.2%, 24.8% (p < 0.001 in each case) in the respective muscles. The activity of neutral Mg (2+)-dependent sphingomyelinase was elevated by 69.5%, 105.9% and 62.3% in the soleus and red and white gastrocnemius, respectively (p < 0.001 for each muscle). The activity of acid sphingomyelinase was stable in the soleus and white gastrocnemius and decreased by 15.7% (p < 0.01) in the red gastrocnemius. CONCLUSION/INTERPRETATION: The results obtained show that insulin deficiency results in elevation in the content of ceramide in skeletal muscles. This indicates that the hormone is involved in regulation of the activity of the sphingomyelin-signalling pathway in the muscles.

Effect of hypothyreosis on the content of ceramides in rat tissues.

Ceramide is the second messenger in the sphingomyelin signalling pathway. A number of extracellular stimuli increase the content of ceramide in the cell. There are some data indicating that the content of ceramide may also be regulated by hormones. The aim of the present study was to examine the effect of hypothyreosis on the content and composition of ceramide in rat tissues. The rats were thyroidectomized and thereafter they received propylthiouracyl in drinking water. The control rats were sham operated. 30 days after thyroidectomy or sham operation the rats were anaesthetized and samples of the liver, white and red vastus lateralis and left ventricle were taken. One set of samples was frozen in liquid nitrogen for analysis of ceramide. Another set of samples was freshly homogenized in chloroform/methanol for further determination of the content of sphingomyelin phosphorous. The content and composition of ceramide-fatty acids was determined by means of gas-liquid chromatography. Twelve ceramides containing different fatty acid residues were identified in both groups. Hypothyreosis reduced the total content of ceramide in each tissue studied: in the heart by 50.9%, in the red vastus by 28.6%, in the white vastus by 29.4% and in the liver by 22%. Concomitantly, the content of individual ceramides was either reduced, stable or even elevated, depending on the tissue. The content of sphingomyelin was elevated in both sections of the vastus lateralis and remained stable in the heart and the liver. The ratio: total content of sphingomyelin to total content of ceramide was elevated in the muscles and remained stable in the liver. This indicates that the reduction in the content of ceramide in the tissues of hypothyroid rats may be a consequence either of a reduction in the formation of ceramide from sphingomyelin, its increased hydrolysis or both. It is concluded that normal thyroid function is needed to maintain the content and composition of ceramide in the tissues.

Effect of acute exercise on the content of free sphinganine and sphingosine in different skeletal muscle types of the rat.

It has previously been shown that prolonged exercise of moderate intensity reduces the content of ceramide in each type of skeletal muscle. This was accompanied by a reduction in the activity of neutral, Mg++-dependent sphingomyelinase (the major enzyme responsible for ceramide formation from sphingomyelin) in the soleus and red gastrocnemius, but not in the white gastrocnemius (A. Dobrzyn and J. Górski, Am. J. Physiol.: Endorcinol. Metab. 282: E277 - E285, 2002). No other data on regulation of ceramide metabolism in contracting muscles are available. The aim of the present study was to examine the content of sphinganine (a key precursor of ceramide on the de novo synthesis route) and the content of sphingosine (the main product of ceramide catabolism) in different skeletal muscle types after two kinds of acute exercise. The experiments were carried out on 30 male Wistar rats, 250 - 280 g of body weight. The rats were divided equally into three groups: 1 - control, 2 - run until exhaustion (1200 m/h, +10 degree incline), 3 - a group in which the sciatic nerve was stimulated 10 min with tetanic pulses (60 pulses/min). Samples were taken of the soleus and of the red and white section of the gastrocnemius. These muscles are composed mostly of the slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively. Lipids were extracted with chloroform/methanol. Sphinganine and sphingosine were quantified by high-performance liquid chromatography. At rest, the content of sphinganine in the soleus was higher than in the red gastrocnemius (p < 0.05), and in the latter, it was higher than in the white gastrocnemius (p < 0.01). Prolonged exercise increased the content of sphinganine approximately 6-fold in each muscle. The resting content of sphingosine in the soleus and in the red gastrocnemius was similar--higher than in the white gastrocnemius (p < 0.001 and p < 0.01, respectively). The content of sphingosine increased over 3-fold in the soleus and nearly 2-fold in the red and white sections of the gastrocnemius. Stimulation of the sciatic nerve increased the content of both compounds approximately 2-fold in each muscle. We conclude that acute exercise increases both de novo synthesis and catabolism of ceramide in skeletal muscles. Accumulation of sphingosine in contracting muscles may contribute to the development of fatigue.

Accumulation of specific ceramides in ischemic/reperfused rat heart; effect of ischemic preconditioning.

Ceramide signalling has been implicated in the mechanism of myocardial ischemia/reperfusion injury (IR). This study tested the hypothesis that ceramides containing a specific amino-linked acyl residue mediate the injury, and that ischemic preconditioning (IPC) affords myocardial protection because it prevents increased ceramide accumulation in IR myocardium. Perfused rat hearts were subjected either to the sham perfusion or to 30 min global ischemia, 30 min ischemia/30 min reperfusion (IR) or were preconditioned prior to the standard IR. The ventricles were harvested for biochemical assay that involved transmethylation of ceramide amino-linked acyl residues, and gas liquid chromatography measurement of acyl methyl esters. Fourteen ceramides containing myrystic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, eicosapentaenoic, behenic, docosapentaenoic, docosahexaenoic or nervonic acid were identified in the myocardium of rats. The total basal ceramide concentration in the myocardium was 135 nmol/g tissue, and it was increased by 14.1% and 48.4% in the ischemia and IR group, respectively. However, in fact, IR increased the accumulation of only 7 out of 14 ceramides identified in the heart (i.e., those containing palmitic, stearic, oleic, linoleic, and arachidonic acid), and the relative magnitude of these increases varied between the particular ceramides and was independent from their basal tissue concentration. IPC improved postischemic hemodynamic recovery and partially prevented the reperfusion-induced increases in these 7 ceramides, while the other ceramides were unaffected by IPC. These results support the role of the specific ceramide signalling in the mechanism of myocardial IR injury. We speculate that by preventing tissue accumulation of certain ceramides, IPC attenuates this signalling, that adds to the mechanism of myocardial protection afforded by IPC.