Partial hepatectomy activates production of the pro-mitotic intermediates of the sphingomyelin signal transduction pathway in the rat liver

Sphingomyelin signal transduction pathway regulates cell cycle through a number of lipid second messengers, which stimulate cell proliferation (sphingosine-1-phosphate), initiate growth arrest or induce apoptosis (sphingosine, ceramide). To asses the functioning of sphingomyelin pathway during liver regeneration after partial hepatectomy in rat (PH) we measured the content of sphingomyelin (SM), ceramide (CER), sphingosine (SPH), sphingosine-1-phosphate (S1P), the activity of neutral Mg(2+)-dependent and acidic sphingomyelinases and ceramidases, in the remnant liver lobes during the first 24h after PH in rat. The activity of acidic ceramidase was highest at 4th hour after PH, whereas the activity of neutral ceramidases peaked at 12th hour after the operation. At these time points the activity Mg(2+)-dependent sphingomyelinase was also elevated, together with the content of SPH, S1P and the ratio of S1P to CER. The activity of acidic sphingomyelinase increased gradually from 4th to 24th hour after the operation. This was accompanied by significant increase in the content of ceramide between 4th and 24th hour and reduction in the content of S1P and S1P to CER ratio. It is concluded that partial hepatectomy induces production of the pro-mitogenic intermediates of sphingomyelin signaling pathway during the first 12h of liver regeneration in rat.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

The activity of sphingomyelin cycle enzymes and concentration of sphingomyelin degradation products in rat liver in dynamics of acute toxic hepatitis

Activity of key enzymes of the sphingomyelin cycle and the content of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of acute toxic hepatitis induced by subcutaneous administration of oil solution of CCl₄. Sphingomyelinase activity significantly increased already on early terms and remained increased over the whole period of observation. Ceramidase activity insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significantly increased. The balance between liver content of ceramide (proapoptotic) and sphingosine-1-phosphate (the antiapoptotic factor) was shifted towards ceramide over the whole observation period. In sphingomyelin molecules there was a significant decrease in the content of the fatty acids C_18:1 and C_22:2, while in ceramide molecules and sphingosine-1-phosphate only the fatty acid C_22:2 changed. In spite of a significant decrease in the content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites insignificantly differed from control. Taking into consideration literature data our results suggest involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes permits realization of repair processes and promotes optimization of cellular community in the liver.     

PPARalpha agonist induces the accumulation of ceramide in the heart of rats fed high-fat diet.

It was shown that high-fat feeding of mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor (PPAR) alpha but not wild type animals leads to the accumulation of ceramide (an important mediator of lipotoxicity) in the heart [Finck et al. 2003 Proc Natl Acad Sci USA]. To investigate the mechanism of this phenomenon we examined the effects of PPARalpha activation on ceramide metabolism in the myocardium. Male Wistar rats were fed either a standard chow or a high-fat diet. Each group was divided into two subgroups: control and treated with selective PPARalpha activator - WY-14643. In the rats fed on the standard diet WY-14643 did not affect the myocardial content of sphingomyelin and ceramide but reduced the content of sphinganine and sphingosine. It also inhibited the activity of neutral sphingomyelinase and increased the activity of acid sphingomyelinase, whereas the activity of ceramidases and serine palmitoyltransferase (SPT) remained stable. High-fat diet itself did not affect the content of the examined sphingolipids. However, it reduced the activity of sphingomyelinases and ceramidases having no effect on the activity of SPT. Administration of WY-14643 to this group significantly increased the content of myocardial free palmitate, ceramide, sphingomyelin and the activity of SPT. Our results demonstrated that PPARalpha activation modulates myocardial ceramide metabolism and leads to the accumulation of ceramide in the heart of the high-fat fed rats due to its increased synthesis de novo.     

Effect of exercise duration on the key pathways of ceramide metabolism in rat skeletal muscles

Ceramide is the key compound on crossroads of sphingolipid metabolism. The content and composition of ceramides in skeletal muscles have been shown to be affected by prolonged exercise. The aim of this study was to examine the effect of exercise on the activity of key enzymes of ceramide metabolism in skeletal muscles. The experiments were carried out on male Wistar rats (200-250 g) divided into four groups: sedentary, exercised for 30 min, 90 min, and until exhaustion. The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of ceramide, sphingosine, sphinganine and sphingosine-1-phosphate were determined in three types of muscle. We have found that the activity and expression of SPT increase gradually in each muscle with duration of exercise. These changes were followed by elevation in the content of sphinganine. These data indicate that exercise increases de novo synthesis of ceramide. The aSMase activity gradually decreased with duration of exercise in each type of muscle. After exhaustive exercise the activity of both isoforms of ceramidase were reduced in each muscle. The ceramide level depends both on duration of exercise and muscle type. The ceramide level in the soleus and white gastrocnemius decreased after 30 min of running. After exhaustive exercise it was elevated in the soleus and red gastrocnemius. It is concluded that exercise strongly affects the activity of key enzymes involved in ceramide metabolism and in consequence the level of sphingolipid intermediates in skeletal muscles.     

Acid sphingomyelinase contributes to evodiamine-induced apoptosis in human gastric cancer SGC-7901 cells

Evodiamine-induced apoptosis has been shown to have anticancer activity by eradication of some carcinoma cell lines. This study was designed to evaluate the effects of evodiamine on the viability of human gastric cancer SGC-7901 cells and to define the cell death pathway. Flow cytometry detection showed that 1.5?μM evodiamine significantly induced SGC-7901 cell apoptosis in a time-dependent manner. This apoptosis was partially inhibited by the pancaspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methylketone, which suggests that evodiamine-induced apoptosis in SGC-7901 cells is partially caspase independent. Further, the total content of sphingomyelin was decreased and expression of acid sphingomyelinase (aSMase) and neutral SMase genes in the SGC-7901cells was upregulated. Protein expression of aSMase, which was exposed to evodiamine, was shown to be increased by western blot analysis and could have been responsible for inducing caspase-independent apoptosis. Our results indicate that evodiamine stimulates upregulation of aSMase expression and hydrolysis of sphingomyelin into ceramide, which might be one of the mechanisms by which apoptosis occurs in SGC-7901 cells.     

Fetal asphyxia induces acute and persisting changes in the ceramide metabolism in rat brain

Fetal asphyctic preconditioning, induced by a brief episode of experimental hypoxia-ischemia, offers neuroprotection to a subsequent more severe asphyctic insult at birth. Extensive cell stress and apoptosis are important contributing factors of damage in the asphyctic neonatal brain. Because ceramide acts as a second messenger for multiple apoptotic stimuli, including hypoxia/ischemia, we sought to investigate the possible involvement of the ceramide pathway in endogenous neuroprotection induced by fetal asphyctic preconditioning. Global fetal asphyxia was induced in rats by clamping both uterine and ovarian vasculature for 30 min. Fetal asphyxia resulted in acute changes in brain ceramide/sphingomyelin metabolic enzymes, ceramide synthase 1, 2, and 5, acid sphingomyelinase, sphingosine-1-phosphate phosphatase, and the ceramide transporter. This observation correlated with an increase in neuronal apoptosis and in astrocyte number. After birth, ceramide and sphingomyelin levels remained high in fetal asphyxia brains, suggesting that a long-term regulation of the ceramide pathway may be involved in the mechanism of tolerance to a subsequent, otherwise lethal, asphyctic event.     

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.     

Role of thyroid hormones in regulation of sphingomyelin metabolism in the liver.

Inhibition of thyroid gland function in rats with mercasolil sharply decreased thyroxine and triiodothyronine levels in blood serum and increased acid sphingomyelinase activity and sphingomyelin content in liver. Thyroxine injected into hypothyroid rats normalized the sphingomyelin content, reduced the free ceramide content, and further increased the acid sphingomyelinase activity in liver. Thyroxine stimulated de novo sphingomyelin synthesis. Changing the thyroid status of the rats did not influence the free sphingosine content. Thyroxine blocks the accumulation of free sphingosine in the liver during activation of sphingomyelinases.     

Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase

Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser(508) as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706-35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.     

Content and structure of ceramide and sphingomyelin and sphingomyelinase activity in mouse hepatoma-22

The relative content of phosphatidylcholine is lower and that of sphingomyelin is higher in transplantable fast growing mouse hepatoma-22, thus decreasing their ratio approximately 2.5-fold versus normal liver. The ceramide content and the neutral sphingomyelinase activity is markedly higher (3- and 6.5-fold, respectively), whereas the acid sphingomyelinase activity is 4-fold lower in hepatoma-22 versus normal liver. The content of saturated fatty acids in ceramide and sphingomyelin of hepatoma-22 is higher than in normal liver. All sphingolipids of hepatoma-22 contain a considerable amount (25-37%) of sphinganine (dihydrosphingosine) along with sphingenine (sphingosine), whereas sphingolipids of normal liver contain predominantly sphingenine (over 95%). These results indicate that the activity of enzymes involved in sphingolipid biosynthesis and catabolism is disturbed in the transplantable mouse hepatoma-22 compared to normal liver.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.     

Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal.

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.     

Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K2 is associated with p53 and independent of the intrinsic apoptotic pathway

Obesity increases the risk for hepatic steatosis. Recent studies have demonstrated that high fat diet (HFD) may affect sphingolipid formation in skeletal muscles, heart, and other tissues. In this work we sought to investigate whether HFD feeding provokes changes in content and fatty acids (FAs) composition of sphingomyelin and ceramide at the level of liver and hepatic nuclei. Furthermore, we investigated whether the ceramide formation is related to the activity of either neutral sphingomyelinase (N-SMase) or acidic sphingomyelinase (A-SMase). Three weeks of HFD provision induced pronounced ceramide and sphingomyelin accumulation in both liver and hepatic nuclei, accompanied by increased activity of N-SMase but not A-SMase. Furthermore, a shift toward greater FAs saturation status in these sphingolipids was also observed. These findings support the conclusion that HFD has a major impact on sphingolipid metabolism not only in the liver, but also in hepatic nuclei.     

Nuclear membrane sphingomyelin-cholesterol changes in rat liver after hepatectomy.

Sphingomyelin and cholesterol play an important role in stabilising the plasma membranes architecture and in many physiological process such as cell growth and differentiation. Degradation of sphingomyelin by exogenous sphingomyelinase induces a decrease of cholesterol due either to an increase of esterification or to a reduced biosynthesis. Variations of sphingomyelin due to the presence of a neutral-sphingomyelinase and of sphingomyelin-synthase have been recently shown in rat liver nuclear membranes. The aim of this research is to study the relation between sphingomyelin and cholesterol in the nuclear membranes following sphingomyelinase activation and during cell proliferation. The nuclear membranes, isolated from liver nuclei, were analysed for their content in protein, nucleic acids, and lipids (sphingomyelin and cholesterol) before and after sphingomyelinase activation and during hepatic regeneration. The activities of nuclear membrane SM-syntase and sphingomyelinase were also determined. The results confirmed that also in the nuclear membranes sphingomyelinase, especially exogenous, causes a strong decrease in cholesterol. The increase observed of sphingomyelin during the first 18 h after hepatectomy followed by a decrease at 24 h, due to the different activity of the enzymes, is accompanied by similar behaviour of cholesterol. This confirms the effect of neutral-sphingomyelinase on cholesterol, due to an increase of esterification process. Changes in cholesterol content modify the nuclear membranes fluidity and, as consequence, mRNA transport as previously shown. It can therefore be concluded that the neutral sphingomyelinase, present in the nuclei, may, across this mechanism, regulate the cell function.     

Sphingomyelinase and cell-permeable ceramide analogs stimulate cellular proliferation in quiescent Swiss 3T3 fibroblasts.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.     

Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin

Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from spingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate. In sum, these studies demonstrate the novel phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from sphingomyelin to ceramide 1-phosphate via the phosphorylation of ceramide.     

Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphingosine kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of sphingosine-1-phosphate (S1P) and that S1P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S1P or increasing endogenous S1P by phorbol ester markedly protected C11 cell line from TNF-induced apoptosis. Conversely, N, N-dimethylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.