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.     

Sphingolipids of transplantable rat nephroma-RA

Contents of sphingolipids (ceramide, sphingomyelin, gangliosides) and the composition of their sphingoid bases were studied in the transplantable rat nephroma-RA and in rat kidneys. The content of sphingomyelin was about 1.3-fold decreased and the content of ceramide was about 1.4-fold increased in the nephroma compared to normal kidneys, and this correlated with a 1.4-fold increased activity of neutral sphingomyelinase; however, the activity of the acidic isoform of the enzyme was virtually unchanged. The content of gangliosides was also increased in the nephroma. Ceramide and sphingomyelin of the nephroma, in addition to sphingosine, contained a significant amount of sphinganine, although a considerable amount of the latter was also found in the renal ceramide. The ratio sphingosine/sphinganine in sphingomyelins changed from 65:1 in kidneys to 5:1 in the nephroma. Thus, the biosynthesis of sphingoid bases seems to be disturbed in the transplantable rat nephroma-RA compared to normal kidneys.     

Dihydroceramide desaturase activity in tumors

Dihydroceramide desaturase activity in the transplantable mouse hepatoma-22, rat hepatoma-27, M1 sarcoma, and RS1 rat cholangiocellular carcinoma has been investigated. It was found that the dihydroceramide desaturase activity in mouse hepatoma-22 is lower than that in normal mouse liver. However, the activity of this enzyme in subcutaneously and intrahepatically transplanted rat hepatoma-27 is increased compared to normal value. Dihydroceramide desaturase activity in subcutaneously and intrahepatically transplanted M1 sarcoma as well as in hepatoma-27 is dependent on the tumor microenvironment. The enzyme activity in RS1 tumor was not revealed. The data indicate that dihydroceramide desaturase activity depends on the tumor type and its microenvironment.     

The changes in the sphingenine/sphinganine ratio in sphingolipids of transplantable rat tumors depends on a transplantation organ

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.     

The Sphingenine/Sphinganine Ratio in Sphingolipids of Transplantable Rat Tumors Depends on a Transplantation Organ

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.     

Exploring the molecular mechanisms of OSU-03012 on vascular smooth muscle cell proliferation

Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein (UCV). We evaluate the sphingolipid composition of UCV and its alteration in preeclampsia. The veins were taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Thin layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed for sphingolipid analyses. The UCV walls of newborns delivered by healthy mothers are abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases are rather low. Preeclampsia is associated with a significant decrease in sphingomyelins and ceramides, whereas the sphingoid bases changed in uncharacteristic manner. The increase in sphinganine and sphingosine 1-phosphate was accompanied with a decrease in sphingosine, hydroxysphinganine and sphinganine 1-phosphate. Stearate is the dominating fatty acid in sphingomyelins and ceramides of both control and preeclamptic veins. Sphingolipids and some sphingoid bases are bioactive molecules which contribute to regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of UCV wall resulting in remodelling of its composition.     

Ablation of Ceramide Synthase 2 Causes Chronic Oxidative Stress Due to Disruption of the Mitochondrial Respiratory Chain

Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids.     

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders

The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.     

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.     

Secondary structure and methylation of DNA in reinoculated tumors and cultures of embryonic cells transformed by oncogenic viruses]

Using the kinetic formaldehyde method the concentration of secondary structure defects (SSD) in the DNAs of ascite leukosis L 1210 cells and cultures of hamster embryonic cells transformed by virus SV-40 and polyoma was evaluated. It was found that this concentration was considerably higher than in the DNAs from normal liver and primary culture of mouse embryonic cells. The occurrence of the defects in malignant cell DNAs is not due to enzymatic degradation of DNA. Using thin-layer chromatography the content of m5C in the DNAs from 17 sources (transformed cell cultures, experimental tumours and liver cells of mouses with Ehrlich ascite carcinoma) were determined. The methylation level for all these DNAs was higher than for normal mouse and rat liver DNAs. No correlation between the SSD concentration and m5C content in the DNAs studied was observed.     

Phospholipid composition of mitochondria and microsomes from some transplantable rat tumors

The phospholipid compositions of mitochondria and microsomes from rat liver, kidney, nephroma RA and sarcoma 45 were investigated. The "lipid dedifferentiation" of hepatoma membranes found earlier was shown to extend also to other tumours. However, this phenomenon may concern some, but not all phospholipids.     

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C₂₀-, C₂₂-, and C₂₄-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.     

Liquid chromatography-tandem mass spectrometric determination of ceramides and related lipid species in cellular extracts

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C(4) ceramide as an internal standard; simple liquid extraction; a short XTerra MS C(18) (3 microm, 50 mm x2.0 mm) column; a gradient mobile phase of 5mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3-->1/2/7); mass spectrometric detection using electrospray ionization. This LC-MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C(2) ceramide and H(2)O(2).     

Differences in Lipid Composition and Proliferative Activity of Rat Hepatoma-27 Depending on the Target Organ

Proliferative activity and lipid composition (phospholipids and gangliosides) were studied in rat hepatoma-27 transplanted subcutaneously or intrahepatically (as models for primary and metastasizing tumors). The mitotic index of subcutaneously transplanted hepatoma far exceeded that of the intrahepatically transplanted tumor. The overall amounts of both phospholipids and gangliosides increased appreciably in the subcutaneously growing hepatoma (in contrast to the intrahepatically growing tumor) in comparison to the control hepatic tissue. The ganglioside composition in the tumors differs from that in the liver: ganglioside GD3 appears, whereas gangliosides GD1b and GT1b decrease in amount in the intrahepatic tumor compared to the control liver and disappear in the subcutaneously transplanted hepatoma. In both tumor types, the amounts of both phosphatidylethanolamine and sphingomyelin exceed the control values. Comparison of these results with previously reported data concerning the phospholipid and ganglioside composition in the regenerating rat liver indicates that the difference in the lipid composition between the subcutaneously and intrahepatically growing hepatomas-27 is due to their different proliferative status and also their microenvironment.     

Enhanced transcription of genes of the intracisternal A particles and B2 elements in mouse tumors

A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.     

Imaging Mass Spectrometry Reveals Acyl-Chain- and Region-Specific Sphingolipid Metabolism in the Kidneys of Sphingomyelin Synthase 2-Deficient Mice

Obesity was reported to cause kidney injury by excessive accumulation of sphingolipids such as sphingomyelin and ceramide. Sphingomyelin synthase 2 (SMS2) is an important enzyme for hepatic sphingolipid homeostasis and its dysfunction is considered to result in fatty liver disease. The expression of SMS2 is also high in the kidneys. However, the contribution of SMS2 on renal sphingolipid metabolism remains unclear. Imaging mass spectrometry is a powerful tool to visualize the distribution and provide quantitative data on lipids in tissue sections. Thus, in this study, we analyzed the effects of SMS2 deficiency on the distribution and concentration of sphingomyelins in the liver and kidneys of mice fed with a normal-diet or a high-fat-diet using imaging mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry. Our study revealed that high-fat-diet increased C18–C22 sphingomyelins, but decreased C24-sphingomyelins, in the liver and kidneys of wild-type mice. By contrast, SMS2 deficiency decreased C18–C24 sphingomyelins in the liver. Although a similar trend was observed in the whole-kidneys, the effects were minor. Interestingly, imaging mass spectrometry revealed that sphingomyelin localization was specific to each acyl-chain length in the kidneys. Further, SMS2 deficiency mainly decreased C22-sphingomyelin in the renal medulla and C24-sphingomyelins in the renal cortex. Thus, imaging mass spectrometry can provide visual assessment of the contribution of SMS2 on acyl-chain- and region-specific sphingomyelin metabolism in the kidneys.     

Effect of a cholesterol-biosynthesis inhibitor on the fatty acid composition of phospholipids in the serum and tissues of rats

1. Changes produced by a cholesterol-biosynthesis inhibitor, trans-1,4-bis-(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944), in the total fatty acids in the liver and brain, and in phospholipids in the serum, liver, heart, brain and lungs from male rats, have been studied. 2. Treatment with AY-9944 produced the following changes in the fatty acid composition: (a) a marked decrease in the percentage of linoleic acid and an increase in oleic acid in the total fatty acids in the liver; (b) in the serum, an overall decrease in the percentage of linoleic acid in neutral lipids and phospholipids; (c) an increased content of linoleic acid in the beta-acyl chain of phosphatidylcholines in the liver and in sphingomyelins in the brain and lungs; (d) an increased content of palmitic acid and oleic acid in the beta-acyl chain of phosphatidylcholine in the liver, heart and lungs; (e) an increased content of phosphatidylcholines and sphingomyelins, together with an increased percentage of saturated fatty acids in these phosphatides in the lungs. 3. Changes in the phosphatides and the production of foam cells in the lungs suggest that AY-9944 may be of use in the study of the alveolar membrane.     

Albumin-based drug delivery as novel therapeutic approach for rheumatoid arthritis

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.     

Phospholipid composition of nuclear membranes and cell nuclei of rat liver and hepatoma 27]

Phospholipid composition of nuclei and nuclear membranes from rat liver and hepatoma-27 were investigated. Hepatoma nuclei and nuclear membranes were found to contain cardiolipin which was absent in the same fractions of rat liver. In the nuclei and nuclear membranes of hepatoma the content of sphingomyelin was higher and that of lecithin is lower than in the corresponding subcellular fractions of rat liver. The content of acid phospholipids was much higher in hepatoma nuclear membranes than those of the normal liver. The data obtained show that the general trend of lipid dedifferentiation which earlier was demonstrated for various membranes of different tumor cells is observed also in the case of nuclear membranes.