Sphingolipid Pattern Research Articles

Lipidomic Profiling Identifies Signatures of Poor Cardiovascular Health.

Ideal cardiovascular health (CVH) is defined for the presence of ideal behavioral and health metrics known to prevent cardiovascular disease (CVD). The association of circulatory phospho- and sphingo-lipids to primary reduction in cardiovascular risk is unclear. Our aim was to determine the association of CVH metrics with the circulating lipid profile of a population-based cohort. Serum sphingolipid and phospholipid species were extracted from 461 patients of the randomly selected prospective Kardiovize study based on Brno, Czech Republic. Lipids species were measured by a hyphenated mass spectrometry technique, and were associated with poor CVH scores, as defined by the American Heart Association. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) species were significantly lower in ideal and intermediate scores of health dietary metric, blood pressure, total cholesterol and blood fasting glucose compared to poor scores. Current smokers presented higher levels of PC, PE and LPE individual species compared to non-smokers. Ceramide (Cer) d18:1/14:0 was altered in poor blood pressure, total cholesterol and fasting blood glucose metrics. Poor cardiovascular health metric is associated with a specific phospho- and sphingolipid pattern. Circulatory lipid profiling is a potential biomarker to refine cardiovascular health status in primary prevention strategies.

A metabolome atlas of the aging mouse brain

The mammalian brain relies on neurochemistry to fulfill its functions. Yet, the complexity of the brain metabolome and its changes during diseases or aging remain poorly understood. Here, we generate a metabolome atlas of the aging wildtype mouse brain from 10 anatomical regions spanning from adolescence to old age. We combine data from three assays and structurally annotate 1,547 metabolites. Almost all metabolites significantly differ between brain regions or age groups, but not by sex. A shift in sphingolipid patterns during aging related to myelin remodeling is accompanied by large changes in other metabolic pathways. Functionally related brain regions (brain stem, cerebrum and cerebellum) are also metabolically similar. In cerebrum, metabolic correlations markedly weaken between adolescence and adulthood, whereas at old age, cross-region correlation patterns reflect decreased brain segregation. We show that metabolic changes can be mapped to existing gene and protein brain atlases. The brain metabolome atlas is publicly available (https://mouse.atlas.metabolomics.us/) and serves as a foundation dataset for future metabolomic studies.

Analysis of the Plasma Metabolome after Trauma, Novel Circulating Sphingolipid Signatures, and In-Hospital Outcomes

Trauma is the leading cause of death and disability for individuals under age 55. Many severely injured trauma patients experience complicated clinical courses despite appropriate initial therapy. We sought to identify novel circulating metabolomic signatures associated with clinical outcomes following trauma. Untargeted metabolomics and circulating plasma immune mediator analysis was performedon plasma collected during 3 post-injury time periods (<6 hours [h], 6 h-24h, day 2-day 5) in critically ill trauma patients enrolled between April 2004 and May 2013 at UPMC Presbyterian Hospital in Pittsburgh, PA. Inclusion criteria were age ≥ 18 years, blunt mechanism, ICU admission, and expected survival ≥ 24 h. Exclusion criteria were isolated head injury, spinal cord injury, and pregnancy. Exploratory endpoints included length of stay (overall and ICU), ventilator requirements, nosocomial infection, and Marshall organ dysfunction (MOD) score. The top 50 metabolites were isolated usingrepeated measures ANOVA and multivariate empirical Bayesian analysis for further study. Eighty-six patients were included for analysis. Sphingolipids were enriched significantly (chi-square, p < 10-6) among the top 50 metabolites. Clustering of sphingolipid patterns identified 3 patient subclasses: nonresponders (no time-dependent change in sphingolipids, n= 41), sphingosine/sphinganine-enhanced (n= 24), and glycosphingolipid-enhanced (n=21). Compared with the sphingolipid-enhanced subclasses, nonresponders had longer mean length of stay, more ventilator days, higher MOD scores, and higher circulating levels of proinflammatory immune mediators IL-6, IL-8, IL-10, MCP1/CCL2, IP10/CXCL10, and MIG/CXCL9 (all p < 0.05), despite similar Injury Severity Scores (p=0.12). Metabolomic analysis identified broad alterations in circulating plasma sphingolipids after blunt trauma. Circulating sphingolipid signatures and their association with both clinical outcomes and circulating inflammatory mediators suggest a possible link between sphingolipid metabolism and the immune response to trauma.

APOL1 polymorphism modulates sphingolipid profile of human podocytes

Apolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-3H]sphingosine. HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.

Farnesoid X receptor activation induces the degradation of hepatotoxic 1-deoxysphingolipids in non-alcoholic fatty liver disease.

Patients with non-alcoholic fatty liver disease (NAFLD) exhibit higher levels of plasma 1-deoxysphingolipids than healthy individuals. The aim of this study was to investigate the role of farnesoid X receptor (FXR) in 1-deoxysphingolipid de novo synthesis and degradation. Mice were fed with a high-fat diet (HFD) to induce obesity and NAFLD, and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Sphingolipid patterns from NAFLD patients and mouse models were assessed by liquid chromatography-mass spectrometry. The molecular mechanism underlying the effect of FXR activation on sphingolipid metabolism was studied in Huh7 cells and primary cultured hepatocytes, as well as in a 1-deoxysphinganine-treated mouse model. 1-deoxysphingolipids were increased in both NAFLD patients and mouse models. FXR activation by OCA protected the liver against oxidative stress, apoptosis, and reduced 1-deoxysphingolipid levels, both in a HFD-induced mouse model of obesity and in 1-deoxysphinganine-treated mice. In vitro, FXR activation lowered intracellular 1-deoxysphingolipid levels by inducing Cyp4f-mediated degradation, but not by inhibiting de novo synthesis, thereby protecting hepatocytes against doxSA-induced cytotoxicity, mitochondrial damage, and apoptosis. Overexpression of Cyp4f13 in cells was sufficient to ameliorate doxSA-induced cytotoxicity. Treatment with the Cyp4f pan-inhibitor HET0016 or FXR knock-down fully abolished the protective effect of OCA, indicating that OCA-mediated 1-deoxysphingolipid degradation is FXR and Cyp4f dependent. Our study identifies FXR-Cyp4f as a novel regulatory pathway for 1-deoxysphingolipid metabolism. FXR activation represents a promising therapeutic strategy for patients with metabolic syndrome and NAFLD.

Sphingolipid serum profiling in vitamin D deficient and dyslipidemic obese dimorphic adults

Recent studies on Saudi Arabians indicate a prevalence of dyslipidemia and vitamin D deficiency (25(OH)D) in both normal weight and obese subjects. In the present study the sphingolipid pattern was investigated in 23 normolipidemic normal weight (NW), 46 vitamin D deficient dyslipidemic normal weight (-vitDNW) and 60 vitamin D deficient dyslipidemic obese (-vitDO) men and women by HPTLC-primuline profiling and LC-MS analyses. Results indicate higher levels of total ceramide (Cer) and dihydroceramide (dhCers C18–22) and lower levels of total sphingomyelins (SMs) and dihydrosphingomyelin (dhSM) not only in -vitDO subjects compared to NW, but also in –vitDNW individuals. A dependency on body mass index (BMI) was observed analyzing specific Cer acyl chains levels. Lower levels of C20 and 24 were observed in men and C24.2 in women, respectively. Furthermore, LC-MS analyses display dimorphic changes in NW, -vitDNW and –vitDO subjects. In conclusion, LC-MS data identify the independency of the axis high Cers, dhCers and SMs from obesity per se. Furthermore, it indicates that long chains Cers levels are specific target of weight gain and that circulating Cer and SM levels are linked to sexual dimorphism status and can contribute to predict obese related co-morbidities in men and women.

Sphingosine 1-Phosphate Receptors and Metabolic Enzymes as Druggable Targets for Brain Diseases.

The central nervous system is characterized by a high content of sphingolipids and by a high diversity in terms of different structures. Stage- and cell-specific sphingolipid metabolism and expression are crucial for brain development and maintenance toward adult age. On the other hand, deep dysregulation of sphingolipid metabolism, leading to altered sphingolipid pattern, is associated with the majority of neurological and neurodegenerative diseases, even those totally lacking a common etiological background. Thus, sphingolipid metabolism has always been regarded as a promising pharmacological target for the treatment of brain disorders. However, any therapeutic hypothesis applied to complex amphipathic sphingolipids, components of cellular membranes, has so far failed probably because of the high regional complexity and specificity of the different biological roles of these structures. Simpler sphingosine-based lipids, including ceramide and sphingosine 1-phosphate, are important regulators of brain homeostasis, and, thanks to the relative simplicity of their metabolic network, they seem a feasible druggable target for the treatment of brain diseases. The enzymes involved in the control of the levels of bioactive sphingoids, as well as the receptors engaged by these molecules, have increasingly allured pharmacologists and clinicians, and eventually fingolimod, a functional antagonist of sphingosine 1-phosphate receptors with immunomodulatory properties, was approved for the therapy of relapsing–remitting multiple sclerosis. Considering the importance of neuroinflammation in many other brain diseases, we would expect an extension of the use of such analogs for the treatment of other ailments in the future. Nevertheless, many aspects other than neuroinflammation are regulated by bioactive sphingoids in healthy brain and dysregulated in brain disease. In this review, we are addressing the multifaceted possibility to address the metabolism and biology of bioactive sphingosine 1-phosphate as novel targets for the development of therapeutic paradigms and the discovery of new drugs.

Regulation of Serum Sphingolipids in Andean Children Born and Living at High Altitude (3775 m).

Recent studies on Andean children indicate a prevalence of dyslipidemia and hypertension compared to dwellers at lower altitudes, suggesting that despite similar food intake and daily activities, they undergo different metabolic adaptations. In the present study, the sphingolipid pattern was investigated in serum of 7 underweight (UW), 30 normal weight (NW), 13 overweight (OW), and 9 obese (O) Andean children by liquid chromatography-mass spectrometry (LC-MS). Results indicate that levels of Ceramides (Cers) and sphingomyelins (SMs) correlate positively with biochemical parameters (except for Cers and Vitamin D, which correlate negatively), whereas sphingosine-1-phosphate (S1P) correlates negatively. Correlation results and LC-MS data identify the axis high density lipoprotein-cholesterol (HDL-C), Cers, and S1P as related to hypoxia adaptation. Specifically UW children are characterized by increased levels of S1P compared to O and lower levels of Cers compared to NW children. Furthermore, O children show lower levels of S1P and similar levels of Cers and SMs as NW. In conclusion, our results indicate that S1P is the primary target of hypoxia adaptation in Andean children, and its levels are associated with hypoxia tolerance. Furthermore, S1P can act as marker of increased risk of metabolic syndrome and cardiac dysfunction in young Andeans living at altitude.

Associations of Mediterranean-style Diet Adherence with Circulating Sphingolipid Patterns (P18-004-19)

ObjectivesBuilding evidence suggests that greater adherence to a Mediterranean-style diet (MD) may reduce the risk of cardiovascular disease, type 2 diabetes, and dementia. However, our understanding of the biological mechanisms explaining these associations is limited. Altered sphingolipid metabolism (e.g., increased ceramides, glycosyl- and lactosyl-ceramides, sphingoid bases, and sphingomyelin (SM)) has been implicated in the development of chronic health conditions. We aimed to quantify the impact of MD adherence on circulating patterns of sphingolipid metabolites. MethodsCross-sectional analyses were conducted in the Boston Puerto Rican Health Study among those with available data (n = 540, 56 y, 74% women). Diet was obtained with a semi-quantitative FFQ. The MD score (MDS) includes 7 beneficial (e.g., fish, monounsaturated/saturated fat ratio (MUFA/SFA)) and 2 moderation components (e.g., meat and dairy) (range 0–9). Metabolomic analysis was conducted on plasma samples using ultra-high-performance liquid chromatography-tandem mass spectrometry. Twenty-six sphingolipids were retained for analyses. Principal components analysis was used to derive 5 sphingolipid patterns. Statistical analyses included multiple linear regression and ANCOVA. ResultsCirculating sphingolipid patterns were characterized by dihydro-SMs and SM species with very-long chain fatty acid groups (Factor 1); glycosyl- and lactosyl-ceramides (Factor 2); ceramides (Factor 3); sphingoid bases, sphinganine-1-phosphate and sphingosine-1-phosphate (Factor 4); and SM species with a sphingadienine group (Factor 5). Each 1-point increase in the MDS was associated with lower Factor 3 z-score (β = −0.07 ± 0.03, P = 0.01), as well as with lower Factor 5 z-score (−0.08 ± 0.02, P = 0.001). We observed that individual MDS components including whole grains, fish, and the MUFA/SFA ratio were associated with the sphingolipid patterns (Table 1). ConclusionsOur results suggest that a Mediterranean-style diet and components of the diet may reduce circulating concentrations of ceramides and certain sphingomyelin species. This may in part explain the protective benefits of this dietary pattern. Longitudinal analyses are needed to clarify the long-term impact of this dietary pattern on sphingolipid metabolism. Funding SourcesNIH grants P01-AG023394 P50-HL105185 and R01-AG027087. Supporting Tables, Images and/or Graphs▪

Identification of sphingolipids as novel noninvasive biomarkers of liver damage caused by chronic hepatitis B

Sphingolipids are key components in the viral life cycle that affects host‐pathogen interactions. In this study, a total of 98 subjects were classified as either healthy control (n=28), chronic hepatitis B (n=23), cirrhosis (n=21), or acute‐on‐chronic liver failure patients (n=26), according to the clinical tests. The serum levels of 43 sphingolipids were detected by high performance liquid chromatography tandem mass spectrometry. Biometrical methods were used to identify and evaluate the potential biomarkers. Sphingolipid patterns differed substantially between the four stages. Sixteen sphingolipids were identified as potential biomarkers. Receiver operating characteristic analysis demonstrated that 15 of them showed significant diagnostic performance (area under the curve (AUC) &gt;; 0.7), especially in distinguishing cirrhosis from chronic hepatitis B (AUC &gt;; 0.9). A marked improvement of diagnostic performance was achieved by combining biomarkers using logistic regression. Significant correlations between sphingolipids, measures of liver damage (MELD and Child‐Pugh scores) and HBV DNA levels were also obtained. Our study revealed that HBV alters sphingolipid metabolism, and sphingolipids hold promise as novel noninvasive biomarkers of HBV caused liver damage. Ministry of Science and Technology of China (2012ZX09301002–006)

Isolation and Characterization of 2 New Human Rotator Cuff and Long Head of Biceps Tendon Cells Possessing Stem Cell–Like Self-Renewal and Multipotential Differentiation Capacity

Background: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Hypothesis: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Study Design: Controlled laboratory study. Methods: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Results: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types—namely, osteoblasts, adipocytes, and skeletal muscle cells. Conclusion: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Clinical Relevance: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

Sphingolipids and brain resident macrophages in neuroinflammation: an emerging aspect of nervous system pathology.

Sphingolipid metabolism is deeply regulated along the differentiation and development of the central nervous system (CNS), and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system. Microglia are resident macrophages of the CNS involved in general maintenance of neural environment. Modulations in microglia phenotypes may contribute to pathogenic forms of inflammation. Since defects in macrophage/microglia activity contribute to neurodegenerative diseases, it will be essential to systematically identify the components of the microglial cell response that contribute to disease progression. In such complex processes, the sphingolipid systems have recently emerged to play important roles, thus appearing as a key new player in CNS disorders. This review provides a rationale for harnessing the sphingolipid metabolic pathway as a potential target against neuroinflammation.

Secondary Alterations of Sphingolipid Metabolism in Lysosomal Storage Diseases

In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal membrane sphingolipid patterns and altered plasma membrane organization. In this paper, we review the potential importance of these alterations to the pathogenesis of these diseases and focus the reader's attention on some secondary alterations of sphingolipid metabolism that have been sporadically reported in the literature. Moreover, we present a detailed analysis of the lipid composition of different central nervous system and extraneural tissues from the acid sphingomyelinase-deficient mouse, the animal model for Niemann-Pick disease type A, characterized by the accumulation of sphingomyelin. Our data show an unexpected, tissue specific selection of the accumulated molecular species of sphingomyelin, and an accumulation of GM3 and GM2 gangliosides in both neural and extraneural tissues, that cannot be solely explained by the lack of acid sphingomyelinase.

Deregulated Sphingolipid Metabolism and Membrane Organization in Neurodegenerative Disorders

Sphingolipids are polar membrane lipids present as minor components in eukaryotic cell membranes. Sphingolipids are highly enriched in nervous cells, where they exert important biological functions. They deeply affect the structural and geometrical properties and the lateral order of cellular membranes, modulate the function of several membrane-associated proteins, and give rise to important intra- and extracellular lipid mediators. Sphingolipid metabolism is regulated along the differentiation and development of the nervous system, and the expression of a peculiar spatially and temporarily regulated sphingolipid pattern is essential for the maintenance of the functional integrity of the nervous system: sphingolipids in the nervous system participate to several signaling pathways controlling neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability and synaptic transmission, and neuron-glia interactions, including the formation and stability of central and peripheral myelin. In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal sphingolipid patterns and altered membrane organization that participate to several events related to the pathogenesis of these diseases. The most impressive consequence of this deregulation is represented by anomalous sphingolipid-protein interactions that are at least, in part, responsible for the misfolding events that cause the fibrillogenic and amyloidogenic processing of disease-specific protein isoforms, such as amyloid beta peptide in Alzheimer's disease, huntingtin in Huntington's disease, alpha-synuclein in Parkinson's disease, and prions in transmissible encephalopathies. Targeting sphingolipid metabolism represents today an underexploited but realistic opportunity to design novel therapeutic strategies for the intervention in these diseases.

Expression of Sialidase Neu2 in Leukemic K562 Cells Induces Apoptosis by Impairing Bcr-Abl/Src Kinases Signaling

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually "switched on" activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45-66 kDa, showed a 50-70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.

Ceramide and sphingomyelin species of fibroblasts and neurons in culture

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

Induction of axonal differentiation by silencing plasma membrane‐associated sialidase Neu3 in neuroblastoma cells

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.

Lack of ceramide generation and altered sphingolipid composition are associated with drug resistance in human ovarian carcinoma cells

PTX (Paclitaxel) is an antimitotic agent used in the treatment of a number of major solid tumours, particularly in breast and ovarian cancer. This study was undertaken to gain insight into the molecular alterations producing PTX resistance in ovarian cancer. PTX treatment is able to induce apoptosis in the human ovarian carcinoma cell line, CABA I. PTX-induced apoptosis in CABA I cells was accompanied by an increase in the cellular Cer (ceramide) levels and a decrease in the sphingomyelin levels, due to the activation of sphingomyelinases. The inhibition of acid sphingomyelinase decreased PTX-induced apoptosis. Under the same experimental conditions, PTX had no effect on Cer and sphingomyelin levels in the stable PTX-resistant ovarian carcinoma cell line, CABA-PTX.The acquisition of the PTX-resistant phenotype is accompanied by unique alterations in the complex sphingolipid pattern found on lipid extraction. In the drug-resistant cell line, the levels of sphingomyelin and neutral glycosphingolipids were unchanged compared with the drug-sensitive cell line. The ganglioside pattern in CABA I cells is more complex compared with that of CABA-PTX cells. Specifically, we found that the total ganglioside content in CABA-PTX cells was approximately half of that in CABA I cells, and GM3 ganglioside content was remarkably higher in the drug-resistant cell line. Taken together our findings indicate that: i) Cer generated by acid sphingomyelinase is involved in PTX-induced apoptosis in ovarian carcinoma cells, and PTX-resistant cells are characterized by their lack of increased Cer upon drug treatment, ii) PTX resistance might be correlated with an alteration in metabolic Cer patterns specifically affecting cellular ganglioside composition.

Metabolism of cerebroside sulfate and subcellular distribution of its metabolites in cultured skin fibroblasts from controls, metachromatic leukodystrophy, and globoid cell leukodystrophy.

With pulse-chase study of 1-[14C]stearic acid-labeled cerebroside sulfate (14C-CS) and subsequent subcellular fractionation by Percoll gradient, the metabolism of CS and translocation of its metabolites in human skin fibroblasts from controls, metachromatic leukodystrophy (MLD), and globoid cell leukodystrophy (GLD) were studied. In control skin fibroblasts, CS was transported to lysosome and metabolized there to galactosylceramide (GalCer) and ceramide (Cer) within 1 h. During the chase period, radioactivity was increased at plasma membrane plus Golgi as phospholipids and no accumulation of GalCer or Cer was found in lysosome. In MLD fibroblasts, 95% of 14C-CS taken up was unhydrolyzed at 24 h-chase and accumulated at not only lysosome but also plasma membrane. In GLD fibroblasts, GalCer was accumulated throughout the subcellular fractions and more accumulated mainly at plasma membrane plus Golgi with longer pulse. This translocation of lipid from lysosome seems to have considerable function, even in lipidosis, which may result in an imbalance of the sphingolipid pattern on the cell surface and these changes might be one of causes of neuronal dysfunction in sphingolipidosis.

The sphingolipid composition of bovine kidney cortex, medulla and papilla

1. 1. The major sphingolipids of bovine kidney cortex, medulla and papilla were prepared, weighed and characterized. Each of these regions had its own sphingolipid pattern. 2. 2. Sphingomyelins were the major sphingolipid class of bovine kidney. Their concentration decreased from cortex to papilla. 3. 3. Free ceramides were present in considerable amounts. They also decreased in concentration from cortex to papilla. 4. 4. The main glycosphingolipids were glucosylceramides, galactosylceramides and galactosylceramide sulphate esters (sulphatides). Diglycosylceramides and triglycosylceramides were smaller fractions, and tetraglycosylceramides were not detected. Gangliosides were not isolated. 5. 5. Galactosylceramides and sulphatides were concentrated in the medulla, while the cortex and papilla had lower concentrations. 6. 6. The level of glucosylceramides was approximately the same in the cortex as in the medulla, but it was much higher in the papilla. 7. 7. Diglycosylceramides and triglycosylceramides, measured together, increased gradually from cortex to papilla. In the cortex their ratio was about 1: 2, in the medulla 1:1, and in the papilla 3:2. 8. 8. A simple thin-layer Chromatographie study on the phospholipid patterns of the cortex and medulla revealed no differences in relative amounts. 9. 9. The (Na +-K +-ATPase, determined as ouabain-sensitive ATPase, had about the same activity in the cortex as in the medulla.