Sphingolipid Classes Research Articles

Ceramide Analysis by Multiple Linked-Scan Mass Spectrometry Using a Tandem Quadrupole Instrument.

Ceramides are a special class of sphingolipids and play a central role in sphingolipid metabolism, and have diverse structures. In this book chapter, tandem quadrupole mass spectrometric approaches applying multiple linked scannings including various constant neutral loss scan (NLS) and precursor ion scan (PIS), the unique applicable feature of a triple-stage quadrupole (TSQ) instrument for analysis of ceramides desorbedas [M-H]- and [M+Li]+ ions are described. Thesemultiple dimensional tandem mass spectrometric approaches are fully adapted to the conventional shotgun lipidomics workflow with minimal or without prior chromatographic separation to profile ceramide molecules, and thus detection of a whole class of ceramide or various specific ceramide subclasses in crude lipid extractcan be achieved. With addition of internal standard(s), semi-quantitation of ceramide in the lipid extract of biological origin is possible. Examples have shown promise in ceramide profiling of several whole lipid extracts from porcine brain, the model Dictyostelium Discoideum cells for cancer study, and skin.

Sphingolipid Properties in Sake Rice Cultivars and Changes During Polishing and Brewing.

Sphingolipids, including ceramide (Cer) and glucosylceramide (GlcCer), have the characteristic structural units called sphingoid bases, and are constituents of cell and vacuole membranes. Plant sphingolipids bear highly diverse base structures and the base composition differs depending on the plant species. It is thought that the composition of sphingolipid classes and sphingoid bases is related to membrane fractions. However, there is little information about differences in sphingolipids among plant cultivars and the changes occurring in sphingolipids during food processing. This study investigated sphingolipids in sake rice (saka-mai) cultivars grown for sake (rice wine), and the changes in sphingolipids during polishing and brewing. In six brown rice samples, there were no large differences of the base composition among Cer or GlcCer of cultivars, whereas there were differences in their sphingolipid contents. When compared to brown rice, highly polished rice contained lower levels of sphingolipids, especially Cer. For three rice brans from different polishing steps, the Cer content was higher in the outer bran than in the inner bran. Sake and sake lees (sake-kasu) were produced by three different starter cultures (shubo preparations: the mixture of koji rice as an enzyme cocktail containing amylases, sake yeast, and adding rice as a carbohydrate source). The Cer/GlcCer ratio in sake and sake lees depended on the starter culture; Cer and GlcCer in sake lees possessed a fungi-specific base, 9-methyl-trans-4,trans-8- sphingadienine. In addition, sake lees had a higher Cer/GlcCer ratio when compared to highly polished rice as a sake source. These results suggest that the sphingolipid content of brown rice differs depending on the rice cultivar; further, the sphingolipids and the sphingolipid composition in sake and sake lees are affected by fungal sphingolipids and self-digestion during brewing.

Effects of a Lacto-Ovo-Vegetarian Diet on the Plasma Lipidome and Its Association with Atherosclerotic Burden in Patients with Coronary Artery Disease-A Randomized, Open-Label, Cross-over Study.

A vegetarian diet has been associated with a lower risk of coronary artery disease (CAD). Plasma triacylglycerols, ceramides, and phosphatidylcholines may improve prediction of recurrent coronary events. We sought to investigate effects of a lacto-ovo-vegetarian diet (VD) on plasma lipidome in CAD patients and simultaneously assess associations of plasma lipids with the extent of coronary atherosclerotic burden. We analyzed 214 plasma lipids within glycerolipid, sphingolipid, and sterol lipid classes using lipidomics from a randomized controlled, crossover trial comprising 31 CAD patients on standard medical therapy. Subjects completed a four-week intervention with VD and isocaloric meat diet (MD), separated by a four-week washout period. The VD increased levels of 11 triacylglycerols and lowered 7 triacylglycerols, 21 glycerophospholipids, cholesteryl ester (18:0), and ceramide (d18:1/16:0) compared with MD. VD increased triacylglycerols with long-chain polyunsaturated fatty acyls while decreased triacylglycerols with saturated fatty acyls, phosphatidylcholines, and sphingomyelins than MD. The Sullivan extent score (SES) exhibited on coronary angiograms were inversely associated with triacylglycerols with long-chain polyunsaturated fatty acyls. Phosphatidylcholines that were lower with VD were positively associated with SES and the total number of stenotic lesions. The VD favorably changed levels of several lipotoxic lipids that have previously been associated with increased risk of coronary events in CAD patients.

Coffee, Black Tea, and Green Tea Consumption in Relation to Plasma Metabolites in an Asian Population.

Coffee and tea are among the most popular beverages in the world. However, the association between habitual coffee, green tea, and black tea consumption with metabolomics profiles in Asian populations remain largely unknown. 158 metabolites (14 amino acids, 45 acylcarnitines, and 99 sphingolipids) in the blood plasma of participants are measured from the population-based Singapore Prospective Study Program cohort using mass spectrometry (MS). Linear regression models are used to obtain the estimates for the association between coffee and tea consumption with metabolite levels, adjusted for potential confounders and false discovery rate (FDR). Coffee consumption is significantly associated with higher levels of 63 sphingolipids (29 sphingomyelins, 32 ceramides, a sphingosine-1-phosphate, and a sphingosine) and lower levels of 13 acylcarnitines and alanine. Black tea consumption is significantly associated with higher levels of eight sphingolipids, and lower levels of an amino acid, whereas green tea is significantly inversely associated with four metabolites (C8:1-OH acylcarnitine, ganglioside GM3 d18:1/16:0, sphingomyelins d18:2/18:0 and d18:1/14:0). Coffee, black tea, and green tea consumption are associated with plasma levels of certain classes of sphingolipids and acylcarnitines in an Asian population, particularly sphingomyelins, which may mediate the health benefits of these beverages.

A Lipidomic Signature Complements Stemness Features Acquisition in Liver Cancer Cells.

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs’ ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

Dihydroceramides: their emerging physiological roles and functions in cancer and metabolic diseases.

Dihydroceramides (DhCers) are a type of sphingolipids that for a long time were regarded as biologically inactive. They are metabolic intermediates of the de novo sphingolipid synthesis pathway, and are converted into ceramides (Cers) with the addition of a double bond. Ceramides are abundant in tissues and have well-established biological functions. On the contrary, dihydroceramides are less prevalent, and despite their hitherto characterization as inert lipids, studies of the past decade began to unravel their implication in various biological processes distinct from those involving ceramides. These processes include cellular stress responses and autophagy, cell growth, pro-death or pro-survival pathways, hypoxia, and immune responses. In addition, their plasma concentration has been related to metabolic diseases and shown as a long-term predictor of type 2 diabetes onset. They are thus important players and potential biomarkers in pathologies ranging from diabetes to cancer and neurodegenerative diseases. The purpose of this mini-review is to highlight the emergence of dihydroceramides as a new class of bioactive sphingolipids by reporting recent advances on their biological characterization and pathological implications, focusing on cancer and metabolic diseases.

Role of ceramides in the pathogenesis of diabetes mellitus and its complications.

Diabetes mellitus (DM) is a systemic metabolic disease that affects 463 million adults worldwide and is a leading cause of cardiovascular disease, blindness, nephropathy, peripheral neuropathy, and lower-limb amputation. Lipids have long been recognized as contributors to the pathogenesis and pathophysiology of DM and its complications, but recent discoveries have highlighted ceramides, a class of bioactive sphingolipids with cell signaling and second messenger capabilities, as particularly important contributors to insulin resistance and the underlying mechanisms of DM complications. Besides their association with insulin resistance and pathophysiology of type 2 diabetes, evidence is emerging that certain species of ceramides are mediators of cellular mechanisms involved in the initiation and progression of microvascular and macrovascular complications of DM. Advances in our understanding of these associations provide unique opportunities for exploring ceramide species as potential novel therapeutic targets and biomarkers. This review discusses the links between ceramides and the pathogenesis of DM and diabetic complications and identifies opportunities for novel discoveries and applications.

Cationic amphiphilic drugs induce elevation in lysoglycerophospholipid levels and cell death in leukemia cells.

Repurposing of cationic amphiphilic drugs (CADs) emerges as an attractive therapeutic solution against various cancers, including leukemia. CADs target lysosomal lipid metabolism and preferentially kill cancer cells via induction of lysosomal membrane permeabilization, but the exact effects of CADs on the lysosomal lipid metabolism remain poorly illuminated. We aimed to systematically monitor CAD-induced alterations in the quantitative lipid profiles of leukemia cell lines in order to chart effects of CADs on the metabolism of various lipid classes present in these cells. We conducted this study on eight cultured cell lines representing two leukemia types, acute lymphoblastic leukemia and acute myeloid leukemia. Mass spectrometry-based quantitative shotgun lipidomics was employed to quantify the levels of around 400 lipid species of 26 lipid classes in the leukemia cell lines treated or untreated with a CAD, siramesine. The two leukemia types displayed high, but variable sensitivities to CADs and distinct profiles of cellular lipids. Treatment with siramesine rapidly altered the levels of diverse lipid classes in both leukemia types. These included sphingolipid classes previously reported to play key roles in CAD-induced cell death, but also lipids of other categories. We demonstrated that the treatment with siramesine additionally elevated the levels of numerous cytolytic lysoglycerophospholipids in positive correlation with the sensitivity of individual leukemia cell lines to siramesine. Our study shows that CAD treatment alters balance in the metabolism of glycerophospholipids, and proposes elevation in the levels of lysoglycerophospholipids as part of themechanism leading to CAD-induced cell death of leukemia cells.

Plasma sphingolipids and risk of cardiovascular diseases: a large-scale lipidomic analysis.

Sphingolipids are a diverse class of lipids with various roles in cell functions and subclasses such as ceramides have been associated with cardiovascular diseases (CVD) in previous studies. We aimed to measure molecularly-distinct sphingolipids via a large-scale lipidomic analysis and expand the literature to an Asian population. We performed a lipidomics evaluation of 79 molecularly distinct sphingolipids in the plasma of 2627 ethnically-Chinese Singaporeans. During a mean follow-up of 12.9years, we documented 152 cases of major CVD (non-fatal myocardial infarction, stroke and cardiovascular death). Total ceramide concentrations were not associated with CVD risk [hazard ratio (HR), 0.99; 95% CI 0.81-1.21], but higher circulating total monohexosylceramides (HR, 1.22; 95% CI 1.03, 1.45), total long-chain sphingolipids (C16-C18) (HR, 1.22; 95% CI 1.02, 1.45) and total 18:1 sphingolipids (HR, 1.21; 95% CI 1.01, 1.46) were associated with higher CVD risk after adjusting for conventional CVD risk factors. Our results do not support the hypothesis that higher ceramide concentrations are linked to higher CVD risk, but suggest that other classes of sphingolipids may affect CVD risk.

Differential Expression of Ormdl Genes in the Islets of Mice and Humans with Obesity

SummaryThe orosomucoid-like (Ormdl) proteins play a critical role in sphingolipid homeostasis, inflammation, and ER stress, all of which are associated with obesity and βcell dysfunction. However, their roles in β cells and obesity remain unknown. Here, we show that islets from overweight/obese human donors displayed marginally reduced ORMDL1-2 expression, whereas ORMDL3 expression was significantly downregulated compared with islets from lean donors. In contrast, Ormdl3 was substantially upregulated in the islets of leptin-deficient obese (ob/ob) mice compared with lean mice. Treatment of ob/ob mice and their islets with leptin markedly reduced islet Ormld3 expression. Ormdl3 knockdown in a β cell line induced expression of pro-apoptotic markers, which was rescued by ceramide synthase inhibitor fumonisin B1. Our results reveal differential expression of Ormdl3 in the islets of a mouse model and humans with obesity, highlight the potential effect of leptin in this differential regulation, and suggest a role for Ormdl3 in β cell apoptosis.

Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterol-enriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

Resolving Sphingolipid Isomers Using Cryogenic Infrared Spectroscopy.

1‐Deoxysphingolipids are a recently described class of sphingolipids that have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1‐deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon–carbon double‐bond (C=C) isomers and hydroxylation regioisomers. Here we demonstrate that cryogenic gas‐phase infrared (IR) spectroscopy of ionized 1‐deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas‐phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome.

Study of enhanced nitrogen removal efficiency and microbial characteristics of an improved two-stage A/O process

ABSTRACT During the cold winter in northern China, the temperature is generally below 8°C, and low water temperature significantly inhibits biological treatment processes, especially the biological denitrification process. To solve this problem, this study proposed an improved two-stage A/O process with built-in submerged biofilm modules. Experimental water was acquired from the Sanbaotun Wastewater Treatment Plant, which is situated in the city of Fushun, Liaoning Province. After one year of experimental research, the improved two-stage A/O process proved to be significantly better than the traditional two-stage A/O process, especially in winter. In the one-year experiment, the average removal rates of COD, TN, and NH4 +-N in the improved two-stage A/O process were 85.2%, 77.6%, and 96.9%, respectively. Microbial properties of the process were studied by means of high-throughput sequencing. High-throughput sequencing was conducted on the biofilm of the improved two-stage A/O terminal aerobic tank and the activated sludge of the conventional two-stage A/O aerobic tank. The result showed that the microbial diversity and abundance of the biofilms were considerably higher than those of the activated sludge during stable operation in winter. Under low-temperature conditions, the main denitrifying bacteria of the improved two-stage A/O process was Terrimonas, belonging to the sphingolipid class of Bacteroides, and the main genus of nitrifying bacteria was Nitrospira, belonging to the nitrite oxidizing bacteria.

Lysosomal Ceramide Metabolism Disorders: Implications in Parkinson's Disease.

Ceramides are a family of bioactive lipids belonging to the class of sphingolipids. Sphingolipidoses are a group of inherited genetic diseases characterized by the unmetabolized sphingolipids and the consequent reduction of ceramide pool in lysosomes. Sphingolipidoses include several disorders as Sandhoff disease, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann Pick disease, Farber disease, and GM2 gangliosidosis. In sphingolipidosis, lysosomal lipid storage occurs in both the central nervous system and visceral tissues, and central nervous system pathology is a common hallmark for all of them. Parkinson’s disease, the most common neurodegenerative movement disorder, is characterized by the accumulation and aggregation of misfolded α-synuclein that seem associated to some lysosomal disorders, in particular Gaucher disease. This review provides evidence into the role of ceramide metabolism in the pathophysiology of lysosomes, highlighting the more recent findings on its involvement in Parkinson’s disease.

The role of ceramides in metabolic disorders: when size and localization matters.

Ceramide accumulation is a hallmark in the manifestation of numerous obesity-related diseases, such as type 2 diabetes mellitus and atherosclerosis. Until the early 2000s, ceramides were viewed as a homogenous class of sphingolipids. However, it has now become clear that ceramides exert fundamentally different effects depending on the specific fatty acyl chain lengths, which are integrated into ceramides by a group of enzymes known as dihydroceramide synthases. In addition, alterations in ceramide synthesis, trafficking and metabolism in specific cellular compartments exert distinct consequences on metabolic homeostasis. Here, we examine the emerging concept of how the intracellular localization of ceramides with distinct acyl chain lengths can regulate glucose metabolism, thus emphasizing their potential as targets in the development of novel and specific therapies for obesity and obesity-associated diseases.

Lipidomics perturbations in the brain of adult zebrafish (Danio rerio) after exposure to chiral ibuprofen

The stereoselective effects of chiral ibuprofen (IBU) were studied using lipidomics by exposing adult zebrafish (Danio rerio) to an environmental concentration of 5 μg/L for 28 days. After treatment with rac-/R-(−)-/S-(+)-IBU, the brain tissue of the zebrafish was harvested to analyze for lipid metabolites by using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Results showed that the six classes of lipids, namely, glycerophospholipids, sterol lipids, prenol lipids, fatty acyls, glycerolipids, and sphingolipids, including 46 biomarkers, were affected after exposure. The different influences on metabolites were observed in the rac-/R-(−)-/S-(+)-IBU-treated samples. The rac-IBU treatment remarkably affected nine lipids. The R-(−)-IBU and S-(+)-IBU treatments had remarkably effects on six and four lipids, respectively. According to the HMDB database and KEGG pathways, nine important lipids were successfully matched to the involved biochemical pathways, such as glycerophospholipid metabolism, arachidonic acid metabolism, and linoleic acid metabolism. Therefore, IBU can cause disorders in the metabolism of the brain lipids of adult zebrafish and affect the composition of biological membranes, inflammatory responses, and cardiovascular and cerebrovascular diseases. The significant difference in the effects of R-(−)-IBU and S-(+)-IBU on lipidomics indicated that chiral IBU has stereoselective toxicity to aquatic organisms. Our study provided new insights into the environmental toxicology and highlighted the hazard of pharmaceutical and personal care product pollution in aquatic environments.

Lipidomic architecture shared by subclinical markers of osteoporosis and atherosclerosis: The Cardiovascular Risk in Young Finns Study.

Studies have shown that osteoporosis and atherosclerosis are comorbid conditions sharing common risk factors and pathophysiological mechanisms. Understanding these is crucial in order to develop shared methods for risk stratification, prevention, diagnosis and treatment. The aim of this study was to apply a system-level bioinformatics approach to lipidome-wide data in order to pinpoint the lipidomic architecture jointly associated with surrogate markers of these complex comorbid diseases. The study was based on the Cardiovascular Risk in Young Finns Study cohort from the 2007 follow-up (n=1494, aged 30-45years, women: 57%). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the serum lipidome, involving 437 molecular lipid species. The subclinical osteoporotic markers included indices of bone mineral density and content, measured using peripheral quantitative computer tomography from the distal and shaft sites of both the tibia and the radius. The subclinical atherosclerotic markers included carotid and bulbus intima media thickness measured with high-resolution ultrasound. Weighted co-expression network analysis was performed to identify networks of densely interconnected lipid species (i.e. lipid modules) associated with subclinical markers of both osteoporosis and atherosclerosis. The levels of lipid species (lipid profiles) of each of the lipid modules were summarized by the first principal component termed as module eigenlipid. Then, Pearson's correlation (r) was calculated between the module eigenlipids and the markers. Lipid modules that were significantly and jointly correlated with subclinical markers of both osteoporosis and atherosclerosis were considered to be related to the comorbidities. The hypothesis that the eigenlipids and profiles of the constituent lipid species in the modules have joint effects on the markers was tested with multivariate analysis of variance (MANOVA). Among twelve studied molecular lipid modules, we identified one module with 105 lipid species significantly and jointly associated with both subclinical markers of both osteoporosis (r=0.24, p-value=2×10-20) and atherosclerosis (r=0.16, p-value=2×10-10). The majority of the lipid species in this module belonged to the glycerolipid (n=60), glycerophospholipid (n=13) and sphingolipid (n=29) classes. The module was also enriched with ceramides (n=20), confirming their significance in cardiovascular outcomes and suggesting their joint role in the comorbidities. The top three of the 37 statistically significant (adjusted p-value<0.05) lipid species jointly associated with subclinical markers of both osteoporosis and atherosclerosis within the module were all triacylglycerols (TAGs) - TAG(18:0/18:0/18:1) with an adjusted p-value of 8.6×10-8, TAG(18:0/18:1/18:1) with an adjusted p-value of 3.7×10-6, and TAG(16:0/18:0/18:1) with an adjusted p-value of 8.5×10-6. This study identified a novel lipid module associated with both surrogate markers of both subclinical osteoporosis and subclinical atherosclerosis. Alterations in the metabolism of the identified lipid module and, more specifically, the TAG related molecular lipids within the module may provide potential new biomarkers for testing the comorbidities, opening avenues for the emergence of dual-purpose prevention measures.

Lipidomics by HILIC-Ion Mobility-Mass Spectrometry.

Lipidomics is a rapidly growing field that enables the characterization of the entire lipidome in cells, tissues, or an organism. Changes in lipid metabolism and homeostasis caused by different disease states or drug treatments can be probed by lipidomics experiments, which can aid our understanding of normal physiology and disease pathology at the molecular level. While current technologies using liquid chromatography coupled with high-resolution mass spectrometry have greatly increased coverage of thelipidome, there are still limitations in resolving the large number of lipid species with similar masses in a narrow mass window. We recently reported that two orthogonal separation techniques, hydrophilic interaction liquid chromatography (HILIC) and ion mobility (IM), enhance the resolution of lipid species based on headgroup polarity and gas-phase size and shape, respectively, of various classes of glycerolipids, glycolipids, phospholipids, and sphingolipids. Here we describe the application of our HILIC-IM-MS lipidomics protocol to the analysis of lipid extracts derived from either tissues or cells, to identify significant changes in the lipidome in response to an internal or external stimulus, such as exposure to environmental chemicals.

P2584Extracellular vesicles are relevant transporters of C16 ceramide and induce apoptosis in endothelial vesicle recipient cells

Abstract Introduction Extracellular vesicles (EVs) have recently been identified as important intercellular transporters of biologically active molecules. Besides microRNAs, which have been shown to mediate an important part of the EV function, various sphingolipids have been shown to be exported into EVs. Among these sphingolipids, ceramides have been gained interest in the cardiovascular context, because elevated plasma levels of d18:1–16:0 Ceramide (C16) have been shown to correlate with an increased risk for cardiovascular events. How ceramides are enriched in endothelial cell derived-EVs and if they are transferred to EV recipient cells to exert specific biological functions is currently unknown. Methods and results Endothelial cell derived large EVs were isolated from human coronary artery endothelial cells (HCAECs) via differential centrifugation of the culture supernatant (1 x 1500 g / 15 min + 2 x 20,000 g / 40 min). EVs were characterized by immunoblotting, electron microscopy and nanoparticle tracking analysis (Size: 252±24 nm). Sphingolipids were extracted from HCAECs and EVs (with and without glycemic injury by 30mmol/L glucose for 72 h) by solid phase extraction and analysed via Q-TOF MS/MS mass spectrometry. Lipidomic analysis revealed an enrichment of all sphingolipid classes in EVs, including C16, which was most abundantly present in EVs. As a next step, we tested if ceramides can be transferred between HCAECs by EVs. Therefore, HCAECs were incubated with NBD-labeled ceramide. NBD-Ceramide uptake into HCAECs and vesicular transfer to native HCAECs were confirmed by fluorescence microscopic imaging. Importantly, the transfer of NBD-Ceramide was abrogated if the vesicles were degraded in Triton-X 1% before incubation with EV recipient cells, which confirms that the transfer is vesicle dependent. Additionally, uptake of C16 into HCAECs and release into EVs was confirmed via mass spectrometry. Viability of C16 treated HCAECs was significantly reduced at concentrations above 5 μM of C16 in an MTT-based assay. In order to assess if the increased vesicular packing and transfer of Ceramides also reduces viability of EV recipient cells, native HCAECs were incubated with EVs from HCAECs which were treated with 2 μM and 20 μM C16. Treatment with C16 enriched vesicles lead to a significant reduction of EV recipient cell viability in a dose dependant manner. Conclusion In the present study, we found that C16 is enriched in EVs and that C16 is transferred through EVs among HCAECs. Depending on the amount of transferred C16, this process reduces the viability of native EV recipient HCAECs. Furthermore, vesicular C16 export is increased after glycemic injury. In comparison to the amounts of free C16, which are necessary to induce a similar reduction in HCAEC viability, the amount of EV transferred C16 is negliable. Hence, vesicular packaging results in a high bioavailability of ceramides leading to a relevant regulation of endothelial cell death. Acknowledgement/Funding BONFOR Progamm University Bonn

Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence.

Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. In this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of thede novoceramide biosynthesis, on the aging phenotype of cortical neurons cultured for 22 days, a setting employed as anin vitromodel of senescence. Our findings indicate that, compared to control cultures, ‘aged’ neurons display dysregulation of [Ca2+]ilevels, mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS positively affected the senescent phenotype, a result associated with recovery of neuronal [Ca2+]isignaling and reduction of mitochondrial dysfunction and ROS generation. The results suggest that thede novoceramide biosynthesis represents a critical intermediate in the molecular and functional cascade leading to neuronal senescence and identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions.