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Background: Endogenous and synthetic cannabinoids have been shown to induce cancer cell death through the accumulation of the sphingolipid, ceramide (Cer). Recently, we have demonstrated that Cer accumulation enhances the induction of immunogenic cell death (ICD). Objectives: The primary objective of this study was to demonstrate that (±) 5-epi CP 55,940 (5-epi), a by-product of the chemical synthesis of the synthetic cannabinoid CP 55,940, induces ICD in colorectal cancer (CRC) cells, and that modulation of the sphingolipid metabolic pathway through inhibition of the sphingosine kinases (SphKs) enhances these effects. Methods: A cell culture model system of human CRC cell lines was employed to measure the cell surface and intracellular production of markers of ICD. The effects of 5-epi, alone and in combination with SphK inhibitors, on production of Cer through the de novo sphingolipid synthesis pathway were measured by Liquid Chromatography - Tandem Mass Spectrometry (LC/MS/MS)-based sphingolipidomic analysis. Cell surface exposure of calreticulin (ectoCRT), a hallmark of ICD, was measured by flow cytometry. Examination of the effects of 5-epi, alone and in combination with SphK inhibitors, on the intracellular signaling pathway associated with ICD was conducted by immunoblot analysis of human CRC cell lines. Results: Sphingolipidomic analysis indicated that 5-epi induces the de novo sphingolipid synthetic pathway. 5-epi dose dependently induces cell surface exposure of ectoCRT, and inhibition of Cer metabolism through inhibition of the SphKs significantly enhances 5-epi-induced ectoCRT exposure in multiple CRC cell lines. 5-epi induces and SphK inhibition enhances activation of the cell death signaling pathway associated with ICD. Conclusions: This study is the first demonstration that cannabinoids can induce the cell surface expression of ectoCRT, and potentially induce ICD. Moreover, this study reinforces our previous observation of a role for Cer accumulation in the induction of ICD and extends this observation to the cannabinoids, agents not typically associated with ICD. Inhibition of SphKs enhanced the 5-epi-induced signaling pathways leading to ICD and production of ectoCRT. Overexpression of SphK1 has previously been associated with chemotherapy resistance. Thus, targeting the SphKs has the potential to reverse chemotherapy resistance and simultaneously enhance the antitumor immune response through enhancement of ICD induction.

Sphingolipids are ubiquitous components in eukaryotic organisms and have attracted attention as physiologically functional lipids. Sphingolipids with diverse structures are present in foodstuffs as these structures depend on the biological species they are derived from, such as mammals, plants, and fungi. The physiological functions of dietary sphingolipids, especially those that improve skin barrier function, have recently been noted. In addition, the roles of dietary sphingolipids in the prevention of diseases, including cancer and metabolic syndrome, have been studied. However, the mechanisms underlying the health-improving effects of dietary sphingolipids, especially their metabolic fates, have not been elucidated. Here, we review dietary sphingolipids, including their chemical structures and contents in foodstuff; digestion, intestinal absorption, and metabolism; and nutraceutical functions, based on the available evidence and hypotheses. Further research is warranted to clearly define how dietary sphingolipids can influence human health.

Background and Objective: Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. Methods: We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. Results: RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. Conclusion: This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

Background: Cancer is a complex disease that derives from the uncontrolled proliferation of cells. Bone cancer is a type of prevalent cancer that occurs both in young and adults. Bone cancer is most common in the long bones of the pelvis, arms and legs. Statistically, more than 200 cases of osteosarcoma have been reported annually in our country. Classical treatment with chemotherapeutics remains ineffective in the cure of this cancer type. Recent studies have shown that ceramide induces apoptosis at its increased levels in the cells. Thus, many studies have been conducted to cause the accumulation of ceramide molecules in the cell by different ways to induce apoptosis. NOE (Noleoylethanolamine) is a specific inhibitor of ceramidase enzymes that hydrolyze intracellular ceramides and prevent apoptosis. Objective: This study investigates the cytotoxic and apoptosis-inducing activities of NOE on human osteosarcoma Saos-2 cells. Methods: Cytotoxic effects were investigated by MTT colorimetric assay. For the detection of morphological and ultrastructural indicators of apoptosis, confocal and TEM techniques were used. Results: Our finding indicated that NOE is effective in the inhibition of the growth of Saos-2 cells. Confocal and TEM findings showed morphological and ultrastructural changes as chromatin condensation, fragmentation of nuclei and mitochondria as well as damaged cytoskeleton and cell shrinkage. Conclusion: The results revealed that NOE exerts its cytotoxicity on Saos-2 cells through changing the ultrastructure and morphology of cells with clear apoptotic sparks.

Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders. ispartof: MOLECULAR THERAPY vol:30 issue:4 pages:1661-1674 ispartof: location:United States status: published

Although lipids are crucial molecules for cell structure, metabolism, and signaling in most organs, they have additional specific functions in the skin. Lipids are required for the maintenance and regulation of the epidermal barrier, physical properties of the skin, and defense against microbes. Analysis of the lipidomeethe totality of lipidseis of similar complexity to those of proteomics or other omics, with lipid structures ranging from simple, linear, to highly complex structures. In addition, the ordering and chemical modifications of lipids have consequences on their biological function, especially in the skin. Recent advances in analytic capability (usually with mass spectrometry), bioinformatic processing, and integration with other dermatological big data have allowed researchers to increasingly understand the roles of specific lipid species in skin biology. In this paper, we review the techniques used to analyze skin lipidomics and epilipidomics.

Dexamethasone (DEX) is a synthetic glucocorticoid with anti-inflammatory properties. We evaluated a potentially protective dexamethasone influence on hepatocellular lipid metabolism and fatty acid (FA) transporters expression. The HepG2 cells were incubated with palmitic acid (PA) and/or dexamethasone in two different time expositions (16 h and 40 h). Intracellular and extracellular lipid and sphingolipid concentrations were estimated by the gas-liquid chromatography and high-performance liquid chromatography, respectively. The protein expression involved in FA uptake and lipid metabolism was determined by immunoblotting. The treatment of HepG2 with dexamethasone and palmitate enhanced lipid transport to the cell via increased especially FABPpm expression and resulted in the increased triacylglycerol (TAG), diacylglycerol (DAG) and ceramide deposition. Dexamethasone with palmitate treatment altered FA composition resulting in the elevated n-3 polyunsaturated fatty acid (PUFA) activity in DAG and TAG and the diminished n-6 PUFA activity in DAG after prolonged exposure. We may speculate that although protective lipid secretion into media and decrease in inflammatory FA precursors dexamethasone treatment exacerbated lipotoxicity in HepG2 cells.

Cancer development is a multistep process in which cells must overcome a series of obstacles before they can become fully developed tumors. First, cells must develop the ability to proliferate unchecked. Once this is accomplished, they must be able to invade the neighboring tissue, as well as provide themselves with oxygen and nutrients. Finally, they must acquire the ability to detach from the newly formed mass in order to spread to other tissues, all the while evading an immune system that is primed for their destruction. Furthermore, increased levels of inflammation have been shown to be linked to the development of cancer, with sites of chronic inflammation being a common component of tumorigenic microenvironments. In this Review, we give an overview of the impact of sphingolipid metabolism in cancers, from initiation to metastatic dissemination, as well as discussing immune responses and resistance to treatments. We explore how sphingolipids can either help or hinder the progression of cells from a healthy phenotype to a cancerous one., This review gives an overview on the impact of sphingolipid metabolism in various cancer types, from initiation to metastatic dissemination, as well as on immune response and resistance to cancer treatments. It explores how sphingolipids can either help or hinder the progression of cells from a healthy to a cancerous phenotype, and discusses the therapeutic potential of targeting sphingolipid metabolism for the treatment of patients with cancer.

Ectopic lipids play a key role in numerous pathologies, including heart disease, stroke, and diabetes. Of all the lipids studied, perhaps the most well understood is cholesterol, a widely used clinical biomarker of cardiovascular disease and a target of pharmacological interventions (e.g., statins). Thousands of studies have interrogated the regulation and action of this disease-causing sterol. As a growing body of literature indicates, a new class of lipid-based therapies may be on the horizon. Ceramides are cholesterol-independent biomarkers of heart disease and diabetes in humans. Studies in rodents suggest that they are causative agents of disease, as lowering ceramides through genetic or pharmacological interventions prevents cardiovascular disease and diabetes. Herein, we discuss the evidence supporting the potential of therapeutics targeting ceramides to treat cardiometabolic disease, contrasting it with the robust datasets that drove the creation of cholesterol-lowering pharmaceuticals.

Ceramide synthases (CerSs) catalyze the formation of ceramides from sphingoid bases and acyl-CoA substrates. Increasing evidence suggests that cancer cells generally exhibit altered sphingolipid metabolism in the tumorigenesis of multiple cancers. However, there is no evidence that CerSs are associated with pancreatic ductal carcinoma (PDAC). In the present study, we examined CerS expression in clinical tissue and conducted data mining to investigate the clinical significance of CerSs in the TCGA-PAAD database. We found that high CerS6 expression positively correlated with progression and predicted worse prognosis in PDAC patients, establishing CerS6 as a potential biomarker for PDAC. Furthermore, CerS6 promoted cell proliferation, colony formation and invasion by producing C16-ceramide and was required for tumor formation. Mechanistically, AKT1 interacted with and phosphorylated FOXP3 at S418, which decreased the binding of FOXP3 to the CERS6 promoter and in turn induced CerS6 expression by reconstituting an activated state on the CERS6 promoter. The AKT1/FOXP3 axis mediated the CerS6 expression and promoted p53 mutant pancreatic tumorigenesis by producing excessive C16-ceramide, which induced the accumulation of mutant p53. Thus, our study explores the relationship between PI3K/AKT signaling and sphingolipid metabolism, revealing an oncogenic role for CerS6, which may represent a potential target for PDAC treatment.

Age-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD) are an unmet health need, with significant economic and societal implications, and an ever-increasing prevalence. Membrane lipid rafts (MLRs) are specialised plasma membrane microdomains that provide a platform for intracellular trafficking and signal transduction, particularly within neurons. Dysregulation of MLRs leads to disruption of neurotrophic signalling and excessive apoptosis which mirrors the final common pathway for neuronal death in ALS, PD and AD. Sphingomyelinase (SMase) and phospholipase (PL) enzymes process components of MLRs and therefore play central roles in MLR homeostasis and in neurotrophic signalling. We review the literature linking SMase and PL enzymes to ALS, AD and PD with particular attention to attractive therapeutic targets, where functional manipulation has been successful in preclinical studies. We propose that dysfunction of these enzymes is upstream in the pathogenesis of neurodegenerative diseases and to support this we provide new evidence that ALS risk genes are enriched with genes involved in ceramide metabolism (P=0.019, OR = 2.54, Fisher exact test). Ceramide is a product of SMase action upon sphingomyelin within MLRs, and it also has a role as a second messenger in intracellular signalling pathways important for neuronal survival. Genetic risk is necessarily upstream in a late age of onset disease such as ALS. We propose that manipulation of MLR structure and function should be a focus of future translational research seeking to ameliorate neurodegenerative disorders.

Recent studies have revealed the importance of cell membrane stability in normal cell function. Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), a lipid modifying enzyme that converts sphingomyelin to ceramide in the cell membrane, is expressed in macrophages and regulates Toll-like receptor (TLR) 4 signaling by altering cell membrane fluidity. SMPDL3b is also expressed in human podocytes, which are involved in the pathogenesis of several glomerular diseases such as diabetic kidney disease, focal segmental glomerulosclerosis, and idiopathic nephrotic syndrome in children; however, the role of SMPDL3b in podocyte innate immunity is unclear. As podocytes are equipped with innate immune systems including TLR3, and viral infections often exacerbate proteinuria in children with idiopathic nephrotic syndrome, we hypothesized that changes in SMPDL3b expression levels could affect anti-viral responses via TLR3 signaling in podocytes, consequently impairing normal podocyte function. To examine the role of SMPDL3b in TLR3 signaling in podocytes, we treated conditionally immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), to activate TLR3 signaling. The cells were then transfected with small interfering RNA against SMPDL3b. Poly IC activated the TLR3 pathway, whereas knockdown of SMPDL3b attenuated poly IC-induced interferon-β/chemokine C-X-C ligand 10 expression in podocytes. To our knowledge, this is the first report demonstrating SMPDL3b involvement in podocyte innate immunity; these results suggest that SMPDL3b is essential for adequate anti-viral responses in podocytes, possibly by modulating lipid metabolism in the cell membrane.

Functional skin barrier requires sphingolipid homeostasis; 3-ketodihydrosphingosine reductase or KDSR is a key enzyme of sphingolipid anabolism catalyzing the reduction of 3-ketodihydrosphingosine to sphinganine. Biallelic mutations in the KDSR gene may cause erythrokeratoderma variabilis et progressive-4, later specified as PERIOPTER syndrome, emphasizing a characteristic periorifical and ptychotropic erythrokeratoderma. We report another patient with compound heterozygous mutations in KDSR, born with generalized harlequin ichthyosis, which progressed into palmoplantar keratoderma. To determine whether patient-associated KDSR mutations lead to KDSR substrate accumulation and/or unrecognized sphingolipid downstream products in stratum corneum (SC), we analyzed lipids of this and previously published patients with non-identical biallelic mutations in KDSR. In SC of both patients, we identified ‘hitherto’ unobserved skin ceramides with an unusual keto-type sphingoid base in lesional and non-lesional areas, which accounted for up to 10% of the measured ceramide species. Furthermore, an overall shorter mean chain length of free and bound sphingoid bases was observed—shorter mean chain length of free sphingoid bases was also observed in lesional psoriasis vulgaris SC, but not generally in lesional atopic dermatitis SC. Formation of keto-type ceramides is probably due to a bottle neck in metabolic flux through KDSR and a bypass by ceramide synthases, which highlights the importance of tight intermediate regulation during sphingolipid anabolism and reveals substrate deprivation as potential therapy.

Sphingolipid-mediated regulation in cancer development and treatment is largely ceramide-centered with the complex sphingolipid metabolic pathways unfolding as attractive targets for anticancer drug discovery. The dynamic interconversion of sphingolipids is tightly controlled at the level of enzymes and cellular compartments in response to endogenous or exogenous stimuli, such as anticancer drugs, including retinoids. Over the past two decades, evidence emerged that retinoids owe part of their potency in cancer therapy to modulation of sphingolipid metabolism and ceramide generation. Ceramide has been proposed as a ‘tumor-suppressor lipid' that orchestrates cell growth, cell cycle arrest, cell death, senescence, autophagy, and metastasis. There is accumulating evidence that cancer development is promoted by the dysregulation of tumor-promoting sphingolipids whereas cancer treatments can kill tumor cells by inducing the accumulation of endogenous ceramide levels. Resistance to cancer therapy may develop due to a disrupted equilibrium between the opposing roles of tumor-suppressor and tumor-promoter sphingolipids. Despite the undulating effect and complexity of sphingolipid pathways, there are emerging opportunities for a plethora of enzyme-targeted therapeutic interventions that overcome resistance resulting from perturbed sphingolipid pathways. Here, we have revisited the interconnectivity of sphingolipid metabolism and the instrumental role of ceramide-biosynthetic and degradative enzymes, including bioactive sphingolipid products, how they closely relate to cancer treatment and pathogenesis, and the interplay with retinoid signaling in cancer. We focused on retinoid targeting, alone or in combination, of sphingolipid metabolism nodes in cancer to enhance ceramide-based therapeutics. Retinoid and ceramide-based cancer therapy using novel strategies such as combination treatments, synthetic retinoids, ceramide modulators, and delivery formulations hold promise in the battle against cancer

Cell surface biochemical changes, notably excessive increase in outer leaflet sphingomyelin (SM) content, are important in cancer initiation, growth, and immune evasion. Innumerable reports describe methods to initiate, promote, or enhance immunotherapy of clinically detected cancer, notwithstanding the challenges, if not impossibility, of identification of tumor-specific, or associated antigens, the lack of tumor cell surface membrane expression of major histocompatibility complex (MHC) class I alpha and β2 microglobulin chains, and lack of expression or accessibility of Fas and other natural killer cell immune checkpoint molecules. Conversely, SM synthesis and hydrolysis are increasingly implicated in initiation of carcinogenesis and promotion of metastasis. Surface membrane SM readily forms inter- and intra- molecular hydrogen bond network, which excessive tightness would impair cell-cell contact inhibition, inter- and intra-cellular signals, metabolic pathways, and susceptibility to host immune cells and mediators. The present review aims at clarifying the tumor immune escape mechanisms, which face common immunotherapeutic approaches, and attracting attention to an entirely different, neglected, key aspect of tumorigenesis associated with biochemical changes in the cell surface that lead to failure of contact inhibition, an instrumental tumorigenesis mechanism. Additionally, the review aims to provide evidence for surface membrane SM levels and roles in cells resistance to death, failure to respond to growth suppressor signals, and immune escape, and to suggest possible novel approaches to cancer control and cure.

A causal relationship between plasma ceramide concentration and respiratory distress symptoms in COVID-19 patients is inferred. In this study, plasma samples of 52 individuals infected with COVID-19 were utilized in a lipidomic analysis. Lipids belonging to the ceramide class exhibited a 400-fold increase in total plasma concentration in infected patients. Further analysis led to the demonstration of concentration dependency for severe COVID-19 respiratory symptoms in a subclass of ceramides. The subclasses Cer(d18:0/24:1), Cer(d18:1/24:1), and Cer(d18:1/22:0) were shown to be increased by 48-, 40-, and 33-fold, respectively, in infected plasma samples and to 116-, 91- and 50-fold, respectively, in plasma samples with respiratory distress. Hence, monitoring plasma ceramide concentration, can be a valuable tool for measuring effects of therapies on COVID-19 respiratory distress patients.

Eryptosis is a physiological mechanism for the clearance of senescent or damaged erythrocytes by phagocytes. Excessive eryptosis is stimulated under several pathologies and associated with endothelial injury and thrombosis. Cigarette smoke (CS) is an established risk factor for vascular diseases and cigarette smokers have high-levels of eryptotic erythrocytes. This study, for the first time, investigates the mechanism by which CS damages red blood cells (RBCs). CS extract (CSE) from commercial cigarettes was prepared and standardized for nicotine content. Cytofluorimetric analysis demonstrated that treatment of human RBCs with CSE caused dose-dependent, phosphatidylserine externalization and cell shrinkage, hallmarks of apoptotic death. CSE did not affect cellular levels of Ca2+, reactive oxygen species (ROS) or glutathione (GSH). Immununoprecipitation and immunoblotting revealed the assembly of the death-inducing signaling complex (DISC) and oligomerization of Fas receptor as well as cleaved caspase-8 and caspase-3 within 6 h from the treatment. At the same time-interval, CSE elicited neutral sphyngomielinase (nSMase) activity-dependent ceramide formation and phosphorylation of p38 MAPK. Through specific inhibitors’ nSMase, caspase-8 or p38 MAPK activities, we demonstrated that p38 MAPK activation is required for caspase-8-mediated eryptosis and that ceramide generation is initiator caspase-dependent. Finally, ex vivo analysis detected phosphorylated p38 MAPK (p-p38) and Fas-associated signaling complex in erythrocytes from cigarette smokers. In conclusion, our study demonstrates that CSE exposure induces in erythrocytes an extrinsic apoptotic pathway involving p38 MAPK-initiated DISC formation followed by activation of caspase-8/caspase-3 via ceramide formation.

The retrograde flow of endometrial tissues deposited into the peritoneal cavity occurs in women during menstruation. Classically (M1) or alternatively (M2) activated macrophages partake in the removal of regurgitated menstrual tissue. The failure of macrophage egress from the peritoneal cavity through the mesothelium leads to chronic inflammation in endometriosis. To study the migration differences of macrophage phenotypes across mesothelial cells, an in vitro model of macrophage egress across a peritoneal mesothelial cell monolayer membrane was developed. M1 macrophages were more sessile, emigrating 2.9-fold less than M2 macrophages. The M1 macrophages displayed a pro-inflammatory cytokine signature, including IL-1α, IL-1β, TNF-α, TNF-β, and IL-12p70. Mass spectrometry sphingolipidomics revealed decreased levels of ceramide-1-phosphate (C1P), an inducer of migration in M1 macrophages, which correlated with its poor migration behavior. C1P is generated by ceramide kinase (CERK) from ceramide, and blocking C1P synthesis via the action of NVP231, a specific CERK chemical inhibitor, prohibited the emigration of M1 and M2 macrophages up to 6.7-fold. Incubation with exogenously added C1P rescued this effect. These results suggest that M1 macrophages are less mobile and have higher retention in the peritoneum due to lower C1P levels, which contributes to an altered peritoneal environment in endometriosis by generating a predominant pro-inflammatory cytokine environment.

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.

Aims Peripheral arterial disease (PAD) and coronary artery disease (CAD) are both caused by atherosclerosis. Serum lipids and lipoproteins are predictive of the development of atherosclerosis but it is not clear if they differ in the two manifestations PAD and CAD. We tested whether a more detailed characterization of the lipid and lipoprotein patterns of PAD and CAD allows a clear differentiation between the two atherosclerotic phenotypes. Methods A cohort of 274 statin-naive patients with either newly diagnosed imaging proven PAD (n = 89) or stable CAD (n = 185) was characterized using NMR- and LC-MS/MS-based advanced lipid and lipoprotein analysis. An independent cohort of 1239 patients with PAD and CAD was used for validation. Results We found a significant difference in markers of inflammation as well as ceramide and phosphatidylcholine levels between PAD and CAD patients. In contrast, basic lipid markers including total cholesterol, low density lipoprotein cholesterol, high density lipoprotein-cholesterol, lipoprotein (a) or detailed lipoprotein profiles did not differ significantly between PAD and CAD patients. Applying ratios and scores derived from ceramides and phosphatidylcholines further improved the discrimination between PAD and CAD. These significant differences were independent from body composition, from the status of smoking or T2DM, and also from apolipoprotein C-III and other inflammatory parameters which were different between CAD and PAD. Conclusion The present study clearly suggests that PAD and CAD differ in terms of their ceramide- and phosphatidylcholine-based lipid pattern but not in lipoprotein characteristics. This article is protected by copyright. All rights reserved.

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno‐associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy., Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. The current study elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.

AimCholesterol-based risk prediction is often insufficient in cardiovascular disease (CVD) patients. Ceramides are a new kind of biomarkers for CVD. The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses only circulating ceramide levels, determined by coupled liquid chromatography–mass spectrometry, to allocate patients into one of four risk categories. This test has recently been modified (CERT2) by additionally including phosphatidylcholine levels.Methods and resultsIn this observational cohort study, we have recruited 999 Austrian patients with CVD and followed them for up to 13 years. We found that CERT and CERT2 both predicted cardiovascular events, cardiovascular mortality, and overall mortality. CERT2 had the higher performance compared to CERT and also to the recent cardiovascular risk score of the ESC/EAS guidelines (Systematic COronary Risk Evaluation (SCORE)) for low-risk European countries. Combining CERT2 with the ESC/EAS-SCORE, predictive capacity was further increased leading to a hazard ratio of 3.58 (2.02–6.36; P ConclusionWe conclude that CERT and CERT2 are powerful predictors of cardiovascular events, cardiovascular mortality, and overall mortality in CVD patients. Including phosphatidylcholine to a ceramide-based score increases the predictive performance and is best in combination with classical risk factors as used in the ESC/EAS-SCORE.

N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.

Osteosarcoma is a bone cancer characterized by the production of osteoid tissue and immature bone from mesenchymal cells. Osteosarcoma mainly affects long bones (femur is most frequently site) and occur in children and young adults with greater incidence. Here, we investigated the role accomplished by polydatin, a natural antioxidative compound, in promoting osteogenic differentiation alone or after radiation therapy on osteosarcoma cells. In vitro, polydatin significantly induced cell cycle arrest in S-phase and enhanced bone alkaline phosphatase activity. Moreover, the differentiation process was paralleled by the activation of Wnt-β-catenin pathway. In combination with radiotherapy, the pretreatment with polydatin promoted a radiosensitizing effect on osteosarcoma cancer cells as demonstrated by the upregulation of osteogenic markers and reduced clonogenic survival of tumor cells. Additionally, we analyzed, by mass spectrometry, the secretion of sphingolipid, ceramides, and their metabolites in osteosarcoma cells treated with polydatin. Overall, our results demonstrate that polydatin, through the secretion of sphingolipids and ceramide, induced osteogenic differentiation, alone and in the presence of ionizing therapy. Future investigations are needed to validate the use of polydatin in clinical practice as a potentiating agent of radiotherapy-induced anticancer effects.

Collaborators : AP-HP / Université de Paris / INSERM COVID-19 research collaboration, AP-HP COVID CDR Initiative, “Entrepôt de Données de Santé” AP-HP Consortium: Pierre-Yves Ancel, Alain Bauchet, Nathanaël Beeker, Vincent Benoit, Mélodie Bernaux, Ali Bellamine, Romain Bey, Aurélie Bourmaud, Stéphane Breant, Anita Burgun, Fabrice Carrat, Charlotte Caucheteux, Julien Champ, Sylvie Cormont, Christel Daniel, Julien Dubiel, Catherine Ducloas, Loic Esteve, Marie Frank, Nicolas Garcelon, Alexandre Gramfort, Nicolas Griffon, Olivier Grisel, Martin Guilbaud, Claire Hassen-Khodja, François Hemery, Martin Hilka, Anne Sophie Jannot, Jerome Lambert, Richard Layese, Judith Leblanc, Léo Lebouter, Guillaume Lemaitre, Damien Leprovost, Ivan Lerner, Kankoe Levi Sallah, Aurélien Maire, Marie-France Mamzer, Patricia Martel, Arthur Mensch, Thomas Moreau, Antoine Neuraz, Nina Orlova, Nicolas Paris, Bastien Rance, Hélène Ravera, Antoine Rozes, Elisa Salamanca, Arnaud Sandrin, Patricia Serre, Xavier Tannier, Jean-Marc Treluyer, Damien Van Gysel, Gaël Varoquaux, Jill Jen Vie, Maxime Wack, Perceval Wajsburt, Demian Wassermann, Eric Zapletal; International audience; Several medications commonly used for a number of medical conditions share a property of functional inhibition of acid sphingomyelinase (ASM), or FIASMA. Preclinical and clinical evidence suggest that the (ASM)/ceramide system may be central to SARS-CoV-2 infection. We examined the potential usefulness of FIASMA use among patients hospitalized for severe COVID-19 in an observational multicenter study conducted at Greater Paris University hospitals. Of 2,846 adult patients hospitalized for severe COVID-19, 277 (9.7%) were taking a FIASMA medication at the time of their hospital admission. The primary endpoint was a composite of intubation and/or death. We compared this endpoint between patients taking vs. not taking a FIASMA medication in time-to-event analyses adjusted for sociodemographic characteristics and medical comorbidities. The primary analysis was a Cox regression model with inverse probability weighting (IPW). Over a mean follow-up of 9.2 days (SD=12.5), the primary endpoint occurred in 104 patients (37.5%) receiving a FIASMA medication, and 1,060 patients (41.4%) who did not. Despite being significantly and substantially associated with older age and greater medical severity, FIASMA medication use was significantly associated with reduced likelihood of intubation or death in both crude (HR=0.71; 95%CI=0.58-0.87; p

MYC is a transcription factor with broad biological functions, notably in the control of cell proliferation. Here, we show that intestinal MYC regulates systemic metabolism. We find that MYC expression is increased in ileum biopsies from individuals with obesity and positively correlates with body mass index. Intestine-specific reduction of MYC in mice improves high-fat-diet-induced obesity, insulin resistance, hepatic steatosis and steatohepatitis. Mechanistically, reduced expression of MYC in the intestine promotes glucagon-like peptide-1 (GLP-1) production and secretion. Moreover, we identify Cers4, encoding ceramide synthase 4, catalysing de novo ceramide synthesis, as a MYC target gene. Finally, we show that administration of the MYC inhibitor 10058-F4 has beneficial effects on high-fat-diet-induced metabolic disorders, and is accompanied by increased GLP-1 and reduced ceramide levels in serum. This study positions intestinal MYC as a putative drug target against metabolic diseases, including non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Background and aims The present study aims to correlate the severity of post-stroke depression (PSD) and serum ceramide concentration. Materials and methods In this two-center case-control study, we prospectively collected clinical and demographical information from age and gender-matched 51 PSD patients, 56 non-post-stroke depression (Non-PSD) patients, and 39 patients with major depression (MD) to perform the suitable biochemical analysis to bring a correlation in causing depression in patients soon after the stroke. The ROC curve method was used to evaluate ceramide’s diagnostic efficacy in all three groups of patients. A follow-up analysis was also conducted based on PSD severity to associate serum ceramide levels and neuropsychiatric symptoms. The severity of the patient's depressive symptoms was assessed by using the self-rating depression scale (SDS). Results In comparison between PSD and MD patients, three serum ceramide species were found to be significantly different. Compared with Non-PSD patients, PSD patients had significantly higher levels of all the four serum ceramides, and increasing levels ofC16:0, C18:0(VS MD) and C16: 0(VS Non-PSD) serve as a diagnostic tool and an independent risk factor in all three categories of patients. Moreover, the follow-up analysis results showed that, as the treatment progressed, the differences in the 3 serum ceramide species were statistically significant. Conclusion There was a stage-specific association between serum ceramides and PSD, and the potential pathophysiological mechanism has to be investigated in future research.

Obesity predisposes individuals to the development of insulin resistance, which is a risk factor for type 2 diabetes, and muscle plays a central role in this phenomenon. Insulin resistance is associated with: (i) a metabolic inflexibility characterized by a reduced impaired switching from free fatty acid (FA) to carbohydrate substrates; and (ii) an ectopic accumulation of triglyceride in skeletal muscle, generating a cellular “lipotoxicity”, but triglyceride per se, does not contribute to insulin resistance (“athlete’s paradox”). A large body of evidence supports the idea that a decreased mitochondrial capacity to oxidize FA leads to an accretion of intracellular triglyceride and an accumulation of acyl‐CoAs, which are used to synthesize diacylglycerol and ceramide. These lipid derivatives activate serine kinases, leading to increase of insulin receptor substrate 1 serine phosphorylation, which impairs insulin signaling. A second model proposes that insulin resistance arises from an excessive mitochondrial FA oxidation. Studies have shown that the type of FA, unsaturated or saturated, is critical in the development of insulin resistance. It should be also stressed that FA oversupply activates inflammatory signals, induces endoplasmic reticulum stress, increases mitochondrial oxidative stress and influences the regulation of genes that contributes to impaired glucose metabolism. These cellular insults are thought to engage stress‐sensitive serine kinases disrupting insulin signaling. In conclusion, reduced dietary lipid intake in association with physical exercise could be a therapeutic option to improve insulin sensitivity., Obesity predisposes individuals to the development of insulin resistance, which is a risk factor for type 2 diabetes, and muscle plays a central role in this phenomenon. The insulin resistance is related to impaired mitochondrial oxidation of fatty acids, resulting to an accumulation of lipid derivatives, which in turn decrease insulin sensitivity. However, excessive mitochondrial fatty acid oxidation might also lead to impaired insulin effect.

Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

The protection mediated by the bioactive sphingolipid sphingosine-1-phosphate (S1P) declines during Alzheimer's disease (AD) progression, especially in patients carrying the apolipoprotein E ε4 (APOE4) isoform. The drug FTY720 mimics S1P bioactivity, but its efficacy in treating AD is unclear. Two doses of FTY720 (0.1 mg / kg and 0.5 mg / kg daily) were given by oral gavage for 15 weeks to transgenic mouse models of familial AD carrying human apolipoprotein E (APOE) APOE3 (E3FAD) or APOE4 (E4FAD). After 12 weeks of treatment, animals were subjected to behavioral tests for memory, locomotion, and anxiety. Blood was withdrawn at different time points and brains were collected for sphingolipids analysis by mass spectrometry, gene expression by RT-PCR and Aβ quantification by ELISA. We discovered that low levels of S1P in the plasma is associated with a higher probability of failing the memory test and that FTY720 prevents memory impairments in E4FAD. The beneficial effect of FTY720 was induced by a shift of the sphingolipid metabolism in the brain towards a lower production of toxic metabolites, like ceramide d18:1/16:0 and d18:1/22:0, and reduction of amyloid-β burden and inflammation. In conclusion, we provide further evidence of the druggability of the sphingolipid system in AD. ispartof: BIOMEDICINE & PHARMACOTHERAPY vol:152 ispartof: location:France status: published

Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products—ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.

Objectives To perform an exploratory study to identify a JDM serum metabolic profile that differs from healthy controls (HCs) and responds to immunosuppressive treatment. Methods Blood was collected from 9 HCs and 10 patients diagnosed with probable (n = 4) or definite (n = 6) JDM based on the criteria of Bohan and Peter for myositis, with 7 of the 10 providing longitudinal samples following initiation of treatment; these patients comprised the treatment-naïve cohort. Sera underwent mass spectroscopy–based measurements of targeted metabolic intermediates, including 15 amino acids, 45 acylcarnitines (ACs), 15 ceramides and 29 sphingomyelins. Principal components analysis reduced metabolites into smaller sets of factors each comprised of correlated metabolic intermediates. Factor scores and metabolite concentrations were compared with HCs using two-sample t-tests while treatment effects were evaluated using paired t-tests. Results Of eight principal components analysis–derived metabolite factors (one AC, two amino acids, three sphingosine and two ceramide), two were significantly associated with JDM: one AC factor containing mostly long-chain ACs (P = 0.049) and one ceramide factor (P Conclusion While additional validation is needed, these lipids have potential as JDM serum diagnostic and/or treatment biomarkers. Additionally, the significant association of long-chain ACs and ceramides with JDM offers insights regarding pathogenesis, implicating dysregulation of mitochondrial fatty acid β-oxidation.

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

Emerging research has linked psychological well-being with many physiological markers as well as morbidity and mortality. In this analysis, the relationship between components of eudaimonic well-being and serum sphingolipids levels was investigated using data from a large national survey of middle-aged American adults (Midlife in the United States). Health behaviors (i.e., diet, exercise, and sleep) were also examined as potential mediators of these relationships. Serum levels of total ceramides - the main molecular class of sphingolipids previously associated with several disease conditions - were inversely linked with environmental mastery. In addition, significant correlations were found between specific ceramide, dihydroceramide, and hexosylceramides species with environmental mastery, purpose in life, and self-acceptance. Using hierarchical regression and mediation analyses, health behaviors appeared to mediate these associations. However, the link between ceramides and environmental mastery was partially independent of health behaviors, suggesting the role of additional mediating factors. These findings point to sphingolipid metabolism as a novel pathway of health benefits associated with psychological well-being. In particular, having a sense of environmental mastery may promote restorative behaviors and benefit health via improved blood sphingolipid profiles.

The skin barrier of melasma is involved in the pathogenesis of melasma. Previous studies have shown that there are differences in the expression of epidermal lipid genes in melasma, but little is known about the epidermis lipid composition of melasma. Compared with the non-lesional skin, the content of total lipids, phosphatidic acid, phosphatidylserine, and ceramide (Cer) increased significantly in the lesional skin. Multivariate data analysis indicated that 40 individual Cer lipid species were responsible for the discrimination. In terms of acyl chain length in Cer, the expressions of very long chain (VLC) (C20-C26) and ultra-long chain (ULC) (>C26) increased significantly in the lesional skin. However, Cer[AH] had negative correlations with the activation of melanocytes in the lesional skin. Some lipid species had lower expression in lesional skin with high activation of melanocytes, as well as the high darkness. The epidermal thickness of lesional skin was higher compared with the non-lesional skin. These results suggest that Cer increased significantly in the lesional skin of melasma, possibly as a compensatory mechanism to maintain skin barrier function. Between different groups of darkness and activation of melanocytes, the change of ceramides might have correlation with the pigmentation progress of melasma.

Background: Understanding how dysregulation in lipid metabolism relates to the severity of Alzheimer‘s disease (AD) pathology might be critical in developing effective treatments. Objective: To identify lipid species in cerebrospinal fluid (CSF) associated with signature AD pathology and to explore their relationships with measures reflecting AD-related processes (neurodegeneration, inflammation, deficits in verbal episodic memory) among subjects at the pre- and early symptomatic stages of dementia. Methods: A total of 60 subjects that had been referred to an Icelandic memory clinic cohort were classified as having CSF AD (n = 34) or non-AD (n = 26) pathology profiles. Untargeted CSF lipidomic analysis was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) for the detection of mass-to-charge ratio (m/z) features. CSF proteins reflecting neurodegeneration (neurofilament light [NFL]) and inflammation (chitinase-3-like protein 1 [YKL-40], S100 calcium-binding protein B [S100B], glial fibrillary acidic protein [GFAP]) were also measured. Rey Auditory Verbal Learning (RAVLT) and Story tests were used for the assessment of verbal episodic memory. Results: Eight out of 1008 features were identified as best distinguishing between the CSF profile groups. Of those, only the annotation of the m/z feature assigned to lipid species C18 ceramide was confirmed with a high confidence. Multiple regression analyses, adjusted for age, gender, and education, demonstrated significant associations of CSF core AD markers (Aβ42: st.β= –0.36, p = 0.007; T-tau: st.β= 0.41, p = 0.005) and inflammatory marker S100B (st.β= 0.51, p = 0.001) with C18 ceramide levels. Conclusion: Higher levels of C18 ceramide associated with increased AD pathology and inflammation, suggesting its potential value as a therapeutic target.

Glioblastoma multiforme (GBM), a malignant, highly aggressive, grade IV brain tumor, which rapidly infiltrates into the nearby tissue, has drawn a significant amount of attention because of its poor prognosis and the limited treatment options available. In GBM, nearly all tumor cells exhibit aberrant cell-surface glycosylation patterns due to the alteration of their biosynthesis or postsynthesis modification process. Since gangliosides (GGs) are acknowledged as tumor- associated antigens, we have carried out here a comprehensive profiling of native ganglioside mixtures extracted and purified from GBM specimens. For this purpose, high performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of GBM-specific structures and the assessment of their roles as tumor markers or possible associated antigens. GG separation by IMS according to the charge state, carbohydrate chain length, degree of sialylation, and ceramide composition led to the identification of no less than 160 distinct components, which represents 3- fold the number of structures identified before. The detected GGs and asialo-GGs were found characterized by a high heterogeneity in their ceramide and glycan compositions, encompassing up five Neu5Ac residues. The tumor was found dominated in equal and high proportions by GD3 and GT1 forms, with a particular incidence of C24:1 fatty acids in the ceramide. By the occurrence of only one mobility feature and the diagnostic fragment ions, the IMS tandem MS conducted using collision-induced dissociation (CID) disclosed for the first time the presence of GT1c(d18:1/24:1) newly proposed here as a potential GBM marker.

Objective:Cardiovascular disease remains a leading cause of mortality worldwide. Ceramide scores have been associated with adverse outcomes in patients with established coronary artery disease. The prognostic value of ceramide score has not been assessed in the general population. We tested the hypothesis that ceramide scores are associated with major adverse cardiac events (MACE) in a community-based cohort with average coronary artery disease burden at enrollment.Approach and results:In a prospective community-based cohort, we performed passive follow-up using a record linkage system to ascertain the composite outcome of MACE, defined as acute myocardial infarction, coronary revascularization (bypass grafting or percutaneous intervention), stroke, or death. Ceramides were analyzed as log-transformed continuous variables, ratios or scores, and quartiles with adjustment for confounders. We analyzed 1131 subjects, 52% females, mean age±(SD) 64±9 years. After a median follow-up of 13.3 years (Q1, 12.7; Q3, 14.4), 486 patients experienced a MACE: myocardial infarction (80), coronary artery bypass surgery (34), percutaneous coronary intervention (62), stroke (94), and all-cause death (362). Ceramide ratios were significantly associated with MACE independently of LDL-c (low-density lipoprotein cholesterol) and conventional coronary artery disease risk factors. Those in the highest quartile of ceramide score had nearly 1.5-fold risk of MACE, hazard ratio, 1.47 (95% CI, 1.12–1.92). There was a dose-response association across quartiles of ceramide ratios and MACE.Conclusions:Elevated ceramide score is a robust predictor of cardiovascular disease and MACE in the community. The risk conferred by the ceramide score has a dose-response behavior and is independent of conventional risk factors.

Introduction: Lactic fermentation products (LFPs) are thought to affect “good” bacteria in the gut. We previously reported that oral administration of LFPs has beneficial therapeutic effects in a mouse model of atopic dermatitis. However, it is unclear how LFPs affect human epidermal cell differentiation, ceramide (Cer), and amino acid production. Objective: The aim of this study was to determine the effects of LFPs on epidermal cell differentiation, by assessing amino acid and Cer production. Methods: A 3-dimensional cultured human epidermis model and normal human epidermal keratinocytes were used. Cytotoxicity tests were performed using alamar Blue. Transepidermal water loss (TEWL) was used as an index to assess barrier function. Keratin 1 (K1), keratin 5 (K5), keratin 10 (K10), involucrin (INV), calpain 1, and transglutaminase (TGase) (markers of differentiation) and profilaggrin (proFLG) and bleomycin hydrolase (amino acid synthesis-related genes) expression levels were quantified by RT-PCR. In addition, TGase protein levels were measured by Western blotting. The intercellular lipid content of the stratum corneum was measured by high-performance thin-layer chromatography. Amino acids were quantified using an amino acid analyzer. Finally, bound water content in the stratum corneum was measured by differential scanning calorimetry. Results: Cell viability did not change, but TEWL was significantly decreased in the cells treated with LFPs compared with the control cells. Treatment with LFPs significantly increased expression of the late-differentiation markers INV and TGase at the RNA level. Furthermore, TGase protein expression was significantly increased by treatment with LFPs. Treating a 3-dimensional cultured epidermis model with LFPs significantly increased the intercellular lipid content of the stratum corneum and production of the amino acid arginine (Arg). The amount of bound water in the stratum corneum was increased significantly in the LFP application group. Conclusion: Treatment with LFPs promotes human epidermal cell differentiation and increases the intercellular content of the free fatty acid, Chol, Cer [NS], Cer [AS], and Cer [AP]. This may result in improved skin barrier function. The increased amount of Arg observed in keratinocytes may help improve water retention.

Summary Background & aims Plasma ceramides have been identified as novel risk factors for metabolic and cardiovascular diseases. We aimed to evaluate the effects of dietary anthocyanins on plasma ceramides and to disentangle whether the alterations in ceramides could be related with those in other cardiometabolic risk factors in the dyslipidemia. Methods In a randomized double-blinded placebo-controlled trial, 176 eligible dyslipidemia subjects were randomly assigned into four groups receiving placebo, 40, 80, or 320 mg/day anthocyanins, respectively for 12 weeks. Results A total of 169 subjects completed the study. After 12-week intervention, dietary anthocyanins dose-dependently reduced plasma concentrations of all six ceramide species in the dyslipidemia subjects (all Ptrend values Conclusions Reductions in plasma Cer 16:0 and Cer 18:0 after 12-week anthocyanin intervention were dose-dependently associated with improvements in plasma lipids and cholesterol efflux capacity in the dyslipidemia. Clinical trial registration The study was registered at ClinicalTrials.gov with the identifier No. NCT03415503.

Background/Aims: Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism. Methods: Lung adenocarcinoma cells and non-pathological human fibroblasts were stimulated with unmethylated Cytosine phosphate Guanosine (CpG), the TLR9 ligand, and S1P-dependent TNF-α release was evaluated by means of ELISA. Immunofluorescence and LC-MS/MS analysis were performed to evaluate/quantify S1P generation following TLR9 activation. Results: We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside membrane receptor S1P1 and S1P3 via the efflux through its membrane transporter SPNS2. Indeed, both the blockade of S1P3 and the transporter SPNS2 significantly reduced the activity of S1P on TNF-α release from lung adenocarcinoma cells. Conclusion: Our study identifies a novel inflammatory pathway in that TLR9 increases the pro-inflammatory cytokine release, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P, adding a novel puzzle piece in TLR9-orchestrated inflammatory pathway and shedding more light on the role of the higher levels of S1P during inflammatory conditions.

Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired β-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-day–dependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.