C2 Ceramide Research Articles

Widely Targeted Lipidomics and Transcriptomics Analysis Revealed Changes of Lipid Metabolism in Spleen Dendritic Cells in Shrimp Allergy.

Shrimp allergy (SA) is pathological type 2 inflammatory immune responses against harmless shrimp protein allergen, which is caused by complex interactions between dendritic cells (DCs) and other immune cells. Lipid metabolism in different DCs states are significantly changed. However, the lipid metabolism of spleen DCs in SA remain ambiguous. In this study, we established a BALB/c mouse shrimp protein extract-induced allergy model to determine the lipid profile of spleen DCs in SA, and the molecular mechanism between lipid metabolism and immune inflammation was preliminarily studied. Spleen DCs were sorted by fluorescence-activated cell sorting, and then widely targeted lipidomics and transcriptomics analysis were performed. Principal component analysis presented the lipidome alterations in SA. The transcriptomic data showed that Prkcg was involved in lipid metabolism, immune system, and inflammatory signaling pathway. In the correlation analysis, the results suggested that Prkcg was positively correlated with triacylglycerol (Pearson correlation coefficient = 0.917, p = 0.01). The lipidomics and transcriptomics integrated pathway analysis indicated the activated metabolic conversion from triacylglycerol to 1,2-diacyl-sn-glycerol and the transmission of lipid metabolism to immune inflammation (from triacylglycerol and ceramide to Prkcg) in SA spleen DCs, and cellular experiments in vitro showed that glyceryl trioleate and C16 ceramide treatment induced immune function alteration in DCs.

Infusion of C20 ceramide induces depressive‐like behavior, increases microglia but not astrocyte expression

Depression is a disease affecting the population worldwide. Depressed patients have shown alteration in the hippocampus suggesting this region can be an important region to study the impact of the development of depressive‐like behavior. Literature has demonstrated depressed patients present an increased levels of long‐chain ceramides. Ceramide is a sphingolipid component of the cellular membrane and play an important role in its structural integrity. To better understand depression mechanisms it is important to assess the glial cells mechanisms, since these cells play an essential role in inflammation, which is increased in depressed patients. Literature has reported interaction between astrocytes and microglia, still their detailed mechanism, their individual response to ceramide signaling, and contribution to depressive‐like behavior development are not clearly understood. Some research has demonstrated that microglia are activated in the presence of C20 and C16 ceramide. Otherwise, data have shown that reactive astrocytes interact with C16 ceramide but not with C24 ceramides. Because of this, we hypothesize that the infusion of long‐chain ceramides (C20) in the ventral hippocampus will increase the development of depressive‐like behavior, will cause an increase in the level of microglia expression but not astrocyte expression. We performed seven infusions of long‐chain ceramides into the ventral hippocampus followed by behavior analysis to measure the development of depressive‐like behavior and tissue collection to analyze inflammatory profile. Results show that rats infused with long‐chain ceramides present development of short‐term anhedonia‐like behavior twenty‐four hours after infusions but no depressive‐like behavior was assessed five days after the last infusion of ceramides. Also, infusion of ceramides induce an increase in microglia expression but no significant differences were assessed in astrocyte expression. These results suggest that C20 ceramide activates the microglia inflammatory pathway but not astrocyte mediated inflammation. There might be different mechanisms inducing inflammation in microglia and astrocytes. This preliminary data can also suggest that microglia mediated inflammation can contribute more to depressive‐like behavior than astrocytes. These results bring speculative significance, but more comprehensive studies can show a higher understanding of ceramide signaling in inflammation and a better understanding of the contribution of microglia and astrocytes to the development of depressive‐like behavior.

Role of NOX2 in Ceramide‐Induced Human Microvascular Endothelial Dysfunction

Endothelial dysfunction, or the loss of nitric oxide (NO)‐mediated dilation to shear stress (flow‐induced dilation; FID), is observed in human adipose arterioles from patients with coronary artery disease (CAD). FID is maintained during CAD by release of the mitochondrial‐derived, pro‐inflammatory dilator, H2O2. Ceramide, a sphingolipid that when elevated in plasma is an independent risk factor for future cardiac events, also induces this transition in human arterioles (10µM, 16‐20hrs). The initial events leading to ceramide‐induced endothelial dysfunction are unknown. Here we hypothesize that activation of NADPH oxidase 2 (NOX2), an endothelial membrane enzyme activated by acute stress, occurs prior to the formation of mitochondrial H2O2 in arterioles exposed to chronic ceramide. Human arterioles (100‐250µm in diameter) were dissected from discarded surgical adipose tissues and prepared for videomicroscopy. Following preconstriction with endothelin‐1, internal diameters were measured in response to increased flow. To determine the minimum exposure time necessary to convert to H2O2‐dependent FID in arterioles exposed to ceramide, microvessels from healthy nonCAD adults were incubated with C2 ceramide for 30 min, 2 hrs, and 4 hrs. FID remained mediated by NO at 30min and 2hrs, however, following a 4hr incubation, FID was significantly impaired in the presence of PEG‐catalase (44.6% of maximal dilator capacity ±13.8 (SEM), n=8, p<0.05, one‐way ANOVA*) compared to vehicle control (83.5%±5.0, n=8). The source of H2O2 generated during FID in arterioles treated for 4hr with ceramide was not mitochondrial as increases in fluorescent intensity of mito peroxy yellow 1 (mitoPY1) were not observed during maximal flow. To test whether NOX2 is the initial source of H2O2 during FID in arterioles exposed to 4hr ceramide, nonCAD arterioles were first treated with the NOX2 inhibitor GSK2795039 (NOX2i, 10‐6 M) or underwent intraluminal administration of siRNA to decrease NOX2 expression prior to treatment with ceramide. FID was reduced in vessels treated with both ceramide and the NOX2 inhibitor in the presence of cPTIO (NO scavenger) (50.2%±30.1, n=4* vs. NOX2 inhibitor and ceramide alone 80.8%±6.4, n=7). L‐NAME also impaired FID in vessels with reduced expression of NOX2 and subsequently treated with ceramide (10.8%±38.9, n=3* compared to siNOX2 and ceramide alone 85.5%±5.8, n=3). This suggests that activation of NOX2 may be the initial event in the conversion of NO‐ to H2O2‐dependent FID due to ceramide and may serve as a potential therapeutic target in those at risk for developing cardiovascular disease due to increased plasma ceramide.

Dihydroceramide- and ceramide-profiling provides insights into human cardiometabolic disease etiology

Metabolic alterations precede cardiometabolic disease onset. Here we present ceramide- and dihydroceramide-profiling data from a nested case-cohort (type 2 diabetes [T2D, n = 775]; cardiovascular disease [CVD, n = 551]; random subcohort [n = 1137]) in the prospective EPIC-Potsdam study. We apply the novel NetCoupler-algorithm to link a data-driven (dihydro)ceramide network to T2D and CVD risk. Controlling for confounding by other (dihydro)ceramides, ceramides C18:0 and C22:0 and dihydroceramides C20:0 and C22:2 are associated with higher and ceramide C20:0 and dihydroceramide C26:1 with lower T2D risk. Ceramide C16:0 and dihydroceramide C22:2 are associated with higher CVD risk. Genome-wide association studies and Mendelian randomization analyses support a role of ceramide C22:0 in T2D etiology. Our results also suggest that (dh)ceramides partly mediate the putative adverse effect of high red meat consumption and benefits of coffee consumption on T2D risk. Thus, (dihydro)ceramides may play a critical role in linking genetic predisposition and dietary habits to cardiometabolic disease risk.

The sphingosine kinase inhibitor SKI-V suppresses cervical cancer cell growth.

Overexpression and/or overactivation of sphingosine kinase 1/2 (SphK1/2) is important for tumorigenesis and progression of cervical cancer. The current study examined the potential activity and signaling mechanisms of SKI-V, a non-lipid small molecule SphK inhibitor, against cervical cancer cells. In different primary and immortalized cervical cancer cells, SKI-V exerted significant anti-cancer activity by inhibiting cell viability, colony formation, proliferation, cell cycle progression and cell migration. Significant apoptosis activation was detected in SKI-V-treated cervical cancer cells. Significantly, SKI-V also provoked programmed necrosis cascade in cervical cancer cells, as it induced mitochondrial p53-cyclophilin-D-adenine nucleotide translocator-1 (ANT1) complexation, mitochondrial membrane potential collapse, reactive oxygen species production and the release of lactate dehydrogenase into the medium. Further, SKI-V blocked SphK activation and induced ceramide accumulation in primary cervical cancer cells, without affecting SphK1/2 expression. SKI-V-induced cytotoxicity in cervical cancer cells was largely inhibited by sphingosine-1-phosphate or the SphK1 activator K6PC-5, but was sensitized by adding the short-chain ceramide C6. Moreover, SKI-V inhibited Akt-mTOR (mammalian target of rapamycin) activation in primary cervical cancer cells, and its cytotoxicity was mitigated by a constitutively-active Akt. In vivo, daily intraperitoneal injection of SKI-V significantly inhibited subcutaneous primary cervical cancer xenograft growth in nude mice. Together, the SphK inhibitor SKI-V suppresses cervical cancer growth in vitro and in vivo.

Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides

BackgroundPro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated.Methods and ResultsA mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1–16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells.ConclusionlEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.Graphical abstract

CERTL reduces C16 ceramide, amyloid-β levels and inflammation in a model of Alzheimer's disease.

Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer's disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. A plasmid expressing CERTL , the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL , and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety and locomotion. At week twelve, brains were investigated for sphingolipid levels by mass spectrometry, plaques and neuroinflammation by immunohistochemistry, gene expression and/or immunoassay. Here, we report that CERTL , binds to APP, modifies Aβ aggregation and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL , decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation and modulates microglia by decreasing their pro-inflammatory phenotype. Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Mechanistic target of rapamycin and an extracellular signaling-regulated kinases 1 and 2 signaling participate in the process of acetate regulating lipid metabolism and hormone-sensitive lipase expression.

ObjectiveAcetate plays an important role in host lipid metabolism. However, the network of acetate-regulated lipid metabolism remains unclear. Previous studies show that mitogen-activated protein kinases (MAPKs) and mechanistic target of rapamycin (mTOR) play a crucial role in lipid metabolism. We hypothesize that acetate could affect MAPKs and/or mTOR signaling and then regulate lipid metabolism. The present study investigated whether any cross talk occurs among MAPKs, mTOR and acetate in regulating lipid metabolism.MethodsThe ceramide C6 (an extracellular signaling-regulated kinases 1 and 2 [ERK1/2] activator) and MHY1485 (a mTOR activator) were used to treat rabbit adipose-derived stem cells (ADSCs) with or without acetate, respectively.ResultsIt indicated that acetate (9 mM) treatment for 48 h decreased the lipid deposition in rabbit ADSCs. Acetate treatment decreased significantly phosphorylated protein levels of ERK1/2 and mTOR but significantly increased mRNA level of hormone-sensitive lipase (HSL). Acetate treatment did not significantly alter the phosphorylated protein level of p38 MAPK and c-Jun aminoterminal kinase (JNK). Activation of ERK1/2 and mTOR by respective addition in media with ceramide C6 and MHY1485 significantly attenuated decreased lipid deposition and increased HSL expression caused by acetate.ConclusionOur results suggest that ERK1/2 and mTOR signaling pathways are associated with acetate regulated HSL gene expression and lipid deposition.

An extra virgin olive oil‐enriched chocolate spread positively modulates insulin‐resistance markers compared with a palm oil‐enriched one in healthy young adults: A double‐blind, cross‐over, randomised controlled trial

AimsTo investigate if extra virgin olive oil (EVOO) or palm oil enriched chocolate spreads consumption leads to different results in terms of plasma ceramides concentration, glucose and lipid metabolism, inflammatory markers and appetite regulation in young healthy subjects.MethodsIn a 2‐week, double‐blind, cross‐over, randomised controlled trial, 20 healthy, normal‐weight subjects with a mean age of 24.2 years (SD: 1.2), consumed chocolate spread snacks (73% of energy [%E] from fat, 20% from carbohydrates and 7% from proteins), providing 570 Kcal/day added to an isocaloric diet. The chocolate spreads were identical, except for the type of fat: EVOO oil, rich in monounsaturated fatty acids (MUFAs), or palm oil, rich in Saturated Fatty Acids (SFAs).ResultsEVOO‐enriched chocolate spread consumption led to better circulating sphingolipids and glucose profile, with reduced plasma ceramide C16:0, ceramide C16:0/ceramide C22:0‐ceramide C24:0 ratio and sphingomyelin C18:0 (P = 0.030, P= 0.032 and P = 0.042, respectively) compared to the palm oil‐enriched chocolate spread diet. HOMA‐IR and plasma insulin were lower, while the Quicki and the McAuley Index were higher after the EVOO diet compared to the palm oil diet (P = 0.046, P = 0.045, P = 0.018 and P = 0.039 respectively). Subjects maintained a stable weight throughout the study. No major significant changes in total cholesterol, triglycerides, HDL, inflammatory markers, and appetite‐regulating hormones/visual analogue scale were observed between the groups.ConclusionsPartially replacing SFAs with MUFAs in a chocolate‐based snack as part of a short‐term isocaloric diet in healthy individuals may limit SFAs detrimental effects on insulin sensitivity and decrease circulating harmful sphingolipids in young adults.

Severity of COVID-19 Patients Predicted by Serum Sphingolipids Signature.

The reason behind the high inter-individual variability in response to SARS-CoV-2 infection and patient’s outcome is poorly understood. The present study targets the sphingolipid profile of twenty-four healthy controls and fifty-nine COVID-19 patients with different disease severity. Sera were analyzed by untargeted and targeted mass spectrometry and ELISA. Results indicated a progressive increase in dihydrosphingosine, dihydroceramides, ceramides, sphingosine, and a decrease in sphingosine-1-phosphate. These changes are associated with a serine palmitoyltransferase long chain base subunit 1 (SPTLC1) increase in relation to COVID-19 severity. Severe patients showed a decrease in sphingomyelins and a high level of acid sphingomyelinase (aSMase) that influences monosialodihexosyl ganglioside (GM3) C16:0 levels. Critical patients are characterized by high levels of dihydrosphingosine and dihydroceramide but not of glycosphingolipids. In severe and critical patients, unbalanced lipid metabolism induces lipid raft remodeling, leads to cell apoptosis and immunoescape, suggesting active sphingolipid participation in viral infection. Furthermore, results indicated that the sphingolipid and glycosphingolipid metabolic rewiring promoted by aSMase and GM3 is age-dependent but also characteristic of severe and critical patients influencing prognosis and increasing viral load. AUCs calculated from ROC curves indicated ceramides C16:0, C18:0, C24:1, sphingosine and SPTLC1 as putative biomarkers of disease evolution.

Palmitate‐mediated induction of neuropeptide Y expression occurs through intracellular metabolites and not direct exposure to proinflammatory cytokines

A contributing factor to the development of obesity is the consumption of a diet high in saturated fatty acids, such as palmitate. These fats induce hypothalamic neuroinflammation, which dysregulates neuronal function and induces orexigenic neuropeptide Y (Npy) to promote food intake. An inflammatory cytokine array identified multiple candidates that could mediate palmitate-induced up-regulation of Npy mRNA levels. Of these, visfatin or nicotinamide phosphoribosyltransferase (NAMPT), macrophage migratory inhibitory factor (MIF), and IL-17F were chosen for further study. Direct treatment of the neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing mHypoE-46 neuronal cell line with the aforementioned cytokines demonstrated that visfatin could directly induce Npy mRNA expression. Preventing the intracellular metabolism of palmitate through long-chain acyl-CoA synthetase (ACSL) inhibition was sufficient to block the palmitate-mediated increase in Npy gene expression. Furthermore, thin-layer chromatography revealed that in neurons, palmitate is readily incorporated into ceramides and defined species of phospholipids. Exogenous C16 ceramide, dipalmitoyl-phosphatidylcholine, and dipalmitoyl-phosphatidylethanolamine were sufficient to significantly induce Npy expression. This study suggests that the intracellular metabolism of palmitate and elevation of metabolites, including ceramide and phospholipids, are responsible for the palmitate-mediated induction of the potent orexigen Npy. Furthermore, this suggests that the regulation of Npy expression is less reliant on inflammatory cytokines per se than palmitate metabolites in a model of NPY/AgRP neurons. These lipid species likely induce detrimental downstream cellular signaling events ultimately causing an increase in feeding, resulting in an overweight phenotype and/or obesity.

Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers.

Prostate cancer is the second most prevalent malignancy worldwide. In the early stages, the development of prostate cancer is dependent on androgens. Over time with androgen deprivation therapy, 20% of prostate cancers progress to a castration-resistant form. Novel treatments for prostate cancers are still urgently needed. Erianin is a plant-derived bibenzyl compound. We report herein that erianin exhibits anti-tumor effects in androgen-sensitive and castration-resistant prostate cancer cells through different mechanisms. Erianin induces endoplasmic reticulum stress-associated apoptosis in androgen-sensitive prostate cancer cells. It also triggers pro-survival autophagic responses, as inhibition of autophagy predisposes to apoptosis. In contrast, erianin fails to induce apoptosis in castration-resistant prostate cancer cells. Instead, it results in cell cycle arrest at the M phase. Mechanistically, C16 ceramide dictates differential responses of androgen-sensitive and castration-resistant prostate cancer cells to erianin. Erianin elevates C16 ceramide level in androgen-sensitive but not castration-resistant prostate cancer cells. Overexpression of ceramide synthase 5 that specifically produces C16 ceramide enables erianin to induce apoptosis in castration-resistant prostate cancer cells. Our study provides both experimental evidence and mechanistic data showing that erianin is a potential treatment option for prostate cancers.

Влияние применения метформина и снижения массы тела на концентрацию церамидов C16:0, C18:0, C24:1 в плазме крови у пациентов с неалкогольной жировой болезнью печени в сочетании с инсулинорезистентностью и сахарным диабетом 2 типа

Актуальность. Церамиды — это жиры семейства сфинголипидов, которые принимают участие в развитии инсулинорезистентности (ИР), неалкогольной жировой болезни печени (НАЖБП) и сахарного диабета 2 типа (СД). Несмотря на стандарты лечения этих заболеваний, на сегодняшний день недостаточно информации относительно их влияния на концентрацию церамидов в плазме крови. В связи с этим мы исследовали, как прием метформина и снижение массы тела влияют на концентрацию церамидов C16:0, C18:0, C24:1 у данных больных. Цель исследования: изучить и сравнить концентрацию церамидов С16:0, С18:0, С24:1 в плазме крови у пациентов с НАЖБП до и после проведения лечения. Материалы и методы. В исследовании приняли участие 50 пациентов с НАЖБП, которые были разделены на две группы: 24 человека с IP и 26 человек с СД 2 типа. В качестве лечения пациентам с ИP рекомендовано было снижение массы тела. Пациенты с СД 2 типа принимали метформин в дозе 500 мг 2 раза в сутки. Контрольную группу составили 10 здоровых лиц. Церамиды определялись с помощью жидкостной хроматографии с масс-спектрометрией (Shimadzu Nexera X2 UHPLC, Shimadzu LCMS-8045 mass-spectrometer). Результаты. При сравнении концентрации церамидов С16:0, С18:0, С24:1 в обеих группах выявлено их достоверное повышение по сравнению с конт­рольной группой (р < 0,001). После проведенного лечения у пациентов обеих групп наблюдалось достоверное уменьшение концентрации церамидов (р < 0,001). Кроме того, было обнаружено, что фракции церамидов С16:0, С18:0, С24:1 в обеих группах коррелировали с основными лабораторными и инструментальными показателями. Выводы. У пациентов с НАЖБП, IP и СД 2 типа концентрация церамидов С16:0, С18:0, С24:1 была повышенной. На фоне соответствующего лечения концентрация церамидов уменьшилась.

Association of plasma ceramides with prevalent and incident type 2 diabetes mellitus in middle and older aged adults

AimsThe role of ceramides in the pathogenesis of type 2 diabetes mellitus (T2DM) is incompletely characterized. Given that ceramides represent therapeutic targets to disrupt the euglycemia-T2DM transition, we aimed to characterize their association with prevalent and incident T2DM in a novel cohort. MethodsWe examined the cross-sectional and longitudinal association of baseline ceramides with prevalent and incident T2DM among 1423 adults (47% women; median (range) baseline age 72 (51–95) years) in the Mayo Clinic Study of Aging cohort. We examined the associations of ceramides with prevalent T2DM (adjusted odds ratio [95% confidence interval]) at baseline and incident T2DM (adjusted hazard ratio [95% confidence interval]) during median follow-up of 6.2 years, after adjusting for demographic and metabolic factors. ResultsAmong 1423 adults, there were 222 prevalent and 37 incident cases of T2DM. In cross-sectional analyses, higher levels of ceramide C16:0 were associated with lower odds of prevalent T2DM (aOR 0.84 [0.71–0.99];P = 0.03) whereas C18:0 (aOR 1.27 [1.06–1.42];P = 0.01), C18:0/16:0 (aOR 1.41 [1.22–1.62]; P < 0.001) and C18:0/24:0 (aOR 1.22 [1.05–1.41]; P = 0.01) were associated with higher odds. In Cox hazard regression models, C18:0/16:0 (aHR 1.63 [1.26–2.10];P < 0.001) and C18:0 (aHR 1.53 [1.12–2.08];P = 0.01) were associated with increased risk of incident T2DM. ConclusionsIn this prospective population-based cohort, ceramides were associated with prevalent T2DM (C16:0,C18:0, C18:0/C16:0 ratio, C18:0/C24:0 ratio) and incident T2DM (C18:0, C18:0/C16:0 ratio) and could suggest targets for the primary and secondary prevention of T2DM.

A Lipid Mixture Enriched by Ceramide NP with Fatty Acids of Diverse Chain Lengths Contributes to Restore the Skin Barrier Function Impaired by Topical Corticosteroid

Introduction: The stratum corneum (SC) is a skin barrier that consists of corneocytes, intercellular lipids, and corneodesmosomes. Ceramides are composed of sphingoid bases linked with various types of fatty acids (FAs), and they are an essential constituent of SC intercellular lipids. Among their subtypes, ceramide NP with a phytosphingosine base is especially important. Most of the previous studies on barrier recovery have focused on a specific ceramide with a single chain FA, not with diverse chain lengths. Skin barrier function is impaired by various factors, including topical corticosteroid. Objective: We evaluated whether a lipid mixture enriched by ceramide NP with FAs of diverse chain lengths (CER [NP]*) can restore the skin barrier function impaired by topical corticosteroid. Methods: Twenty-seven healthy adult male volunteers were recruited. Topical corticosteroid was applied on both volar forearms of volunteers. Then, the test cream containing a lipid mixture with CER (NP)* was applied on the left forearm, and a vehicle cream without a lipid mixture was applied on the right forearm of each subject. The functional parameters of the skin barrier were compared before and after the treatment. Epidermal differentiation markers, hyaluronic acid synthase 3 (HAS3), cytokine levels, and the lipid profiles in the SC were analyzed. Results: The functional parameters of the skin barrier, such as barrier recovery rate, SC integrity, and SC hydration were significantly improved in the test cream-applied site compared to the vehicle cream-applied sites. Filaggrin and HAS3 levels were significantly higher in the sites applied with the test cream. Interleukin (IL)-1α levels were also significantly increased in these sites. IL-2, IL-6, IL-10, and IL-13 levels were significantly decreased in the test cream-applied sites. Lipid analyses showed that C18, C20, and total ceramide NP levels significantly increased in the sites where the test cream was applied. Also, C16, C18, C20, C24, and total ceramide NP levels were significantly elevated in the test cream-applied sites after acute barrier disruption. Conclusion: Our results demonstrate that a lipid mixture enriched by CER (NP)* could recover the barrier function impaired by topical corticosteroid.

Effect of Korean Red Ginseng on Plasma Ceramide Levels in Postmenopausal Women with Hypercholesterolemia: A Pilot Randomized Controlled Trial.

Cardiovascular disease (CVD) is a crucial cause of death in postmenopausal women. Plasma ceramide concentrations are correlated with the development of atherosclerosis and are significant predictors of CVD. Here, we conducted a 4-week, double-blinded, placebo-controlled clinical pilot study to investigate the effect of Korean red ginseng (KRG) on serum ceramide concentrations in 68 postmenopausal women with hypercholesterolemia. Patients were randomly assigned to two groups: the experimental group (n = 36) received KRG and the control (n = 32) group received placebo, 2 g each, once daily. Serum ceramides were measured using liquid chromatography–tandem mass spectrometry at baseline and study completion, with changes in serum ceramide levels as the primary end point. We detected significantly greater mean changes in C16 ceramide levels (d18:1/16:0: −6.4 ± 6.3 pmol/mL vs. 14.6 ± 6.8 pmol/mL, respectively, p = 0.040; d18:1/22:0: −20.8 ± 24.4 pmol/mL vs. 71.1 ± 26.2 pmol/mL, respectively, p = 0.020). Additionally, changes in the median C16 (d18:1/16:0) and C22 (d18:1/22:0) ceramide levels were significantly greater in KRG-group subjects with metabolic syndrome than those without. Therefore, we found that KRG decreases the serum levels of several ceramides in postmenopausal women with hypercholesterolemia, suggesting it may be beneficial for preventing CVD in these individuals.

Neuroprotective role of sphingolipid rheostat in excitotoxic retinal ganglion cell death

The glutamate excitotoxicity has been suggested as a factor involved in the loss of retinal neuronal cells, including retinal ganglion cell (RGC), in various retinal degenerative diseases including ischemia-reperfusion injury, diabetic retinopathy, and glaucoma. Excitotoxic RGC death is caused not only by direct damage to RGCs but also by indirect damage due to the inflammation of retinal glial cells. Sphingosine 1-phosphate (S1P) and ceramides are bioactive sphingolipids which have been shown to possess important physiological roles in cellular survival and apoptosis, and the balance between S1P and ceramide, sphingolipid rheostat, has been suggested to be important for determining cellular fate. Therefore, we conducted the present study to clarify the neuroprotective role of sphingolipid rheostat in excitotoxic RGC death in vivo and in vitro. Acute RGC death was induced by intravitreal N-methyl-d-aspartate (NMDA) injection in the mouse. The mRNA expression of sphingosine kinase (SphK1/SphK2) was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of SphK1/2, S1P, S1P-receptor (S1PR), glial fibrillary acidic protein (GFAP), Iba1, and CD31 were examined by immunostaining. Retinal sphingolipids and ceramides were quantified by liquid chromatography with tandem mass spectrometry. The neuroprotective effect of the sphingosine kinase inhibitor (SKI) on RGC death was assessed by RGC count and Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Further, the in vitro effect of SKI was investigated using rat primary cultured RGCs and glial cells. In addition, MG5 cells and A1 cells, which were mouse microglia and astrocyte cell-line, were also used. The expression of cleaved-caspase-3, GFAP, and Iba1 in RGCs, primary glial cells, MG5 cells, and A1 cells was assessed by immunostaining. NMDA injection resulted in mRNA upregulation of SphK1; however, SphK2 was reduced in the mouse retina. SphKs, S1P, S1PR1, S1PR2, and GFAP expression increased in the early-stage NMDA group, whereas S1P and GFAP were higher in the late-stage NMDA + SKI group. In the NMDA group, S1P expression was lower whereas sphingosine, C20, C22, and C24 ceramides showed higher levels. The proportion of very-long-chain ceramide was elevated in the NMDA group but reduced in the NMDA + SKI group. SKI treatment significantly increased RGC survival in retinal wholemount analysis and decreased apoptosis in the ganglion cell layer and inner nuclear layer. In vitro, SKI suppressed excitotoxic RGC death, cleaved-caspase-3 expression, and activated glial cells. The findings in the present study provide the first evidence demonstrating the involvement of sphingolipid rheostat in the neuroprotection against excitotoxic RGC death. Therefore, regulation of sphingolipid rheostat might serve as a potential therapy for retinal degenerative disease.

Cocaine Self‐administration Modulates Brain Ceramide Metabolism

Introduction Chronic cocaine exposure produces neuroadaptations of many plasma membrane integrated proteins including dopamine transporters that contribute to relapse; however, the mechanisms remain elusive. A few studies show that chronic cocaine exposure alters blood levels of lipids including fatty acids and cholesterol and these changes are correlated with vulnerability to relapse. To date, little information is available on chronic cocaine modulation of brain lipids. Neuronal membrane predominantly consists of sphingolipids along with cholesterol and glycerophospholipids. Among sphingolipids, ceramide is a key membrane structural component and highly concentrated. Ceramide not only influences membrane rigidity but also acts as a signaling molecule. A change in ceramide content in cultured cells alters the activity of dopamine transporters. The purpose of this project was to investigate if cocaine self-administration modulated brain ceramide content, which may underlie functional changes of dopamine transporters. Methods Male Sprague-Dawley rats were trained and allowed to self-administer cocaine (1.5 mg/kg/infusion, 40 injections/6hrs/day) for 5 days. Rat prefrontal cortex (PFC) and striatum were dissected 18 hrs after the last cocaine infusion along with saline controls. Samples underwent a Bligh & Dyer method of lipid extraction. A triple quadrupole mass spectrometer was used to measure the content of ceramide species (C16, C18, C20 and C24) in these brain regions. A 50 × 2.0 mm, 3 µm reversed-phase column was used with a flow rate set to 0.8 mL/min. Samples underwent a five minute gradient consisting of mobile phase A (0.1% formic acid and 25 mM ammonium acetate in water) and mobile phase B (100% acetonitrile). The C17 ceramide, not present in the brain, was used as an internal standard for data analysis. Moreover, the expression and activity of enzymes involved in ceramide metabolism including neural sphingomyelinase 2, which converts sphingomyelin to ceramide on the membrane, and ceramide synthesis 1 & 2, which are primarily responsible for the synthesis of brain ceramide species, were analyzed by western blot and activity assay. Results We optimized lipid extraction method and mass spectrometry condition so that ceramide species in the brain can be clearly identified and quantitated. We found that cocaine SA alters brain ceramide content in a brain region and species-dependent manner. The C18 ceramide was confirmed as the most abundant ceramide species compared to the others. Cocaine SA also changed the expression and/or the activity of sphingomyelinase 2, ceramide synthesis 1&2. The present study provides evidence that cocaine SA regulates brain ceramide content by influencing ceramide synthesis in both sphingomyelin hydrolysis and de novo synthesis pathways. Our data suggests that cocaine SA modulates brain ceramide homeostasis. Given the important role of ceramide in regulation of membrane fluidity and acting as a signaling molecule, brain ceramide remodeling highlights a potential new mechanism underlying chronic cocaine-mediated functional changes of membrane proteins.

Investigation of the Dual Functional Role of Ceramide in the Human Microcirculation

Increased plasma ceramide levels and microvascular dysfunction are both independent risk factors for major adverse cardiac events (MACE). We have previously shown that chronic exposure to exogenous ceramide promotes microvascular endothelial dysfunction, defined as hydrogen peroxide (H2O2)-mediated flow-induced dilation (FID) as opposed to dilation due to formation and release of endothelial nitric oxide (NO). Interestingly, ceramide and its metabolites (e.g. sphingosine-1-phosphate; S1P) have also been shown to stimulate production of NO. Our previous studies indicate that activation of the ceramide-producing enzyme neutral sphingomyelinase (NSmase) in necessary for NO-mediated FID. We therefore hypothesized that formation of S1P is responsible for cellular increases in NO from both acute exogenous administration as well as endogenous formation of ceramide from shear. Human resistance arterioles (100-200µm) were dissected from adipose tissue collected from healthy patients and were prepared for videomicroscopy. Increasing doses of exogenous C2 ceramide (10-9 to 10-5 M) were administered in the absence or presence of the NO scavenger c-PTIO (1µM) and a sphingosine kinase inhibitor (SpKi, 5µM). We observed a dose-response increase in arteriolar dilation from ceramide (46.9%±10.3 of maximal dilator capacity±SEM, n=3) that was impaired by c-PTIO (15%±6.0, n=3). Ceramide-induced dilation was also decreased in the presence of the SpKi (20.8%±4.0, n=3). To examine the role of S1P formation in NO-mediated FID, SpKi was administered to healthy arterioles (5µM, 30 min) prior to initiating flow (pressure gradient 5-100cm H2O) and a dramatic decrease in overall dilation was observed (7.7%±8.0, n=3). Together these findings suggest that NO generated from ceramide is primarily due to the formation of S1P, a process also critical to maintain FID in arterioles from healthy individuals.

Cerebrospinal Fluid C18 Ceramide Associates with Markers of Alzheimer's Disease and Inflammation at the Pre- and Early Stages of Dementia.

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.