Ceramide Profile Research Articles

Amelioration of lactic acid sensations in sensitive skin by stimulating the barrier function and improving the ceramide profile.

We determined whether compensating ceramides in the stratum corneum (SC) may ameliorate the impaired barrier function and subsequently attenuate the enhanced skin sensitivity. Treatment for 4 weeks with the ceramide complex cream or the placebo cream significantly ameliorated the intensity of lactic acid sensations in 39 female subjects with sensitive skin, the degree of which was attenuated to a greater extent at 1 week by the ceramide complex cream compared with the placebo cream. The amelioration of skin sensations was accompanied by a significant increase in total ceramide content in the SC elicited by the ceramide complex cream that was significantly more effective than the placebo cream at 4 weeks. Consistently, TEWL and conductance values were significantly decreased or increased at 1 and 4 weeks, respectively, to a greater extent by the ceramide complex cream than by the placebo cream. TEWL levels were significantly correlated with the increased levels of SC total ceramide in the ceramide complex cream-treated skin but not in the placebo cream-treated skin. Thus, the amelioration of lactic acid sensations by topical application of a ceramide complex cream, provides a deep insight into the pathophysiology of sensitive skin as a reduced barrier function-dependent sub-clinical sensory response.

Determination of fatty acid and sphingoid base composition of eleven ceramide subclasses in stratum corneum by UHPLC/scheduled-MRM.

Ceramides play a key role in skin barrier function in homeostatic and pathological conditions and can be sampled non-invasively through stratum corneum collection. To develop a novel UHPLC/Scheduled MRM method for the identification and relative distribution of eleven classes of ceramides, which are separated by UHPLC and determined by their specific retention times. The precise composition of the fatty acid and sphingoid base parts of each individual ceramide is determined via mass fragmentation. More than 1000 human and pig ceramides were identified. Three human and minipig ceramide classes, CER[AS], CER[NS] and CER[EOS] have been investigated in depth. Sphingoid bases were characterized by a prevalence of chain lengths with sizes from C16 to C22, whereas fatty acids were mainly observed in the range of C22-C26. Overall, the ceramide profiles between human and minipig stratum corneum were similar. Differences in the CER[AS] and CER[NS] classes included a more homogeneous distribution of fatty acids (16-30 carbon atoms) in minipig, whereas in human longer fatty acid chains (> 24 carbon atoms) predominated. The method will be useful for the analysis of healthy and pathological skin in various specie, and the measurement of the relative distribution of ceramides as biomarkers for pharmacodynamic studies.

634 Skin ceramide profile in children with atopic dermatitis

634 Skin ceramide profile in children with atopic dermatitis

Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration.

Macrophage aging is pathogenic in diseases of the elderly, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. However, the role of microRNAs, which modulate immune processes, in regulating macrophage dysfunction and thereby promoting age-associated diseases is underexplored. Here, we report that microRNA-150 (miR-150) coordinates transcriptomic changes in aged murine macrophages, especially those associated with aberrant lipid trafficking and metabolism in AMD pathogenesis. Molecular profiling confirmed that aged murine macrophages exhibit dysregulated ceramide and phospholipid profiles compared with young macrophages. Of translational relevance, upregulation of miR-150 in human peripheral blood mononuclear cells was also significantly associated with increased odds of AMD, even after controlling for age. Mechanistically, miR-150 directly targets stearoyl-CoA desaturase-2, which coordinates macrophage-mediated inflammation and pathologic angiogenesis, as seen in AMD, in a VEGF-independent manner. Together, our results implicate miR-150 as pathogenic in AMD and provide potentially novel molecular insights into diseases of aging.

Applying a vernix caseosa based formulation accelerates skin barrier repair by modulating lipid biosynthesis

Restoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties.

Ceramides in tracheal aspirates of preterm infants: Marker for bronchopulmonary dysplasia.

BackgroundIn an experimental mouse model we showed that ceramides play a role in the pathogenesis of bronchopulmonary dysplasia (BPD) and are a potential target for therapeutic intervention. We investigated whether ceramides are detectable in tracheal aspirates (TAs) of preterm infants and differ between infants with or without BPD.MethodsInfants born ≤ 32 weeks of gestational age in need of mechanical ventilation in the first week of life were included. TAs were obtained directly after intubation and at day 1, 3, 5, 7, and 14. Ceramide concentrations were measured by tandem mass spectrometry. At 36 weeks postmenstrual age BPD was defined as having had ≥ 28 days supplemental oxygen.Results122 infants were included, of which 14 died and 41 developed BPD. All infants showed an increase in ceramides after the first day of intubation. The ceramide profile differed significantly between preterm infants who did and did not develop BPD. However, the ceramide profile had no additional predictive value for BPD development over GA at birth, birth weight and total days of mechanical ventilation.ConclusionsCeramides are measurable in TAs of preterm born infants and may be an early marker for BPD development.

X – linked Hypohidrotic Ectodermal Dysplasia and atopic eczema – case report and discussion on mechanisms of eczema

X-linked Hypohidrotic Ectodermal Dysplasia (XHED) is an inherited disorder that involves defective development of tissues derived from embryonic ectoderm and it is generally suspected by the triad of hypohidrosis, hypotrichosis and hypodontia. We report a four-year-old boy with a persistent and severe eczema, anodontia, a sparse, thin and blonde hair, periorbital wrinkling with hyperpigmentation and absence of sweating. Laboratory results showed elevation of total serum IgE and IgE specific to house dust mites and grass pollen. Skin biopsy revealed absence of eccrine and sebaceous glands and hair follicles. The genetic molecular study disclosed a c.463C>T (p.Arg155Cys) mutation of the EDA1 gene, consistent with X-linked HED. Although XHED has a favorable prognosis, eczema is a major problem and the eczematous characteristics of patient’s skin resemble those of atopic dermatitis. Ceramide profile, reduction of natural moisturizing factors due to hypohidrosis and skin barrier dysfunction elicited by airborne proteins likely contribute to persistent and difficult-to-control AD-like eczema. As a consequence of the rarity of the disease, obtaining a significant number of clinical studies to clarify and validate the pathways involved in skin barrier dysfunction and the consequent eczematous lesions in HED patients will probably remain a challenge.

Sphingosine-1-Phosphate Receptor 5 Modulates Early-Stage Processes during Fibrogenesis in a Mouse Model of Systemic Sclerosis: A Pilot Study.

Systemic sclerosis (SSc) is a rare multi-organ autoimmune disease characterized by progressive skin fibrosis. Inflammation, type 2 immunity, and fibrogenic processes are involved in disease development and may be affected by sphingolipids. However, details about early-stage pathophysiological mechanisms and implicated mediators remain elusive. The sphingolipid sphingosine-1-phosphate (S1P) is elevated in the sera of SSc patients, and its receptor S1P5 is expressed in skin tissue. Nevertheless, almost nothing is known about the dermatological contribution of S1P5 to inflammatory and pro-fibrotic processes leading to the pathological changes seen in SSc. In this study, we observed a novel effect of S1P5 on the inflammatory processes during low-dose bleomycin (BLM)-induced fibrogenesis in murine skin. By comparing 2-week-treated skin areas of wild-type (WT) and S1P5-deficient mice, we found that S1P5 is important for the transcriptional upregulation of the Th2 characteristic transcription factor GATA-3 under treatment-induced inflammatory conditions, while T-bet (Th1) and FoxP3 (Treg) mRNA expression was regulated independently of S1P5. Additionally, treatment caused a regulation of S1P receptor 1 and S1P receptor 3 mRNA as well as a regulation of long-chain ceramide profiles, which both differ significantly between the genotypes. Despite S1P5-dependent differences regarding inflammatory processes, similar macroscopic evidence of fibrosis was detected in the skin histology of WT and S1P5-deficient mice after 4 weeks of subcutaneous BLM treatment. However, at the earlier 2-week point in time, the mRNA data of pro-collagen type 1 and SMAD7 indicate a pro-fibrotic S1P5 contribution in the applied SSc mouse model. In conclusion, we propose that S1P5 plays a role as a novel modulator during the early phase of BLM-caused fibrogenesis in murine skin. An immediate relationship between dermal S1P5 expression and fibrotic processes leading to skin alterations, such as formative for SSc pathogenesis, is indicated but should be studied more profound in further investigations. Therefore, this study is an initial step in understanding the role of S1P5-mediated effects during early stages of fibrogenesis, which may encourage the ongoing search for new therapeutic options for SSc patients.

Technical note: Characterization of ceramide in bovine lipoproteins

The hepatic synthesis and export of ceramide is enhanced in diabetic monogastrics. Moreover, ceramide in lipoproteins can mediate the development of insulin resistance. We have previously demonstrated that circulating ceramide increases during the progression of insulin resistance in postpartum dairy cows. Considering that the origins of circulating ceramide required investigation, our objective was to develop a method to characterize the ceramide profile of lipoprotein fractions collected from dairy cows. Serum was collected from 4 nonpregnant and nonlactating Holstein dairy cows. Serum lipoproteins were isolated using size exclusion chromatography by fast protein liquid chromatography (SEC-FPLC). Measurement of triacylglycerol (TAG), phospholipid, total cholesterol, and protein was performed using standard colorimetry practices. Following lipid extraction, fractions were analyzed using electrospray ionization tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model. Lipoprotein isolation using SEC-FPLC and subsequent colorimetric analyses confirmed the presence of 4 distinct fractions: TAG-rich, low density (LDL), and large (buoyant) and small (dense) high density lipoprotein (HDL) subclasses. As expected, the fraction representing mixed very low density lipoproteins and chylomicrons primarily contained TAG. Low density lipoprotein fractions were equally enriched with cholesterol and phospholipid. Buoyant HDL contained elevated levels of cholesterol, phospholipid, and protein. In contrast, the fraction containing dense HDL primarily contained protein. Our method revealed that LDL are enriched with ceramides. Ceramides were also compartmentalized to a lesser extent within both HDL subclasses and TAG-rich lipoproteins. Comparable to whole serum, C16:0-ceramide was the predominant ceramide quantified in all lipoprotein subclasses. Interestingly, the proportion of C24:0-ceramide to total ceramide was elevated in TAG-rich lipoproteins, relative to all other lipoprotein subclasses. We conclude that SEC-FPLC coupled with mass spectrometry is a means to quantify ceramides in lipoprotein fractions. Moreover, ceramides are enriched within bovine LDL, and lipoprotein ceramide profiles reflect levels observed in whole serum. Future investigation will need to determine the biological importance of lipoprotein ceramides with distinct C-chains at amide residues.

Sphingolipids in marine microalgae: Development and application of a mass spectrometric method for global structural characterization of ceramides and glycosphingolipids in three major phyla

Sphingolipid compositions are crucial for the structural and physiological properties of microalgae membranes. In the present study, we developed a quadrupole time-of-flight (Q-TOF) mass spectrometric method based on MSE data collection for the identification of sphingolipids with high efficiency, selectivity, sensitivity and mass accuracy and applied this method for precise structural identification and quantitative profiling of ceramides and glycosphingolipids in total lipid extracts from 17 strains of microalgae, including 11 strains of diatom, 3 strains of dinoflagellate and 3 strains of haptophyta. Using this method, four species of sphingolipids including 27 ceramides, 13 monosaccharide ceramides, 18 disaccharide ceramides and 18 trisaccharide ceramides were identified. The compositions of sphingolipid-included glycosyl moieties, long chain bases and N-acyl chains showed a significant difference among different microalgae categories. Some long chain bases including d19:2, d19:3 and d19:4, glycosyl moieties including disaccharide and trisaccharide, and N-acyl chains such as 14:0, 14:1, 24:0, 24:1, h18:1, h19:1 and h22:0–2 can be chosen as the molecular signature for microalgae from three major phyla. This methodology will be useful for a wide range of physiological and pathological studies of sphingolipids. Furthermore, the diversity of sphingolipid structure could provide a new criterion for microalgae chemotaxonomy.

Lipid functions in skin: Differential effects of n-3 polyunsaturated fatty acids on cutaneous ceramides, in a human skin organ culture model

Ceramides are important for skin health, with a multitude of species found in both dermis and epidermis. The epidermis contains linoleic acid-Ester-linked Omega-hydroxylated ceramides of 6-Hydroxy-sphingosine, Sphingosine and Phytosphingosine bases (CER[EOH], CER[EOS] and CER[EOP], respectively), that are crucial for the formation of the epidermal barrier, conferring protection from environmental factors and preventing trans-epidermal water loss. Furthermore, a large number of ceramides, derivatives of the same sphingoid bases and various fatty acids, are produced by dermal and epidermal cells and perform signalling roles in cell functions ranging from differentiation to apoptosis.Supplementation with the n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown promise as therapeutic agents in a number of inflammatory skin conditions, altering the lipid profile of the skin and production of bioactive lipids such as the eicosanoids, docosanoids and endocannabinoids. In this study we wished to investigate whether EPA and DHA could also affect the ceramide profile in epidermis and dermis, and, in this way, contribute to formation of a robust lipid barrier and ceramide-mediated regulation of skin functions.Ex vivo skin explants were cultured for 6days, and supplemented with EPA or DHA (50μM). Liquid chromatography coupled to tandem mass spectrometry with electrospray ionisation was used to assess the prevalence of 321 individual ceramide species, and a number of sphingoid bases, phosphorylated sphingoid bases, and phosphorylated ceramides, within the dermis and epidermis.EPA augmented dermal production of members of the ceramide families containing Non-hydroxy fatty acids and Sphingosine or Dihydrosphingosine bases (CER[NS] and CER[NDS], respectively), while epidermal CER[EOH], CER[EOS] and CER[EOP] ceramides were not affected. DHA did not significantly affect ceramide production. Ceramide-1-phosphate levels in the epidermis, but not the dermis, increased in response to EPA, but not DHA.This ex vivo study shows that dietary supplementation with EPA has the potential to alter the ceramide profile of the skin, and this may contribute to its anti-inflammatory profile. This has implications for formation of the epidermal lipid barrier, and signalling pathways within the skin mediated by ceramides and other sphingolipid species. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Pleiotropic Effect of Human ApoE4 on Cerebral Ceramide and Saturated Fatty Acid Levels.

Apolipoprotein E (ApoE) is known for its role in lipid trafficking and the ɛ4 allele is a risk factor for late onset Alzheimer's disease (AD). Recently, aberrant ceramide and fatty acid (FA) levels have been implicated in AD. To determine the specific effects of human ApoE4 (hE4) on cerebral ceramide and FA content during chow or a high fat/high cholesterol (HFHC) diet. Cerebral ceramide and FA profiles were determined by LC-MSMS in 15-month-old female wild-type (WT), ApoE-knockout (E0), and hE4-knockin mice fed chow or a HFHC diet for 3 months. mRNA levels of genes involved in ceramide and FA metabolism were determined by qPCR. Similar to E0, hE4 mice displayed lower cerebral total ceramide, Cer16 : 0, and Cer24 : 1 levels than WT mice on both diets. Akin to WT mice, hE4 mice had lower total and saturated FA levels on chow than E0 mice. The HFHC diet significantly increased total and saturated FA levels in hE4 mice. Chow-fed hE4 mice showed lower mRNA levels of ceramide synthase (CerS) 6, acid sphingomyelinase, and of most ceramide and FA transporters than WT and E0 mice. The HFHC diet downregulated the expression of CerSs in hE4 and WT mice, and of ceramide and FA transporters in WT mice, but not in E0 mice. hE4 reduced cerebral ceramide levels to levels observed in E0 mice independent of diet. The HFHC diet increased cerebral FA levels in hE4 mice. This was associated with alterations in the expression of ceramide and FA transporters specifically in hE4 mice.

Acid Ceramidase Deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy

Acid Ceramidase Deficiency (Farber disease, FD) is an ultra-rare Lysosomal Storage Disorder that is poorly understood and often misdiagnosed as Juvenile Idiopathic Arthritis (JIA). Hallmarks of FD are accumulation of ceramides, widespread macrophage infiltration, splenomegaly, and lymphocytosis. The cytokines involved in this abnormal hematopoietic state are unknown. There are dozens of ceramide species and derivatives, but the specific ones that accumulate in FD have not been investigated. We used a multiplex assay to analyze cytokines and mass spectrometry to analyze ceramides in plasma from patients and mice with FD, controls, Farber patients treated by hematopoietic stem cell transplantation (HSCT), JIA patients, and patients with Gaucher disease. KC, MIP-1α, and MCP-1 were sequentially upregulated in plasma from FD mice. MCP-1, IL-10, IL-6, IL-12, and VEGF levels were elevated in plasma from Farber patients but not in control or JIA patients. C16-Ceramide (C16-Cer) and dhC16-Cer were upregulated in plasma from FD mice. a-OH-C18-Cer, dhC12-Cer, dhC24:1-Cer, and C22:1-Cer-1P accumulated in plasma from patients with FD. Most cytokines and only a-OH-C18-Cer returned to baseline levels in HSCT-treated Farber patients. Sphingosines were not altered. Chitotriosidase activity was also relatively low. A unique cytokine and ceramide profile was seen in the plasma of Farber patients that was not observed in plasma from HSCT-treated Farber patients, JIA patients, or Gaucher patients. The cytokine profile can potentially be used to prevent misdiagnosis of Farber as JIA and to monitor the response to treatment. Further understanding of why these signaling molecules and lipids are elevated can lead to better understanding of the etiology and pathophysiology of FD and inform development of future treatments.

Profile and quantification of human stratum corneum ceramides by normal-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: development of targeted lipidomic method and application to human stratum corneum of different age groups.

Skin, the largest organ of the human body, serves as the primary barrier to the external environment. Ceramides are one of the main constituents of stratum corneum (SC), playing an important role in skin barrier function. Therefore, comprehensive profiling and quantification of SC ceramide is important. Herein, a new targeted lipidomic method for human SC ceramide profiling and quantification is presented and tested. Normal-phase high-performance liquid chromatography coupled with dynamic multiple reaction monitoring mass spectrometry (NP-HPLC-dMRM-MS) was used to separate ceramides into subclasses and then characterize different ceramides within each subclass on the basis of their characteristics. In total, 483 ceramides were quantified in a single run within 20min, covering 12 subclasses as well as some glycosylated ceramides not previously reported. Each subclass had typical standard substances (if available) that served to establish representative standard curves and were used for related substances with no standards. Linearity range, limit of quantification (LOQ), limit of detection (LOD), precision, accuracy, stability, and matrix effects were validated. dMRM increased sensitivity and accuracy greatly compared with common MRM (cMRM). This method was successfully applied to the study of human SC from different age groups. A total of 193 potential biomarkers were found to indicate age differences between children and adults. This method is an innovative approach for high-throughput quantification of SC ceramide. Graphical Abstract Method establishment (MRM spectra by the established method) and method application (score scatter plots of authentic samples).

The roughness of lip skin is related to the ceramide profile in the stratum corneum.

The structure of skin on the lips is different from skin at other areas of the body; the water content of the stratum corneum (SC) and the barrier function there is low. Therefore, the lips can easily become dry and rough. The SC plays an important role in the barrier and water-holding functions of the skin. Above all, ceramides (CERs) are important SC lipids which maintain SC functions. The purpose of this study was to clarify the relationship between the degree of lip roughness and the CER profile. Forty-one Japanese female subjects with normal skin (age range 22-52 years; mean 34.1) were enrolled in this study. The degree of lip roughness was scored, the values of capacitance and transepidermal water loss (TEWL) were measured and CER profiles of the SC were analysed using tape-stripping. The degree of lip roughness was related to the capacitance values but not the TEWL values. The roughness level correlated with the levels of CER[NH], CER[NP], CER[AH], CER[EOS] and CER[EOH] and the average carbon numbers of CER[NDS], CER[NH] and CER[NP] but not with the level of total CERs. The capacitance values of the lips were related not only with the levels of total CERs but also with the levels of CER[NH], CER[NP], CER[AH], CER[AP], CER[EOS], CER[EOH] and CER[EOP] and with the average carbon numbers of CER[NDS], CER[NS], CER[NH] and CER[NP]. The TEWL values of the lips were related not only with the levels of total CERs but also with the levels of CER[NH], CER[AH], CER[AP], CER[EOS] and CER[EOH]. The relationship between lip roughness and the CER profile was clarified for the first time. The results suggest that not only the level of total CERs but also the specific CER species and their carbon numbers affect the maintenance of SC function.

Metabolic profiling and quantification of ceramide by liquid chromatography mass spectrometry

ObjectiveCeramides are putative mediators of insulin resistance and lipotoxicity; an up‐regulation of ceramide biosynthesis has been well described in rodent models of metabolic disease. A precise, robust and specific LC/MS method was developed to quantify ceramide profiles and support determinations of ceramide flux. The LC/MS method was used to determine the effects of diet‐induced obesity (DIO) in C57B1/6J mice ±treatment with myriocin (1mg/kg, over 7 days), an inhibitor of ceramide synthesis.MethodPlasma and tissue samples were collected from mice (lean, high‐fat fed ± myriocin) at various times following administration of stable isotope tracers. The ceramides were extracted with organic solvents and analyzed using electrospray ionization tandem mass spectrometry; concentrations were determined by normalization to an internal standard (d31‐c16:0 ceramide).ResultsIn contrast to lean mice, DIO‐mice demonstrated an increased concentration and synthesis of circulating ceramides. Myriocin treatment inhibited ceramide synthesis and led to a normalization of circulating levels.ConclusionLC/MS analyses can enable characterization of circulating concentrations of ceramide subspecies and enable stable isotope determinations of in vivo kinetics.Support or Funding InformationAll authors are employed by Merck.

Protective effects of fish oil on pre-diabetes: a lipidomic analysis of liver ceramides in rats.

A high intake of fat and sucrose can dramatically increase bioactive lipids such as ceramides in tissues. Ceramides regulate several steps in the insulin signal pathway. The effects of n-3 PUFA on insulin resistance are inconsistent, especially in liver. We investigated the effect of n-3 PUFA (EPA/DHA 1 : 1) from fish oil on hepatic ceramides in a pre-diabetic animal model. Three groups of rats were fed standard feed, high fat high sucrose feed (HFHS) or HFHS enriched with n-3 PUFA. We investigated by lipidomic analysis how supplementation of a HFHS diet with n-3 PUFA modifies the hepatic ceramide profile triggered by a HFHS diet. Our results show that n-3 PUFA modified the ceramide profile of the liver and reduced their total content in pre-diabetic rats. Significant linear correlations were observed between ceramides and biochemical insulin parameters. Long chain ceramide 18:1/18:0 showed a positive correlation with the HOMA index. Very long chain ceramide 18:1/24:0 showed a negative correlation with insulin and the HOMA index. Finally, very long chain ceramide 18:1/20:0 correlated negatively with glucose levels, plasmatic insulin levels and the HOMA index. In conclusion, the modulation of the ceramide profile in pre-diabetic rats may explain the protective action of n-3 PUFA against liver insulin resistance (IR) caused by an HFHS diet. We confirm the protective role of very long chain ceramide 18:1/24:0 and the harmful role of long chain ceramide 18:1/18:0 in the pre-diabetic state and propose ceramide 18:1/20:0 as a biomarker of early liver IR in rats.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Myristic acid potentiates palmitic acid-induced lipotoxicity and steatohepatitis associated with lipodystrophy by sustaning de novo ceramide synthesis

Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy.

Chronic Voluntary Ethanol Consumption Induces Favorable Ceramide Profiles in Selectively Bred Alcohol-Preferring (P) Rats.

Heavy alcohol consumption has detrimental neurologic effects, inducing widespread neuronal loss in both fetuses and adults. One proposed mechanism of ethanol-induced cell loss with sufficient exposure is an elevation in concentrations of bioactive lipids that mediate apoptosis, including the membrane sphingolipid metabolites ceramide and sphingosine. While these naturally-occurring lipids serve as important modulators of normal neuronal development, elevated levels resulting from various extracellular insults have been implicated in pathological apoptosis of neurons and oligodendrocytes in several neuroinflammatory and neurodegenerative disorders. Prior work has shown that acute administration of ethanol to developing mice increases levels of ceramide in multiple brain regions, hypothesized to be a mediator of fetal alcohol-induced neuronal loss. Elevated ceramide levels have also been implicated in ethanol-mediated neurodegeneration in adult animals and humans. Here, we determined the effect of chronic voluntary ethanol consumption on lipid profiles in brain and peripheral tissues from adult alcohol-preferring (P) rats to further examine alterations in lipid composition as a potential contributor to ethanol-induced cellular damage. P rats were exposed for 13 weeks to a 20% ethanol intermittent-access drinking paradigm (45 ethanol sessions total) or were given access only to water (control). Following the final session, tissues were collected for subsequent chromatographic analysis of lipid content and enzymatic gene expression. Contrary to expectations, ethanol-exposed rats displayed substantial reductions in concentrations of ceramides in forebrain and heart relative to non-exposed controls, and modest but significant decreases in liver cholesterol. qRT-PCR analysis showed a reduction in the expression of sphingolipid delta(4)-desaturase (Degs2), an enzyme involved in de novo ceramide synthesis. These findings indicate that ethanol intake levels achieved by alcohol-preferring P rats as a result of chronic voluntary exposure may have favorable vs. detrimental effects on lipid profiles in this genetic line, consistent with data supporting beneficial cardioprotective and neuroprotective effects of moderate ethanol consumption.