Ceramide Profile Research Articles

ARTICLE: Alteration to the Skin Barrier Integrity Following Broad-Spectrum UV Exposure in an Ex Vivo Tissue Model.

Dynamic changes to the skin barrier’s molecular structure and ceramide profile are well-documented in skin conditions such as atopic dermatitis and psoriasis. Pathological and environmental factors have been shown to impair barrier integrity and demonstrate shifts in ceramide composition in the skin. However, the relationship between acute and prolonged sun exposure and its effects on skin barrier homeostasis is insufficiently investigated. This study aims to uncover new scientific evidence to elucidate the relationship of UV irradiation with the skin barrier using an ex vivo tissue model following simulated UVA/UVB exposure. Fresh ex vivo human skin pretreated either with or without a broad-spectrum sunscreen was exposed to either a physiological or elevated UV condition. Following eight days in culture, structural and molecular changes were evaluated. UV irradiated skin displayed epidermal cell death and altered expression of key barrier proteins. TEM analysis demonstrated disruption to adherens junctions and dissociation between tissue layers following both physiological and extensive UV exposures. An effective broad-spectrum sunscreen containing essential skin ceramides completely protected the skin from such changes. This is one of the first works demonstrating a clear correlation of altered skin barrier integrity using a physiologically relevant dose in an ex vivo tissue model. Our findings also further support the additional importance and benefits of sun protection among the consumers. J Drugs Dermatol. 20(4 Suppl):s23-28. doi:10.36849/JDD.S589D.

The efficacy of synthetic pseudo-ceramide for dry and rough lips.

OBJECTIVELips can easily become dry and rough, one reason being the characteristics of their ceramide (CER) profile. Lips have lower levels of total ceramides, higher percentages of CER[NS] and CER[AS], and lower percentages of CER[NP] and CER[NH] than skin in other regions of the body. The purpose of this study was to clarify the effects of synthetic pseudo‐ceramide (pCer; Cetyl‐PG hydroxyethyl palmitamide) to improve the dryness and roughness of the lips of healthy subjects in a formulation that exclude an occlusive effect.METHODSThirty‐one Japanese female subjects with normal skin (age range 21–37 years; mean 28.6) were enrolled in this study. A four‐week continuous use test was conducted using samples with or without 0.5% or 2.0% pCer. The degree of lip roughness was scored, and values of capacitance, transepidermal water loss (TEWL) and lip surface elasticity were measured. Endogenous CER profiles and absorption levels of pCer in the stratum corneum (SC) were analysed in tape‐stripped skin specimens.RESULTSTreatment with the pCer‐2.0% sample significantly improved the visual roughness score after 2 and 4 weeks compared to 0 weeks and compared to the Blank. Moreover, an improvement in TEWL was observed after 4 weeks of treatment with the pCer‐2.0% sample. CER[NP] showed a significant increase in pCer‐2.0% treated lips after 4 weeks compared to the Blank. Both pCer‐0.5% and pCer‐2.0% were significantly absorbed after 2 weeks compared with the Blank.CONCLUSIONThe effect of the synthetic pseudo‐ceramide pCer to improve the roughness of lips was shown excluding the effect of occlusiveness derived from the formulation for the first time. Since the improvement of TEWL and absorption of pCer was observed, we concluded that pCer was first absorbed in rough lip areas, improved the ceramide profile and consequently restored the barrier function.

ARTICLE: Alteration to the Skin Barrier Integrity Following Broad-Spectrum UV Exposure in an Ex Vivo Tissue Model

Dynamic changes to the skin barrier’s molecular structure and ceramide profile are well-documented in skin conditions such as atopic dermatitis and psoriasis. Pathological and environmental factors have been shown to impair barrier integrity and demonstrate shifts in ceramide composition in the skin. However, the relationship between acute and prolonged sun exposure and its effects on skin barrier homeostasis is insufficiently investigated. This study aims to uncover new scientific evidence to elucidate the relationship of UV irradiation with the skin barrier using an ex vivo tissue model following simulated UVA/UVB exposure. Fresh ex vivo human skin pretreated either with or without a broad-spectrum sunscreen was exposed to either a physiological or elevated UV condition. Following eight days in culture, structural and molecular changes were evaluated. UV irradiated skin displayed epidermal cell death and altered expression of key barrier proteins. TEM analysis demonstrated disruption to adherens junctions and dissociation between tissue layers following both physiological and extensive UV exposures. An effective broad-spectrum sunscreen containing essential skin ceramides completely protected the skin from such changes. This is one of the first works demonstrating a clear correlation of altered skin barrier integrity using a physiologically relevant dose in an ex vivo tissue model. Our findings also further support the additional importance and benefits of sun protection among the consumers. J Drugs Dermatol. 20(4 Suppl):s23-28. doi:10.36849/JDD.S589D

Cutting Edge of the Pathogenesis of Atopic Dermatitis: Sphingomyelin Deacylase, the Enzyme Involved in Its Ceramide Deficiency, Plays a Pivotal Role.

Atopic dermatitis (AD) is characterized clinically by severe dry skin and functionally by both a cutaneous barrier disruption and an impaired water-holding capacity in the stratum corneum (SC) even in the nonlesional skin. The combination of the disrupted barrier and water-holding functions in nonlesional skin is closely linked to the disease severity of AD, which suggests that the barrier abnormality as well as the water deficiency are elicited as a result of the induced dermatitis and subsequently trigger the recurrence of dermatitis. These functional abnormalities of the SC are mainly attributable to significantly decreased levels of total ceramides and the altered ceramide profile in the SC. Clinical studies using a synthetic pseudo-ceramide (pCer) that can function as a natural ceramide have indicated the superior clinical efficacy of pCer and, more importantly, have shown that the ceramide deficiency rather than changes in the ceramide profile in the SC of AD patients plays a central role in the pathogenesis of AD. Clinical studies of infants with AD have shown that the barrier disruption due to the ceramide deficiency is not inherent and is essentially dependent on postinflammatory events in those infants. Consistently, the recovery of trans-epidermal water loss after tape-stripping occurs at a significantly slower rate only at 1 day post-tape-stripping in AD skin compared with healthy control (HC) skin. This resembles the recovery pattern observed in Niemann–Pick disease, which is caused by an acid sphingomyelinase (aSMase) deficiency. Further, comparison of ceramide levels in the SC between before and after tape-stripping revealed that whereas ceramide levels in HC skin are significantly upregulated at 4 days post-tape-stripping, their ceramide levels remain substantially unchanged at 4 days post-tape-stripping. Taken together, the sum of these findings strongly suggests that an impaired homeostasis of a ceramide-generating process may be associated with these abnormalities. We have discovered a novel enzyme, sphingomyelin (SM) deacylase, which cleaves the N-acyl linkage of SM and glucosylceramide (GCer). The activity of SM deacylase is significantly increased in AD lesional epidermis as well as in the involved and uninvolved SC of AD skin, but not in the skin of patients with contact dermatitis or chronic eczema, compared with HC skin. SM deacylase competes with aSMase and β-glucocerebrosidase (BGCase) to hydrolyze their common substrates, SM and GCer, to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine (GSP), respectively, instead of ceramide. Consistently, those reaction products (SPC and GSP) accumulate to a greater extent in the involved and uninvolved SC of AD skin compared with chronic eczema or contact dermatitis skin as well as HC skin. Successive chromatographies were used to purify SM deacylase to homogeneity with a single band of ≈43 kDa and with an enrichment of >14,000-fold. Analysis of a protein spot with SM deacylase activity separated by 2D-SDS-PAGE using MALDI-TOF MS/MS allowed its amino acid sequence to be determined and to identify it as the β-subunit of acid ceramidase (aCDase), an enzyme consisting of α- and β-subunits linked by amino-bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ≈56 and ≈13 kDa and the β-subunit at ≈43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ≈43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with an apparent size of ≈40 kDa upon gel chromatography in contrast to aCDase activity with an apparent size of ≈50 kDa in untreated recombinant human aCDase. These results provide new insights into the essential role of SM deacylase as the β-subunit aCDase that causes the ceramide deficiency in AD skin.

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations.

The stratum corneum (SC) of the epidermis acts as a skin permeability barrier, and abnormalities in SC formation lead to several skin disorders. Lipids, especially the epidermis-specific ceramide classes ω-O-acylceramides (acylceramides) and protein-bound ceramides, are essential for skin barrier formation. Ceramide synthase 3 (CERS3) is involved in the synthesis of acylceramides and protein-bound ceramides, and CERS3 mutations cause autosomal recessive congenital ichthyosis. In the present study, we measured ceramide synthase activity and performed comprehensive SC ceramide profiling in an ichthyosis patient with compound heterozygous CERS3 mutations: nonsense mutation p.Arg75* and missense mutation p.Arg229His. The activity of p.Arg75* and p.Arg229His mutant CERS3 proteins was reduced to 4% and 56%, respectively, of the wild-type protein. In the patient's SC, acylceramide levels were greatly reduced, but the levels of protein-bound ceramides remained almost unchanged. Non-acylated ceramide levels were also affected in the patient; in particular, the levels of ceramides composed of sphingosine and non-hydroxy or α-hydroxy fatty acid were substantially higher than in healthy controls. These results suggest that a reduction in acylceramide levels alone leads to ichthyosis. Although protein-bound ceramides are synthesized from acylceramides, levels of acylceramides and protein-bound ceramides are not necessarily correlated.

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

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

Ceramide ratios are affected by cigarette smoke but not heat-not-burn or e-vapor aerosols across four independent mouse studies

AimsSmoking is an important risk factor for the development of chronic obstructive pulmonary disease and cardiovascular diseases. This study aimed to further elucidate the role of ceramides, as a key lipid class dysregulated in disease states. Main methodsIn this article we developed and validated LC–MS/MS method for ceramides (Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1(15Z)) for the absolute quantification. We deployed it together with proteomics and transcriptomic analysis to assess the effects of cigarette smoke (CS) from the reference cigarette as well as aerosols from heat-not-burn (HnB) tobacco and e-vapor products in apolipoprotein E-deficient (ApoE−/−) mice over several time points. Key findingsIn the lungs, CS exposure substantially elevated the ratios of Cer(d18:1/24:0) and Cer(d18:1/24:1) to Cer(d18:1/18:0) in two independent ApoE−/− mouse inhalation studies. Data from previous studies, in both ApoE−/− and wild-type mice, further confirmed the reproducibility of this finding. Elevation of these ceramide ratios was also observed in plasma/serum, the liver, and—for the Cer(d18:1/24:1(15Z)) to Cer(d18:1/18:0) ratio—the abdominal aorta. Also, the levels of acid ceramidase (Asah1) and glucocerebrosidase (Gba)—lysosomal enzymes involved in the hydrolysis of glucosylceramides—were consistently elevated in the lungs after CS exposure. In contrast, exposure to HnB tobacco product and e-vapor aerosols did not induce significant changes in the ceramide profiles or associated enzymes. SignificanceOur work in mice contributes to the accumulating evidence on the importance of ceramide ratios as biologically relevant markers for respiratory disorders, adding to their already demonstrated role in cardiovascular disease risk assessment in humans.

Serum phospholipidomics reveals altered lipid profile and promising biomarkers in multiple sclerosis

Multiple sclerosis is a neurodegenerative disease causing disability in young adults. Alterations in metabolism and lipid profile have been associated with this disease. Several studies have reported changes in the metabolism of arachidonic acid and the profile of fatty acids, ceramides, phospholipids and lipid peroxidation products. Nevertheless, the understanding of the modulation of circulating lipids at the molecular level in multiple sclerosis remains unclear. In the present study, we sought to assess the existence of a distinctive lipid signature of multiple sclerosis using an untargeted lipidomics approach. It also aimed to assess the differences in lipid profile between disease status (relapse and remission). For this, we used hydrophilic interaction liquid chromatography coupled with mass spectrometry for phospholipidomic profiling of serum samples from patients with multiple sclerosis. Our results demonstrated that multiple sclerosis has a phospholipidomic signature different from that of healthy controls, especially the PE, PC, LPE, ether-linked PE and ether-linked PC species. Plasmalogen PC and PE species, which are natural endogenous antioxidants, as well as PC and PE polyunsaturated fatty acid esterified species showed significantly lower levels in patients with multiple sclerosis and patients in both remission and relapse of multiple sclerosis. Our results show for the first time that the serum phospholipidome of multiple sclerosis is significantly different from that of healthy controls and that few phospholipids, with the lowest p-value, such as PC(34:3), PC(36:6), PE(40:10) and PC(38:1) may be suitable as biomarkers for clinical applications in multiple sclerosis.

Stratum corneum lipidomics analysis reveals altered ceramide profile in atopic dermatitis patients across body sites with correlated changes in skin microbiome.

Atopic dermatitis (AD) is driven by the interplay between a dysfunctional epidermal barrier and a skewed cutaneous immune dysregulation. As part of the complex skin barrier dysfunction, abnormalities in lipid organization and microbiome composition have been described. We set out to systematically investigate the composition of the stratum corneum lipidome, skin microbiome and skin physiology parameters at three different body sites in patients with AD and healthy volunteers. We analysed tape strips from different body areas obtained from 10 adults with AD and 10 healthy volunteers matched for FLG mutation status for 361 skin lipid species using the Metabolon mass spectrometry platform. 16S rRNA data were available from all probands. Our study showed that the lipid composition differs significantly between body sites and between AD patients and healthy individuals. Ceramide species NS was significantly higher in AD patients compared to healthy volunteers and was also higher in AD patients with a FLG mutation compared to AD patients without a FLG mutation. The correlation analysis of skin lipid alterations with the microbiome showed that Staphylococcus colonization in AD is positively correlated with ceramide subspecies AS, ADS, NS and NDS. This is the first study to reveal site-specific lipid alterations and correlations with the skin microbiome in AD.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio‐reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non–small‐cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR‐101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1‐positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Supplementation with eicosapentaenoic acid and linoleic acid increases the production of epidermal ceramides in in vitro canine keratinocytes

Few studies have investigated the effects of essential fatty acids on the production of epidermal ceramide (CER) in canine keratinocytes. To investigate the effects of eicosapentaenoic acid (EPA) and linoleic acid (LA) supplementation on the production of CERs using an in vitro canine keratinocyte culture system. Canine keratinocyte cells (MSCEK) were incubated with high Ca2+ [1.8mM calcium chloride (CaCl2 )] serum-free medium, supplemented with 3µM EPA and 15µM LA when the cells showed confluency. On Day 8 of application, lipid analysis using high-performance thin layer chromatography and real-time PCR for detecting glucosylceramide synthase and ceramidase were performed. It was revealed that the amounts of CER (EOS) (combination of ω-hydroxy fatty acids and sphingosines), CER [EOP] (combination of ω-hydroxy fatty acids and phytosphingosines) and a mixture of CER [NS] (combination of nonhydroxy fatty acids and sphingosines) and [NDS] (combination of nonhydroxy fatty acids and dihydrosphingosines), as well as total CERs, were significantly increased in cells incubated with EPA and LA compared to those of the vehicle, with increased mRNA expression of glucosylceramide synthase. These findings suggest that EPA and LA can potentially alter the CER profile of the skin and this may contribute to its epidermal barrier function in canine skin.

Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS

Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC/MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from human and mouse SC. Phytosphingosine- and 6-hydroxy sphingosine-type ceramides, which both contain an additional hydroxyl group, were abundant in the human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, phytosph-ingosine- and 6-hydroxy sphingosine-type ceramides were present at ∼1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ∼90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy or ω-hydroxy FA were abundant in mice. The hydroxylated β-carbon in β-hydroxy ceramides was in the (R) configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased β-hydroxy ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the ER, is a substrate for β-hydroxy ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.

Treatment with Synthetic Pseudoceramide Improves Atopic Skin, Switching the Ceramide Profile to a Healthy Skin Phenotype

Little is known about the pathophysiological linkages between altered ceramide profiles in the stratum corneum (SC) of patients with atopic dermatitis and their impaired skin barrier and water-holding functions. We studied those characteristics following topical treatment with a designed synthetic pseudoceramide (pCer) and analyzed that pathophysiological linkage by microanalyzing ceramides using normal phase liquid chromatography-electrospray ionization mass spectrometry. Four weeks of treatment with pCer significantly reduced skin symptoms, accompanied by significant decreases in transepidermal water loss and increases in water content. In the SC ceramide profiles, ceramides containing nonhydroxy fatty acids and 6-hydroxysphingosines (Cer[NH]) and ceramides containing nonhydroxy fatty acids and phytosphingosines (Cer[NP]) increased, whereas ceramides containing nonhydroxy fatty acids and sphingosines (Cer[NS]) and ceramides containing a-hydroxy fatty acids and sphingosines (Cer[AS]) decreased, with larger alkyl chain lengths in Cer[NS], distinctly representing a switch from an atopic dermatitis to a healthy skin phenotype. The level of pCer that penetrated into the SC was significantly correlated with the SC water content but not with transepidermal water loss. The level and the average carbon chain length of Cer[NS] were closely correlated with the pCer level in the SC. These findings indicate that the penetrated pCer contributes to shift the ceramide profile from an atopic dermatitis to a healthy skin phenotype. Taken together, the observed clinical efficacy of treatment with pCer provides a deep insight into the pathogenesis of atopic dermatitis as a ceramide-deficient disease.

Lipidomic Analysis Reveals Specific Differences between Fibroblast and Keratinocyte Ceramide Profile of Patients with Psoriasis Vulgaris.

Ceramides are important lipid metabolites for primal skin functions. There is increasing evidence that alteration of the profile and metabolism of ceramides is associated with skin diseases, such as psoriasis vulgaris. Most studies have reported alteration in ceramide content in the stratum corneum, but these have been scarcely reported for other skin layers. In the present work, we aimed to explore changes in the ceramide profile of fibroblasts and keratinocytes in patients with psoriasis vulgaris and healthy subjects. Using the reversed-phase liquid chromatography-quadrupole-time-of-flight-tandem-mass spectrometry (RPLC-QTOF-MS/MS) platform, we identified ceramide containing non-hydroxy fatty acid ([N]), α-hydroxy fatty acid ([A]), and esterified ω-hydroxy fatty acid ([EO]) and 3 sphingoid bases, dihydrosphingosine ([DS]), sphingosine ([S]), and phytosphingosine ([P]). We found that in the keratinocytes of patients with psoriasis, CER[NS], CER[NP], CER[AS], CER[ADS], CER[AP] and CER[EOS] tended to be expressed at higher relative levels, whereas CER[NDS] tended to be expressed with lower levels than in healthy subjects. In the case of fibroblasts, significant differences were observed, mainly in the three ceramide classes (CER[AS], CER[ADS] and CER[EOS]), which were expressed at significantly higher levels in patients with psoriasis. The most significant alteration in the fibroblasts involved elevated levels of CER[EOS] that contained ester-linked fatty acids. Our findings provide insights into the ceramide profile in the dermis and epidermis of patients with psoriasis and contribute for the research in this field, focusing on the role of keratinocyte-fibroblast crosstalk in the development of psoriasis vulgaris.

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.

Preliminary evidence for reduced adipose tissue inflammation in vegetarians compared with omnivores

BackgroundThere are links between obesity and inflammation that may relate activation of pro-inflammatory pathways by dietary factors. Because dietary fat intake of vegetarians is thought to be more beneficial than that of omnivores, we hypothesized that obese vegetarians would have less adipose tissue inflammation and lower intramyocellular ceramide concentrations than equally obese omnivores.MethodsEight obese vegetarian (1 male) and 8 obese omnivore volunteers (1 male) completed a Food Frequency Questionnaire, underwent body composition measures, subcutaneous adipose tissue and muscle biopsies. We used immunohistochemistry to measure adipose macrophage (ATM) and senescent cells. Plasma free fatty acid (FFA), adipose FA and muscle ceramide profiles were measured using liquid chromatography/mass spectrometry. Student t tests were used for the comparison of primary outcomes; univariate regression analysis was used to test for associations between dietary patterns and ATMs (secondary analysis).ResultsThere were no differences in age (38 ± 8 vs. 39 ± 8 years), BMI (32.2 ± 2.6 vs. 33.3 ± 1.9 kg/m2) or percent body fat (44 ± 8 vs. 45 ± 4) between the vegetarians and omnivores. Vegetarians consumed 42% (P = 0.02) less saturated fat and 50% (P = 0.04) less cholesterol than the omnivores. Plasma FFA of vegetarians had lesser proportions of palmitic acid (24 ± 3 vs. 29 ± 4%, P = 0.02) and vegetarians had fewer femoral pro-inflammatory ATMs than omnivores (3.6 ± 2.8 vs. 7.9 ± 4.4 per 100 adipocytes, respectively; P = 0.02). Omnivores had 50% greater (P = 0.01) expression of TNF mRNA in abdominal fat. We found no significant between group differences in muscle ceramide concentrations.ConclusionsAlthough the sample size is small, these results may indicate that dietary patterns play a role in adipose tissue inflammation, as reflected by reduced number of femoral ATMs in obese vegetarians than obese omnivores.

Effects of abomasal infusions of fatty acids and one-carbon donors on hepatic ceramide and phosphatidylcholine in lactating Holstein dairy cows

Our objectives were to (1) determine whether the abomasal infusion of behenic acid (C22:0) elevated hepatic ceramide relative to palmitic acid (C16:0) or docosahexaenoic acid (C22:6n-3) infusion; (2) assess whether the abomasal infusion of choline chloride or l-serine elevated hepatic phosphatidylcholine (PC) in cows abomasally infused with C16:0; and (3) characterize the PC lipidome in cows abomasally infused with C22:6n-3, relative to C16:0 or C22:0 infusion. In a 5 × 5 Latin square design, 5 rumen-cannulated Holstein cows (214 ± 4.9 DIM; 3.2 ± 1.1 parity) were enrolled in a study with 6-d periods. Abomasal infusates consisted of (1) palmitic acid (PA; 98% C16:0); (2) PA + choline chloride (PA+C; 50 g/d choline chloride); (3) PA + l-serine (PA+S; 170 g/d l-serine); (4) behenic acid (BA; 92% C22:0); and (5) an algal oil rich in docosahexaenoic acid (DHA; 44% C22:6n-3). Emulsion infusates provided 301 g/d of total fatty acids containing a minimum of 40 g/d of C16:0. Cows were fed a corn silage-based diet. Milk was collected on d -2, -1, 5, and 6. Blood was collected and liver biopsied on d 6 of each period. Although we did not detect differences in milk yield, milk fat yield and content were lower in cows infused with DHA relative to PA. Plasma triacylglycerol concentrations were lower with DHA treatment relative to PA or BA. Cows infused with DHA had lower plasma insulin concentrations relative to cows infused with PA only. For objective 1, hepatic ceramide-d18:2/22:0 was highest in cows infused with BA relative to other treatments. For objective 2, plasma free choline concentrations were greater in PA+C cows relative to PA; however, we did not observe this effect with PA+S. Plasma total PC concentrations were similar for all treatments. Regarding the hepatic lipidome, a total of 18 hepatic PC were higher (e.g., PC-16:1/18:2) and 25 PC were lower (e.g., PC-16:0/22:6) with PA+C infusion relative to PA. In addition, 17 PC were higher (e.g., PC-20:3/22:5) and 21 PC were lower (e.g., PC-18:0/22:6) with PA+S infusion relative to PA. For objective 3, hepatic concentrations of many individual saturated PC (e.g., PC-18:0/15:0) were lower with DHA relative to other treatments. Hepatic concentrations of highly unsaturated PC with very-long-chain fatty acids (e.g., PC-14:0/22:6) were higher in DHA-infused cows relative to PA, PA+C, PA+S, or BA. The abomasal infusion of emulsions containing palmitic acid, palmitic acid with choline chloride or serine, behenic acid, or docosahexaenoic acid influence the hepatic ceramide and PC profiles of lactating cows.

Very Long-Chain C24:1 Ceramide Is Increased in Serum Extracellular Vesicles with Aging and Can Induce Senescence in Bone-Derived Mesenchymal Stem Cells.

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

CerS1-Derived C18:0 Ceramide in Skeletal Muscle Promotes Obesity-Induced Insulin Resistance

Skeletal muscle accumulates ceramides in obesity, which contribute to the development of obesity-associated insulin resistance. However, it remained unclear which distinct ceramide species in this organcontributes to instatement of systemic insulinresistance. Here, ceramide profiling of high-fat diet(HFD)-fed animals revealed increased skeletal muscle C18:0 ceramide content, concomitant withincreased expression of ceramide synthase (CerS)1. Mice lacking CerS1, either globally or specifically in skeletal muscle (CerS1ΔSkM), exhibit reduced muscle C18:0 ceramide content and significant improvements in systemic glucose homeostasis. CerS1ΔSkM mice exhibit improved insulin-stimulated suppression of hepatic glucose production, and lack of CerS1 in skeletal muscle improves systemic glucose homeostasis via increased release of Fgf21 from skeletal muscle. In contrast, muscle-specific deficiency of C16:0 ceramide-producing CerS5 and CerS6 failed to protect mice from obesity-induced insulin resistance. Collectively, these results reveal the tissue-specific function of distinct ceramide species during the development of obesity-associated insulin resistance.