Increase In Ceramide Research Articles

Reinforcement of barrier function and scalp homeostasis by Senkyunolide A to fight against dandruff.

Senkyunolide-A (SENKY) can be isolated from Apium graveolens seed oil obtained using supercritical CO2 extraction. SENKY and its parent compounds, the N-butyl phthalides, have been demonstrated to protect cells from CO poisoning, to prevent diabetes mellitus and to decrease cancer cell proliferation. This study was undertaken to evaluate in vitro and in vivo the effect of SENKY on epidermal function improvement, Malassezia effect control, scalp soothing and dandruff reduction via skin protection-related pathways. DNA-array and proteomic studies were performed on human keratinocytes, sebocytes and skin explants to demonstrate SENKY activities. Two clinical evaluations were performed under dermatologist control on 106 volunteers, with greasy or dry scalp, experiencing dandruff, itching and redness. Volunteers tested a shampoo followed, or not, by a leave-on, containing SENKY, or their placebos. Dandruff severity and redness were scored on the scalp. Moisturization and sebum release were recorded using relevant measuring apparatus. Itching and scratching evaluations came from volunteers' self-declarations. DNA-array studies on keratinocytes showed a clear regulation of skin barrier functions and epidermis defence pathways. Upregulation of epidermal differentiation complex genes was observed. These preliminary observations were reinforced by immunocytochemistry and immunohistochemistry studies showing a significant increase of involucrin, filaggrin, loricrin, SPRR, LC3B and ceramide 2 productions. Tight-junctions and corneodesmosomes were significantly reinforced both in keratinocyte cultures (corneodesmosin, claudin, ZO-1) and in skin explants (desmoglein). DNA-array studies also demonstrated upregulation of genes involved in detoxification and anti-inflammation pathways. Proteomic studies revealed that hBD2 production was increased in keratinocytes in contact with SENKY, whereas IL-8, PGE-2 and TLR-9 releases were repressed as well as sebocyte lipid production. Clinical evaluations confirmed that after 3 weeks, SENKY significantly reduced dandruff intensity, redness, itching and scalp histamine content compared to placebo and beginning of treatment. For the first time, SENKY has been shown to promote scalp homoeostasis by reinforcing barrier and defence functions at both gene and protein levels. It reduces irritation and redness in promoting detoxification and anti-inflammation pathways while controlling the niche of Malassezia. Applied on scalp, SENKY significantly reduces the formation of dandruff and soothes the scalp.

Effects of meal ingestion on intramyocellular ceramide concentrations and fractional de novo synthesis in humans.

We investigated the effects of meal ingestion on intramyofibrillar (IMF) and subsarcolemmal (SS) ceramide metabolism in volunteers ranging from lean to obese. Thirty-eight women and men underwent a steady-state meal ingestion protocol that included a 6.5-h infusion of [U-13C]palmitate and muscle biopsies 1.5 and 6.5 h after starting the tracer infusion. We measured IMF and SS sphingolipid concentrations and the contribution of plasma palmitate to intramyocellular C16:0 ceramide by use of LC-MS-MS. In response to meal ingestion SS C24 ceramide concentrations, but not C14-C20 concentrations, increased significantly. IMF ceramide concentrations did not change. The increases in SS C24 ceramides were negatively related to parameters of insulin resistance. The fractional contribution of plasma palmitate to intramyocellular C16:0 ceramides in both IMF and SS fractions was inversely related to overweight status (β = -0.432, P = 0.0095 and β = -0.443, P = 0.0058, respectively). These data indicate that meal ingestion has differing effects on SS ceramide subspecies and suggest that the fractional de novo synthesis of intramyocellular ceramide from plasma palmitate in the postprandial condition is reduced in those who are overweight.

Bioactive sphingolipid mediators promote corneal epithelial homeostasis and wound healing

PurposeTo investigate sphingolipid expression profile in alkali‐burned cornea and subsequently evaluate effects of the selected species on corneal epithelial wound healing.MethodsAll experiments were performed using porcine cornea organ culture or porcine corneal epithelial cells (PCEC). For sphingolipid profiling samples were spiked with internal standards, extracted by Bligh&Dyer method and analyzed using liquid chromatography‐mass spectrometry. PCEC viability/proliferation and migration were assessed using MTT and Boyden chamber assays, respectively. Epithelial wound healing in cornea organ culture was followed by fluorescein staining. Molecular mechanisms underlying the action of selected sphingolipids were studied using iTRAQ‐based quantitative proteomics.ResultsSphingolipid profiling of alkali‐burned corneas revealed early decrease in sphingosine 1‐phosphate and lysosphingomyelin with concomitant increase of ceramide 1‐phosphate. We tested 7 lipids from aforesaid subclasses for their ability to affect proliferation/viability and migration of PCEC. 2 lipids: A, B improved viability/proliferation of alkali‐injured PCEC, induced PCEC chemotaxis as well as promoted re‐epithelialization after alkali burn in cornea organ culture. Another lipid: C potently improved viability/proliferation of healthy PCEC in a wide range of concentrations. In response to A/B/C treatments PCEC displayed differential expression of 114/375/47 proteins, majority with binding and catalytic activity functions. Proteins ↑ upon A/B were involved mainly in proliferation, adhesion and glycolysis, while upon C ‐ in maintenance of corneal epithelial homeostasis.ConclusionsWe identified 2 sphingolipids that facilitate cornea re‐epithelialization by promoting proliferation and migration of corneal epithelial cells and another that promotes healthy corneal epithelial cell phenotype.

Mass Spectrometric Imaging of Ceramide Biomarkers Tracks Therapeutic Response in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a serious public health problem and the leading cause of death in children and young adults. It also contributes to a substantial number of cases of permanent disability. As lipids make up over 50% of the brain mass and play a key role in both membrane structure and cell signaling, their profile is of particular interest. In this study, we show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and reproducibility to demonstrate the anatomical distribution of 50 μm diameter microdomains that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3 days post injury with and without treatment. Adult male Sprague-Dawley rats received one strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides in CCI animals compared to control as well as significant reduction in ceramides in CCI treated animals, demonstrating therapeutic effect of a peptide agonist. The data also suggests the presence of diffuse changes outside of the injured area. These results shed light on the extent of biochemical and structural changes in the brain after traumatic brain injury and could help to evaluate the efficacy of treatments.

Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma.

Abstract 2017: The role of ceramide in the dual cytotoxic and metabolic stimulative effects of sigma-2 receptors

Abstract Sigma-2 receptors are highly overexpressed in cancer cells compared to normal cells, as well as in proliferative versus quiescent cells. Sigma-2 receptors have been of interest as an antineoplastic target, as sigma-2 agonists cause cell death via a variety of pathways. Recently, a new class of sigma-2 agonists has been discovered, with ligands such as CM764, that do not induce cell death but that affect cellular metabolism, as shown by increased MTT reduction, increased ATP level, and HIF-1α stabilization. Previously, our lab has shown that treatment of breast tumor cells with sigma-2 receptor agonists CB-184 and BD737 caused dose dependent increases in ceramide and simultaneous decreases in sphingomyelin, suggesting that sigma-2 receptors modulate the sphingolipid pathway. The ceramide pathway has been shown to play both prosurvival and pro-apoptotic roles in cancer cells. Here, we examine the role of ceramide in sigma-2 receptor mediated pathways in human SK-N-SH neuroblastoma cells. A 24 h treatment of SK-N-SH neuroblastoma cells with 20 μM exogenous C6-ceramide caused less than 20% cell death. Interestingly, this compared to over 50% cell death in MCF-7 breast adenocarcinoma cells and PANC-1 pancreatic cancer cells, indicating a differential role of ceramide in cell death pathways across cell lines. SK-N-SH cells were treated with 10 μM of either CM572, a classic sigma-2 agonist that causes cell death, or CM764, a sigma-2 agonist that stimulates metabolism. Fumonisin B1 is a potent inhibitor of ceramide synthase. Treatment of cells with fumonisin B1 (10 μM) did not significantly alter CM572-induced cytotoxicity. Conversely, treatment with fumonisin B1 did significantly inhibit the MTT reduction caused by CM764. Taken together, these results suggest that ceramide formation may play more of a role in the metabolic stimulative effect of sigma-2 receptors, as opposed to the cytotoxic effect. Decreased levels of ceramide have been shown to increase cellular resistance to apoptosis. Therefore, it has been of interest to increase intracellular ceramide levels in drug resistant cancer cells in order to sensitize the resistant cells to chemotherapeutics. Multidrug resistance (MDR) in cancer cells is often attributed to the overexpression of particular ABC transporters that effectively pump drugs out of the cells. One such protein is the multidrug resistance-associated protein 1 (MRP1). It has been shown that increased expression of drug efflux pumps also decreases cell sensitivity to ceramide. Treatment with CM572 caused a dose-dependent decrease in MRP1 protein levels, while treatment with CM764 had little effect on MRP1. We will examine how this modulation of MRP1 affects sensitivity to C6-ceramide. We will also examine whether combination of C6-ceramide with sigma-2 receptor ligands augments the cytotoxic and/or metabolic stimulative effects of sigma-2 receptors. Citation Format: Cheri Z. Liu, Ellen Sukharevsky, Wayne D. Bowen. The role of ceramide in the dual cytotoxic and metabolic stimulative effects of sigma-2 receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2017. doi:10.1158/1538-7445.AM2017-2017

Tolerogenic versus Immunogenic Lipidomic Profiles of CD11c+ Immune Cells and Control of Immunogenic Dendritic Cell Ceramide Dynamics.

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell-cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70-/- cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation.

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity.

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2+/+ and CerS2-/- mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2-/- mice developed more severe disease than CerS2+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2-/- mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2-/- mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2-/- mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4+ and Th17+ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.

Insulin alters brain lipid profile and mitochondrial function

Alzheimer's disease (AD), the most common neurodegenerative disorder worldwide, is characterized by increased levels of neurofibrillary tangles and amyloid‐beta plaques. Evidence has previously implicated the potent role that insulin resistance plays in the development and progression of AD, suggesting to some that AD represents a distinct type of diabetes. Additionally, disparate lines of evidence find a role for alterations in brain mitochondrial function in AD etiology. We sought to test a unifying hypothesis—namely, that hyperinsulinemia promotes accrual of the sphingolipid ceramide, which, as we've previously found, pathologically alters mitochondrial function. Via daily insulin injections, we induced hyperinsulinemia in ApoE4 mice. In addition to insulin, one group received myriocin injections to inhibit ceramide biosynthesis. We observed significant increases in brain ceramides in the insulin‐treated group, which correlated with disrupted brain mitochondrial function. However, the group receiving myriocin alone, or, importantly, myriocin with insulin had normal lipid profiles and apparently normal mitochondrial bioenergetics. Altogether, our findings suggest a causative role for insulin in AD etiology via an insulin‐induced upregulation of ceramide biosynthesis and accrual in the brain.

Milk Fermented by Lactic Acid Bacteria Enhances the Absorption of Dietary Sphingomyelin in Rats.

Supplementation with sphingomyelin has been reported to prevent disease and maintain good health. However, intact sphingomyelin and ceramides are poorly absorbed compared with glycerolipids. Therefore, if the bioavailability of dietary sphingomyelin can be increased, supplementation would be more effective at lower doses. The aim of this study in rats was to evaluate the effect of fermented milk on the bioavailability of dietary sphingomyelin in rats. After the rats had fasted for 15h, test solutions were administrated orally. Blood samples were collected from the tail vein before and 90, 180, 270, and 360min after administration. Compared with sphingomyelin/milk phospholipids concentrate (MPL) alone, co-ingestion of sphingomyelin/MPL with fermented milk caused an approximate twofold significant increase in serum ceramides containing d16:1 sphingosine with 16:0, 22:0, 23:0 and 24:0 fatty acids, which was derived from the ingested sphingomyelin. While nonfat milk also increased the serum levels of these ceramides, fermented milk was more effective. Co-ingestion of the upper layer of fermented milk or exopolysaccharide concentrate prepared from fermented milk significantly increased serum ceramide levels. X-ray diffraction analysis also showed addition of fermented milk or EPS concentrate to sphingomyelin eliminated the characteristic peak of sphingomyelin. This study demonstrated for the first time that co-ingestion of dietary sphingomyelin and fermented milk, compared with ingestion of dietary sphingomyelin alone, caused a significant increase in the absorption of sphingomyelin. Our results indicate exopolysaccharides in fermented milk may contribute to inhibition of sphingomyelin crystallization, resulting in enhanced absorption of dietary sphingomyelin in rats.

Novel Lipidomic Biomarkers in Hyperlipidemia and Cardiovascular Diseases: An Integrative Biology Analysis.

Lipidomics is a new frontier of omics research and offers much promise for new-generation biomarkers for common complex phenotypes such as hyperlipidemia (HL) and cardiovascular diseases (CVDs). HL is a disorder characterized by increased levels of blood lipids and is a well-established risk factor for CVD. Traditional clinical markers for prognosis of hyperlipidemic individuals are inadequate to forecast or diagnose cardiac events. In this expert review, lipidomic profiles from recent HL and CVD studies were compared with the normolipidemic profile prepared from the Standard Reference Material. Our analysis showed that palmitoyl-lysophosphatidylcholine [LPC(16:0)], the most abundant LPC species in normolipidemic plasma, decreases in HL causative conditions such as high-fat diet, obesity, and diabetes. This is accompanied by increase in free fatty acids (FFAs) and ceramides (Cers). HL was also found to be characterized by increase in small-chain, saturated fatty acid content of diacylglycerols, triacylglycerols, and phosphatidylcholines (PCs). These factors were also associated with increased CVD risk. The decrease in LPC(16:0) in HL and CVD is consistent with its role in regulation of peroxisome proliferator-activated receptor alpha, an approved HL drug target that impacts the uptake and oxidation of fatty acids. FFAs are involved in endothelial-dependent nitric oxide production and activation of nuclear factor κB signaling. Cers control uptake and anabolic catabolism of nutrients in tissues. However, additional studies are required to establish the range of normal and disease levels of the identified lipids in different populations and conditions. In all, these observations underscore that lipidomics deserves greater research attention from the biomarker and precision medicine research communities.

Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Valproate Induces the Unfolded Protein Response by Increasing Ceramide Levels

Bipolar disorder (BD), which is characterized by depression and mania, affects 1-2% of the world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproate (VPA) is one of the most widely used drugs for the treatment of BD, but the therapeutic mechanism of action of this drug is not understood. This knowledge gap has hampered the development of effective treatments. To identify candidate pathways affected by VPA, we performed a genome-wide expression analysis in yeast cells grown in the presence or absence of the drug. VPA caused up-regulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for ceramide synthesis. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. In agreement with increased fatty acid elongase gene expression, VPA increased levels of phytoceramide, especially those containing C24-C26 fatty acids. Consistent with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects were rescued by supplementation of inositol and similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide.

Abstract 2479: Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation

Abstract Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants. Conclusions: ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Julio Garcia, A A. Qudeimat, Jacqueline M. Kraveka. Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2479.

Frontline Science: Sphingosine rescues burn-injured mice from pulmonary Pseudomonas aeruginosa infection.

Burn patients with concomitant pulmonary Pseudomonas aeruginosa (PA) infection have mortality rates as high as 50%, despite antibiotic therapy. Sphingosine is generated from ceramide via ceramidase and has been reported to have antimicrobial properties. We observed a reduction in sphingosine and a concurrent increase in ceramide in bronchial epithelial cells after burn injury. After PA inoculation, these mice had a significant decrease in survival compared to noninjured mice. However, when injured mice were pretreated with sphingosine or neutral ceramidase and subsequently infected, mortality and bacterial levels were robustly reduced. We further observed that sphingosine directly kills PA. Together, these results demonstrate that reduction in sphingosine is associated with an increased susceptibility to pulmonary infection after burn injury. Restoration of sphingosine levels through direct sphingosine administration or conversion of the increased ceramide to sphingosine by neutral ceramidase reduces mortality and mitigates pulmonary infection after burn injury.

Targeting ceramide metabolism in obesity.

Obesity is a major health concern that increases the risk for insulin resistance, type 2 diabetes (T2D), and cardiovascular disease. Thus, an enormous research effort has been invested into understanding how obesity-associated dyslipidemia and obesity-induced alterations in lipid metabolism increase the risk for these diseases. Accordingly, it has been proposed that the accumulation of lipid metabolites in organs such as the liver, skeletal muscle, and heart is critical to these obesity-induced pathologies. Ceramide is one such lipid metabolite that accumulates in tissues in response to obesity, and both pharmacological and genetic strategies that reduce tissue ceramide levels yield salutary actions on overall metabolic health. We will review herein why ceramide accumulates in tissues during obesity and how an increase in intracellular ceramide impacts cellular signaling and function as well as potential mechanisms by which reducing intracellular ceramide levels improves insulin resistance, T2D, atherosclerosis, and heart failure. Because a reduction in skeletal muscle ceramide levels is frequently associated with improvements in insulin sensitivity in humans, the beneficial findings reported for reducing ceramides in preclinical studies may have clinical application in humans. Therefore, modulating ceramide metabolism may be a novel, exciting target for preventing and/or treating obesity-related diseases.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

The ORMDL3-ceramide axis may be a novel therapeutic target for the control of allergic airway disease

Abstract Asthma, defined as a chronic inflammatory condition characterized by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern. The WHO estimates that asthma affects more than 230 million people worldwide. There is a strong genetic component to asthma and numerous genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as an asthma associated gene. Surprisingly however, the mechanism by which ORMDL3 contributes to asthma pathogenesis is not well understood. The yeast ortholog of ORMDL3 is a negative regulator of serine palmitoyltransferase (SPT), the rate limiting step in de novo ceramide synthesis, yet elevations of ceramide rather than its reduction have been linked to lung inflammation. Thus, we examined the role of ORMDL3 in asthma immunopathology. Consistent with its role in yeast, we show that decreasing expression of ORMDL3 in lung epithelial cells and macrophages increases ceramide and conversely, modest increases in ORMDL3 decrease ceramide levels. In a house dust mite (HDM) mouse model of allergic airway disease, allergen challenge induced expression of ORMDL3 and resulted in a concomitant increase in lung ceramide. Intriguingly, the use of specific drugs, which inhibit ceramide synthesis, prevented HDM-induced airway hyperreactivity (AHR) and suppressed airway inflammation. Nasal administration of the orally available FDA approved prodrug FTY720/fingolimod reduced both ORMDL3 expression and ceramide production while mitigating airway inflammation, hyperreactivity, and mucus hypersecretion in HDM challenged mice. Thus the ORMDL3 -ceramide pathway may be a novel therapeutic target for the control of allergic asthma.

Lactosylceramide contributes to mitochondrial dysfunction in diabetes

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.