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We have investigated the role of ceramide in the cellular adaptation to folate stress induced by Aldh1l1, the enzyme involved in the regulation of folate metabolism. Our previous studies demonstrated that Aldh1l1, similar to folate deficiency, evokes metabolic stress and causes apoptosis in cancer cells. Here we report that the expression of Aldh1l1 in A549 or HCT116 cells results in the elevation of C16-ceramide and a transient up-regulation of ceramide synthase 6 (CerS6) mRNA and protein. Pretreatment with ceramide synthesis inhibitors myriocin and fumonisin B1 or siRNA silencing of CerS6 prevented C16-ceramide accumulation and rescued cells supporting the role of CerS6/C16-ceramide as effectors of Aldh1l1-induced apoptosis. The CerS6 activation by Aldh1l1 and increased ceramide generation were p53-dependent; this effect was ablated in p53-null cells. Furthermore, the expression of wild type p53 but not transcriptionally inactive R175H p53 mutant strongly elevated CerS6. Also, this dominant negative mutant prevented accumulation of CerS6 in response to Aldh1l1, indicating that CerS6 is a transcriptional target of p53. In support of this mechanism, bioinformatics analysis revealed the p53 binding site 3 kb downstream of the CerS6 transcription start. Interestingly, ceramide elevation in response to Aldh1l1 was inhibited by silencing of PUMA, a proapoptotic downstream effector of p53 whereas the transient expression of CerS6 elevated PUMA in a p53-dependent manner indicating reciprocal relationships between ceramide and p53/PUMA pathways. Importantly, folate withdrawal also induced CerS6/C16-ceramide elevation accompanied by p53 accumulation. Overall, these novel findings link folate and de novo ceramide pathways in cellular stress response.

The Wickerhamomyces ciferrii strain NRRL Y-1031 F-60-10A is a well-known producer of tetraacetylphytosphingosine (TAPS) and used for the biotechnological production of sphingolipids and ceramides. It was our aim to gain new biological insights into the sphingolipid metabolism by employing a dual platform mass spectrometry strategy. The first step comprised metabolic (15)N-labeling in combination with label-free proteomics using high resolution LTQ Orbitrap mass spectrometry. Then selected reaction monitoring tandem mass spectrometry served for the targeted quantification of sphingoid base biosynthesis enzymes. The non-producer strain NRRL Y-1031-27 served as a reference strain. In total, 1697 proteins were identified, and 123 enzymes of main metabolic pathways were observed as differentially expressed. Major findings were: 1) the likely presence of higher carbon flux in NRRL Y-1031 F-60-10A, resulting in higher availability of pyruvate and acetyl-CoA; 2) indications of oleaginous yeast-like behavior in NRRL Y-1031 F-60-10A; and 3) approx. 2-fold higher abundance of eight sphingolipid biosynthesis enzymes in NRRL Y-1031 F-60-10A. Taken together, in NRRL Y-1031 F-60-10A, the levels of glycolytic enzymes apparently gear carbon flux towards higher acetyl-CoA synthesis rates, while simultaneously reducing protein biosynthesis in the process. Thereby, C2 building blocks for acyl-moieties and downstream sphingoid base acetylation are provided to maintain TAPS production., Biological Significance: First quantitative proteome study for a phytosphingosine-producing yeast., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced "mild" traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5-5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology.

Background: To determine whether ceramide is responsible for the induction of p53-independent early or late apoptosis in response to high- and low-Linear-Energy-Transfer (LET) irradiation., Methods: Four cell lines displaying different radiosensitivities and p53-protein status were irradiated with photons or 33.4 or 184 keV/μm carbon ions. The kinetics of ceramide production was quantified by fluorescent microscopy or High-Performance-Liquid-Chromatogaphy and the sequence of events leading to apoptosis by flow cytometry., Results: Regardless of the p53-status, both low and high-LET irradiation induced an early ceramide production in radiosensitive cells and late in the radioresistant. This production strongly correlated with the level of early apoptosis in radiosensitive cells and delayed apoptosis in the radioresistant ones, regardless of radiation quality, tumor type, radiosensitivity, or p53-status. Inhibition of caspase activity or ceramide production showed that, for both types of radiation, ceramide is essential for the initiation of early apoptosis in radiosensitive cells and late apoptosis following mitotic catastrophe in radioresistant cells., Conclusions: Ceramide is a determining factor in the onset of early and late apoptosis after low and high-LET irradiation and is the mediator of the p53-independent-apoptotic pathway. We propose that ceramide is the molecular bridge between mitotic catastrophe and the commitment phase of delayed apoptosis in response to irradiation.

Ceramide-1-phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an antiapoptotic molecule released from cells that are damaged and "leaky." As reported recently, C1P promotes migration of hematopoietic cells. In this article, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for the first time that C1P is upregulated in damaged tissues and chemoattracts bone marrow (BM)-derived multipotent stromal cells, endothelial progenitor cells, and very small embryonic-like stem cells. Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal cell-derived factor-1 from BM-derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization., (Copyright © 2012 AlphaMed Press.)

Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1β and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia.

Present study shows that drug resistant human breast cancer cells are enriched in cancer stem-like cells (CSCs) and express elevated levels of Stat-3 signaling mediators, which contribute to CSC enrichment. Simvastatin (SVA) and gamma-tocotrienol (γT3) eliminate enriched CSCs and suppress expression of Stat-3 signaling mediators via inhibition of the mevalonate pathway and activation of de novo ceramide synthesis pathway, respectively. Combination of SVA+γT3 at low doses enhanced these actions via inhibition of the mevalonate pathway. Data demonstrate that SVA and γT3 alone or in combination possess the ability to eliminate CSCs in drug resistant human breast cancer cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Ceramides play an essential role in modulating immune signaling pathways and proinflammatory cytokine production in response to infectious pathogens, stress stimuli, or chemotherapeutic drugs. In this study, we demonstrated that Aggregatibacter actinomycetemcomitans, the pathogen for aggressive periodontitis, induced de novo synthesis of ceramide in Raw 264.7 cells. In addition, we identified that fenretinide, a synthetic retinoid, suppressed the de novo synthesis of ceramide induced by A. actinomycetemcomitans. Moreover, fenretinide attenuated interleukin (IL)-1β, IL-6, and cyclooxygenase-2 mRNA expression induced by A. actinomycetemcomitans. Fenretinide also decreased IL-1β, IL-6, and prostaglandin E2 proinflammatory cytokine levels in Raw 264.7 cells induced by A. actinomycetemcomitans. However, fenretinide had no significant effects on tumor necrosis factor alpha mRNA or protein levels. Furthermore, we showed that fenretinide inhibited the janus kinase-signal transducer and activator of transcription, phosphatidylinositol 3-kinase-Akt, protein kinase C, and nuclear factor-kappaB signaling pathways, whereas fenretinide up-regulated the mitogen-activated protein kinase signaling pathways after bacterial stimulation. This study emphasizes the de novo ceramide synthesis pathway in response to bacterial stimulation and demonstrates the anti-inflammatory role of fenretinide in the bacteria-induced immune response.

Cell injury associated with reactive oxygen species (ROS) has been reported in various muscular disorders. We found that a Cichorium intybus (Cii) extract reduced H(2)O(2)-induced viability loss in C2C12 myoblasts, inhibited oxidative stress-induced apoptosis and increased intracellular heat shock protein 70 (Hsp 70) expression. Cii also inhibited the level of intracellular ceramide. These results indicate that Cii may prevent skeletal muscle atrophy by inducing the expression of Hsp 70 and inhibiting the level of ceramide.

Sphingolipid de novo biosynthesis is related with metabolic diseases. However, the mechanism is still not quite clear. Sphingolipids are ubiquitous and critical components of biological membranes. Their biosynthesis starts with soluble precursors in the endoplasmic reticulum and culminates in the Golgi complex and plasma membrane. The interaction of sphingomyelin, cholesterol, and glycosphingolipid drives the formation of plasma membrane rafts. Lipid rafts have been shown to be involved in cell -signaling, lipid and protein sorting, and membrane trafficking. It is well known that toll-like receptors, class A and B scavenger receptors, and insulin receptor are located in lipid rafts. Sphingomyelin is also a reservoir for other sphingolipids. So, sphingomyelin has important impact in cell -signaling through its structural role in lipid rafts or its catabolic inter-mediators, such as ceramide and glycoceramide. In this chapter, we will discuss both aspects.

Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs.

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.

Aims: Chronic alcohol abuse causes steatohepatitis with insulin resistance, which impairs hepatocellular growth, survival and metabolism. However, growing evidence supports the concept that progressive alcohol-related liver injury may be mediated by concurrent mal-signaling through other networks that promote insulin resistance, e.g. pro-inflammatory, pro-ceramide and endoplasmic reticulum (ER) stress cascades., Methods: Using the Long Evans rat model of chronic ethanol feeding, we characterized the histopathologic and ultrastructural features of steatohepatitis in relation to biochemical and molecular indices of tissue injury, inflammation, insulin resistance, dysregulated lipid metabolism and ER stress., Results: Chronic steatohepatitis with early chicken-wire fibrosis was associated with enlargement of mitochondria and disruption of ER structure by electron microscopy, elevated indices of lipid storage, lipid peroxidation and DNA damage, increased activation of pro-inflammatory cytokines, impaired signaling through the insulin receptor (InR), InR substrate-1, Akt, ribosomal protein S6 kinase and proline-rich Akt substrate 40 kDa, glycogen synthase kinase 3β activation and constitutive up-regulation of ceramide and ER stress-related genes. Liquid chromatography coupled with tandem mass spectrometry demonstrated altered ceramide profiles with higher levels of C14 and C18, and reduced C16 species in ethanol-exposed livers., Conclusion: The histopathologic and ultrastructural abnormalities in chronic alcohol-related steatohepatitis are associated with persistent hepatic insulin resistance and pro-inflammatory cytokine activation, dysregulated lipid metabolism with altered ceramide profiles and both ER and oxidative stress. Corresponding increases in lipid peroxidation, DNA damage and protein carbonylation may have contributed to the chronicity and progression of disease. The findings herein suggest that multi-pronged therapeutic strategies may be needed for effective treatment of chronic alcoholic liver disease in humans.

Scope: This study further examines mechanisms involved in the pro-apoptotic action of gamma-tocopherol (γT) and gamma-tocotrienol (γT3) in human breast cancer cell lines., Methods and Results: γT upregulates phospho-JNK (pJNK), CCAAT/enhancer-binding protein homologous protein (CHOP), and death receptor-5 (DR5) protein expression as detected by Western blot assays. siRNA knockdown of JNK, CHOP, or DR5 shows that γT-induced apoptosis is JNK/CHOP/DR5 signaling dependent, which is similar to γT3-mediated apoptotic signaling. Furthermore, both γT and γT3 induce increased levels of cellular ceramides and dihydroceramides as determined by LC-MS/MS analyses. Inhibition of de novo ceramide synthesis using chemical inhibitors blocked the ability of γT and γT3 to induce apoptosis as detected by Annexin V-FITC/PI assay and to activate JNK/CHOP/DR5 pro-apoptotic signaling thereby demonstrating the involvement of de novo ceramide synthesis in γT- and γT3-induced apoptosis., Conclusion: Taken together, data show that both γT and γT3 induce apoptosis via de novo ceramide synthesis dependent activation of JNK/CHOP/DR5 pro-apoptotic signaling., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ceramides (Cer) and glucosylceramides (GlcCer) play an important role in moisturizing the epidermis. Dietary GlcCer has been reported to improve transepidermal water loss (TEWL). However, the effect of GlcCer on epidermal Cer and GlcCer has not been well established. Therefore, we prepared a GlcCer-rich fraction (GCFr) from rice and evaluated its effect on TEWL and epidermal Cer and GlcCer in mice. In addition, we examined the effect of GlcCer (d18:2) contained in GCFr on the changes in Cer and GlcCer in a human epidermal equivalent. Oral dosing of GCFr (3 and 10 mg/[kg·day]) improved TEWL treated with sodium dodecyl sulfate. In the skin, epidermal Cer 1 was increased, and GlcCer (esterified ω-hydroxy fatty acid and sphingosine [EOS]) and a complex mixture of GlcCer (NS), (NP), and (C24,26-AS), known as GlcCer A/B were decreased by the GCFr. These changes were accompanied with the enhancement of glucosylceramide synthase (GCSase) and glucocerebrosidase expression. On the other hand, GlcCer (d18:2) increased Cer 1, Cer 2, GlcCer (EOS), and GlcCer A/B in a human epidermal equivalent accompanied with expression of GCSase and epidermal maturation markers. These results suggest that oral dosing of rice-derived GlcCer can compensate for epidermal loss of Cer by enhancing epidermal GlcCer metabolism. Rice-derived GlcCer may improve epidermal water loss and barrier function.

Background: The yeast Orm1/2 proteins regulate ceramide biosynthesis., Results: Depletion of the mammalian Orm1/2 homologues, ORMDL1-3, eliminates the negative feedback of exogenous ceramide on ceramide biosynthesis in HeLa cells., Conclusion: ORMDL proteins are the primary regulators of ceramide biosynthesis in mammalian cells., Significance: Therapeutically manipulating levels of the pro-death lipid, ceramide, requires a molecular understanding of its regulation. The mammalian ORMDL proteins are orthologues of the yeast Orm proteins (Orm1/2), which are regulators of ceramide biosynthesis. In mammalian cells, ceramide is a proapoptotic signaling sphingolipid, but it is also an obligate precursor to essential higher order sphingolipids. Therefore levels of ceramide are expected to be tightly controlled. We tested the three ORMDL isoforms for their role in homeostatically regulating ceramide biosynthesis in mammalian cells. Treatment of cells with a short chain (C6) ceramide or sphingosine resulted in a dramatic inhibition of ceramide biosynthesis. This inhibition was almost completely eliminated by ORMDL knockdown. This establishes that the ORMDL proteins mediate the feedback regulation of ceramide biosynthesis in mammalian cells. The ORMDL proteins are functionally redundant. Knockdown of all three isoforms simultaneously was required to alleviate the sphingolipid-mediated inhibition of ceramide biosynthesis. The lipid sensed by the ORMDL-mediated feedback mechanism is medium or long chain ceramide or a higher order sphingolipid. Treatment of permeabilized cells with C6-ceramide resulted in ORMDL-mediated inhibition of the rate-limiting enzyme in sphingolipid biosynthesis, serine palmitoyltransferase. This indicates that C6-ceramide inhibition requires only membrane-bound elements and does not involve diffusible proteins or small molecules. We also tested the atypical sphingomyelin synthase isoform, SMSr, for its role in the regulation of ceramide biosynthesis. This unusual enzyme has been reported to regulate ceramide levels in the endoplasmic reticulum. We were unable to detect a role for SMSr in regulating ceramide biosynthesis. We suggest that the role of SMSr may be in the regulation of downstream metabolism of ceramide.

Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Arsenic trioxide (ATO) has shown anticancer activity against a variety of solid tumor models through induction of apoptosis, promotion of cellular differentiation, and inhibition of cellular invasive ability. The present study investigated the role of ceramide in regulating the invasive activity of hepatoma carcinoma HCCLM3 cells during ATO treatment. We found that ATO treatment inhibited HCCLM3 cell invasion and downregulated matrix metalloproteinase-9 (MMP-9) protein levels in a concentration-dependent manner. ATO also dose dependently induced the generation and accumulation of ceramide in HCCLM3 cells. Blockage of intracellular ceramide production through the inhibition of de novo ceramide synthesis or the hydrolysis of sphingomyelin increased the invasive ability and upregulated MMP-9 protein levels. The findings of this study indicated that ATO induced ceramide production through de novo ceramide synthesis and the hydrolysis of sphingomyelin and suggested that ceramide accumulation in response to ATO stimuli may play an important role in cancer therapy.

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300 μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dysregulation of ceramide synthesis has been associated with metabolic disorders such as atherosclerosis and diabetes. We examined the changes in lipid homeostasis and gene expression in Huh7 hepatocytes when the synthesis of ceramide is perturbed by knocking down serine pal mitoyltransferase subunits 1, 2, and 3 (SPTLC123) or dihydroceramide desaturase 1 (DEGS1). Although knocking down all SPTLC subunits is necessary to reduce total ceramides significantly, depleting DEGS1 is sufficient to produce a similar outcome. Lipidomic analysis of distribution and speciation of multiple lipid classes indicates an increase in phospholipids in SPTLC123-silenced cells, whereas DEGS1 depletion leads to the accumulation of sphingolipid intermediates, free fatty acids, and diacylglycerol. When cer amide synthesis is disrupted, the transcriptional profiles indicate inhibition in biosynthetic processes, downregulation of genes involved in general endomembrane trafficking, and upregulation of endocytosis and endosomal recycling. SPTLC123 silencing strongly affects the expression of genes involved with lipid metabolism. Changes in amino acid, sugar, and nucleotide metabolism, as well as vesicle trafficking between organelles, are more prominent in DEGS1-silenced cells. These studies are the first to provide a direct and comprehensive understanding at the lipidomic and transcriptomic levels of how Huh7 hepatocytes respond to changes in the inhibition of ceramide synthesis.

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.

Following the initial discovery that adipose tissue actively synthesizes and secretes cytokines, obesity-induced inflammation has been implicated in the etiology of a host of disease states related to obesity, including cardiovascular disease and type II diabetes. Interestingly, a growing body of evidence similarly implicates sphingolipids as prime instigators in these same diseases. From the recent discovery that obesity-related inflammatory pathways modulate sphingolipid metabolism comes a novel perspective—sphingolipids may act as the dominant mediators of deleterious events stemming from obesity-induced inflammation. This paradigm may identify sphingolipids as an effective target for future therapeutics aimed at ameliorating diseases associated with chronic inflammation.

DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs., (Copyright © 2012. Published by Elsevier B.V.)

Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS) that each use acyl-CoAs of defined chain length for N-acylation of the sphingoid long chain base. CerS mRNA expression and enzymatic activity do not always correlate with the sphingolipid acyl chain composition of a particular tissue, suggesting post-translational mechanism(s) of regulation of CerS activity. We now demonstrate that CerS activity can be modulated by dimer formation. Under suitable conditions, high M(r) CerS complexes can be detected by Western blotting, and various CerS co-immunoprecipitate. CerS5 activity is inhibited in a dominant-negative fashion by co-expression with catalytically inactive CerS5, and CerS2 activity is enhanced by co-expression with a catalytically active form of CerS5 or CerS6. In a constitutive heterodimer comprising CerS5 and CerS2, the activity of CerS2 depends on the catalytic activity of CerS5. Finally, CerS dimers are formed upon rapid stimulation of ceramide synthesis by curcumin. Together, these data demonstrate that ceramide synthesis can be regulated by the formation of CerS dimers and suggest a novel way to generate the acyl chain composition of ceramide (and downstream sphingolipids), which may depend on the interaction of CerS with each other.

Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-α (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sonic hedgehog (SHh) signaling is important in the pathogenesis of various human cancers, such as medulloblastomas, and it has been identified as a valid target for anticancer therapeutics. The SHh inhibitor cyclopamine induces apoptosis. The bioactive sphingolipid ceramide mediates cell death in response to various chemotherapeutic agents; however, ceramide's roles/mechanisms in cyclopamine-induced apoptosis are unknown. Here, we report that cyclopamine mediates ceramide generation selectively via induction of neutral sphingomyelin phosphodiesterase 3, SMPD3 (nSMase2) in Daoy human medulloblastoma cells. Importantly, short interfering RNA-mediated knockdown of nSMase2 prevented cyclopamine-induced ceramide generation and protected Daoy cells from drug-induced apoptosis. Accordingly, ectopic wild-type N-SMase2 caused cell death, compared with controls, which express the catalytically inactive N-SMase2 mutant. Interestingly, knockdown of smoothened (Smo), a target protein for cyclopamine, or Gli1, a downstream signaling transcription factor of Smo, did not affect nSMase2. Mechanistically, our data showed that cyclopamine induced nSMase2 and cell death selectively via increased nitric oxide (NO) generation by neuronal-nitric oxide synthase (n-NOS) induction, in Daoy medulloblastoma, and multiple other human cancer cell lines. Knockdown of n-NOS prevented nSMase2 induction and cell death in response to cyclopamine. Accordingly, N-SMase2 activity-deficient skin fibroblasts isolated from homozygous fro/fro (fragilitas ossium) mice exhibited resistance to NO-induced cell death. Thus, our data suggest a novel off-target function of cyclopamine in inducing apoptosis, at least in part, by n-NOS/NO-dependent induction of N-SMase2/ceramide axis, independent of Smo/Gli inhibition., (©2012 AACR)

Depletion of intracellular glutathione (GSH) is the prime hallmark of the progression of apoptosis. Previously, we reported that curcumin induces reactive oxygen species (ROS)-mediated depletion of GSH, which leads to caspase-dependent and independent apoptosis in mouse fibroblast cells (F. Thayyullathil et al., Free Radic. Biol. Med.45, 1403-1412, 2008). In this study, we investigated the antileukemic potential of curcumin in vitro, and we further examined the molecular mechanisms of curcumin-induced apoptosis in human leukemic cells. Curcumin suppresses the growth of human leukemic cells via ROS-independent GSH depletion, which leads to caspase activation, inhibition of sphingomyelin synthase (SMS) activity, and induction of ceramide (Cer) generation. Pretreatment of leukemic cells with carbobenzoxy-Val-Ala-Asp fluoromethylketone, a universal inhibitor of caspases, abrogates the SMS inhibition and Cer generation, and in turn prevents curcumin-induced cell death. Curcumin treatment of leukemic cells also downregulates the expression of the inhibitor of apoptosis proteins (IAPs), phospho-Akt, c-Myc, and cyclin D1. Extracellular supplementation with GSH attenuates curcumin-induced depletion of GSH, caspase-dependent inhibition of SMS, Cer generation, and downregulation of IAPs, whereas, L-D-buthionine sulfoximine, a widely used inhibitor of GSH synthesis, potentiates GSH depletion, Cer generation, and apoptosis induced by curcumin. Taken together, our findings provide evidence suggesting for the first time that GSH regulates caspase-dependent inhibition of SMS activity, Cer generation, and apoptosis induced by curcumin in human leukemic cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H(+)-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase-mediated production of O(2)(-.) in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H(+)-ATPase apparently attenuated FasL-caused pH decrease. V1 H(+)-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H(+)-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H(+)-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ectopic lipids in peripheral tissues have been implicated in attenuating insulin action in vivo. The botanical extract of Artemisia dracunculus L. (PMI 5011) improves insulin action, yet the precise mechanism is not known. We sought to determine whether the mechanism by which PMI 5011 improves insulin signaling is through regulation of lipid metabolism. After differentiation, cells were separately preincubated with free fatty acids (FFAs) and ceramide C2, and the effects on glycogen content, insulin signaling, and ceramide profiles were determined. The effect of PMI 5011 on ceramide accumulation and ceramide-induced inhibition of insulin signaling was evaluated. FFAs resulted in increased levels of total ceramides and ceramide species in L6 myotubes. Saturated FFAs and ceramide C2 inhibited insulin-stimulated phosphorylation of protein kinase B/Akt and reduced glycogen content. PMI 5011 had no effect on ceramide formation or accumulation but increased insulin sensitivity via restoration of Akt phosphorylation. PMI 5011 also attenuated the FFA-induced upregulation of a negative inhibitor of insulin signaling, i.e., protein tyrosine phosphatase 1B (PTP1B), and increased phosphorylation of PTP1B. PMI 5011 attenuates the reduction in insulin signaling induced by ceramide accumulation, but the mechanism of improved insulin signaling is independent of ceramide formation.

Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Lag1p and Lac1p catalyse ceramide synthesis in Saccharomyces cerevisiae. This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to S. cerevisiae where these proteins are functionally redundant, C. albicans Lag1p (CaLag1p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of C. albicans. Deletion of CaLAG1 decreased expression of the hypha-specific HWP1 and ECE1 genes. Moreover, overexpression of CaLAG1 induced pseudohyphal growth in this organism under non-hypha-inducing conditions, suggesting that CaLag1p is necessary for relaying signals to induce hypha-specific gene expression. Analysis of ceramide and sphingolipid composition revealed that CaLag1p predominantly synthesizes ceramides with C24:0/C26:0 fatty acid moieties, which are involved in generating inositol-containing sphingolipids, whereas CaLac1p produces ceramides with C18:0 fatty acid moieties, which are precursors for glucosylsphingolipids. Thus, our study demonstrates that CaLag1p and CaLac1p have distinct substrate specificities and physiological roles in C. albicans., (© 2011 Blackwell Publishing Ltd.)

The objectives of this study were to identify a plant extract that would improve stratum corneum functions and to elucidate the mechanism(s) involved. Based on the information that stratum corneum functions depend on the level of ceramide in the stratum corneum, we identified a Eucalyptus extract that was able to increase the level of ceramide in human keratinocytes in culture and in human stratum corneum and that improves the stratum corneum water holding and barrier functions. Addition of the Eucalyptus extract to human keratinocytes in culture increased the level of ceramide in a dose-dependent manner and also increased the biosynthesis of ceramide, glucosylceramide and sphingomyelin. Topical application of the Eucalyptus extract on the dry skin of human subjects induced by acetone and diethylether treatment resulted in a significant increase in ceramide level in the stratum corneum, a significant improvement in its water-holding function and an improvement in its barrier function. The addition of macrocarpal A, one of the main components of the Eucalyptus extract, to human keratinocytes in culture increased the level of ceramide and the mRNA expression of serine palmitoyltransferase, acid sphingomyelinase, neutral sphingomyelinase, glucosylceramide synthase and glucocerebrosidase in a dose-dependent manner. Our results indicate that the increased content of ceramides in the stratum corneum may underlie the therapeutic effect of the Eucalyptus extract. Our results also indicate the possibility that macrocarpal A is the key component that stimulates the synthesis of ceramide in the stratum corneum., (© 2011 The Authors. ICS © 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Oligodendroglial injury is a pathological hallmark of many human white matter diseases, including multiple sclerosis (MS) and periventricular leukomalacia (PVL). Critical regulatory mechanisms of oligodendroglia destruction, however, remain incompletely understood. Ceramide, a bioactive sphingolipid pivotal to sphingolipid metabolism pathways, regulates cell death in response to diverse stimuli and has been implicated in neurodegenerative disorders. We report here that ceramide accumulates in reactive astrocytes in active lesions of MS and PVL, as well as in animal models of demyelination. Serine palmitoyltransferase, the rate-limiting enzyme for ceramide de novo biosynthesis, was consistently upregulated in reactive astrocytes in the cuprizone mouse model of demyelination. Mass spectrometry confirmed the upregulation of specific ceramides during demyelination, and revealed a concomitant increase of sphingosine and a suppression of sphingosine-1-phosphate, a potent signaling molecule with key roles in cell survival and mitogenesis. Importantly, this altered sphingolipid metabolism during demyelination was restored upon active remyelination. In culture, ceramide acted synergistically with tumor necrosis factor, leading to apoptotic death of oligodendroglia in an astrocyte-dependent manner. Taken together, our findings implicate that disturbed sphingolipid pathways in reactive astrocytes may indirectly contribute to oligodendroglial injury in cerebral white matter disorders., (© 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.)

Sphingosine 1-phosphate (S1P) and ceramide have been implicated in both autophagy and apoptosis. However, the roles of these sphingolipid metabolites in the links between these two processes are not completely understood. Depletion of S1P phosphohydrolase-1 (SPP1), which degrades intracellular S1P, induces the unfolded protein response and endoplasmic reticulum stress-induced autophagy (Lépine, S., Allegood, J. C., Park, M., Dent, P., Milstien, S., and Spiegel, S. (2011) Cell Death Differ. 18, 350-361). Surprisingly, however, treatment with doxorubicin, which by itself also induced autophagy, markedly reduced the extent of autophagy mediated by depletion of SPP1. Concomitantly, doxorubicin-induced apoptosis was greatly enhanced by down-regulation of SPP1. Autophagy and apoptosis seemed to be sequentially linked because inhibiting autophagy with 3-methyladenine also markedly attenuated apoptosis. Moreover, silencing Atg5 or the three sensors of the unfolded protein response, IRE1α, ATF6, and PKR-like eIF2α kinase (PERK), significantly decreased both autophagy and apoptosis. Doxorubicin stimulated calpain activity and Atg5 cleavage, which were significantly enhanced in SPP1-depleted cells. Inhibition or depletion of calpain not only suppressed Atg5 cleavage, it also markedly decreased the robust apoptosis induced by doxorubicin in SPP1-deficient cells. Importantly, doxorubicin also increased de novo synthesis of the pro-apoptotic sphingolipid metabolite ceramide. Elevation of ceramide in turn stimulated calpain; conversely, inhibiting ceramide formation suppressed Atg5 cleavage and apoptosis. Hence, doxorubicin switches protective autophagy in SPP1-depleted cells to apoptosis by calpain-mediated Atg5 cleavage.

Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase-1 (HO-1) as indicators of possible Cav-1-mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD.

Autoproteolytic cleavage of the inactive acid ceramidase (AC) precursor into the active heterodimer exposes a free cysteine residue, leading us to study whether AC could be regulated by one or more members of the cystatin family. Co-expression of the full-length AC and cystatin SA (cysSA) cDNAs led to significant reduction of AC activity in the transfected cells. Expression of cysSA also inhibited endogenous AC activity in cells and increased ceramide. Conversely, cysSA siRNA expression led to elevated AC activity and reduction in ceramide. The effects of cysSA siRNA expression could be reversed by the addition of recombinant cysSA into the culture media. These results were consistent with detection of a physical interaction between AC and cysSA, assessed by co-immunoprecipitation and nickel-nitrilotriacetic acid affinity chromatography, and further supported by co-localization of the endogenous proteins using confocal microscopy. In vitro kinetic analysis of purified, recombinant AC and cysSA confirmed the transfection results and suggested a non-competitive type of inhibition with a K(i) in the low micromolar range. Processing of the AC precursor into the active form was not affected by cysSA expression, suggesting that it likely inhibits AC by allosteric interference. Computer modeling and expression studies identified several potential inhibitory domains in cysSA, including a small "AC-like" domain (identical to the AC cleavage site, TICT). Small peptides, synthesized with combinations of this and a "cystatin-like" domain (QXVXG), exhibited significant AC inhibition as well. Such peptide-based AC inhibitors could potentially be used to regulate AC activity in cancer cells that are known to overexpress this enzyme alone and in combination with conventional anti-cancer drugs.

As obesity and type 2 diabetes are becoming an epidemic in westernized countries, the incidence and prevalence of obesity- and diabetes-related co-morbidities are increasing. In type 2 diabetes ectopic lipid accumulation in the heart has been associated with cardiac dysfunction and apoptosis, a process termed lipotoxicity. Since cardiovascular diseases are the main cause of death in diabetic patients, diagnosis and treatment become increasingly important. Although ischaemic heart disease is a major problem in diabetes, non-ischaemic heart disease (better known as diabetic cardiomyopathy) becomes increasingly important with respect to the impairment of cardiac function and mortality in type 2 diabetes. The underlying aetiology of diabetic cardiomyopathy is incompletely understood but is beginning to be elucidated. Various mechanisms have been proposed that may lead to lipotoxicity. Therefore, this review will focus on the mechanisms of cardiac lipid accumulation and its relation to the development of cardiomyopathy.

Scope: Molecules involved in skin function are greatly affected by nutritional conditions. However, the mechanism linking high-fat (HF) diets with these alterations is not well understood. This study aimed to investigate the molecular changes in skin function that result from HF diets., Methods and Results: Sprague-Dawley rats were fed HF diets for 28 days. The skin levels of ceramide, lipids and mRNAs involved in lipid metabolism were evaluated using TLC, oil red O staining and quantitative PCR, respectively. The serum adiponectin concentration was determined by ELISA. HF diets led to reduced ceramide levels and lowered skin lipid content. They also decreased mRNA levels of serine palmitoyltransferase (SPT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the skin and those of peroxisome proliferator-activated receptor-α -PPAR-α), which upregulates SPT and HMG-CoA reductase expression. The HF diets reduced the serum concentration of adiponectin, which acts upstream of PPAR-α. Finally, these diets led to increased mRNA levels of carnitine palmitoyltransferase-1, the rate-limiting enzyme that acts in β-oxidation., Conclusion: Our study suggests that HF diets reduce ceramide and lipid synthesis in the skin by reducing levels of SPT and HMG-CoA reductase through lowered adiponectin and PPAR-α activity. Additionally, they decrease lipid content by enhancing β-oxidation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Ceramide may be synthesized de novo or generated by sphingomyelinase-dependent hydrolysis of sphingomyelin., Areas Covered: The role of ceramide, ceramide-sensitive signaling and ion channels in β-cell apoptosis, lipotoxicity and amyloid-induced β-cell death., Expert Opinion: Ceramide participates in β-cell dysfunction and apoptosis after exposure to TNFα, IL-1β and IFN-γ, excessive amyloid and islet amyloid polypeptide or non-esterified fatty acids (lipotoxicity). Knockout of sphingomyelin synthase 1, which converts ceramide to sphingomyelin, leads to impairment of insulin secretion. Increased ceramidase activity or pharmacological inhibition of ceramide synthetase, inhibits β-cell apoptosis. Ceramide contributes to endoplasmatic reticulum (ER) stress, decreased mitochondrial membrane potential in insulin-secreting cells and mitochondrial release of cytochrome c into the cytosol, which are all triggers of apoptotic cell death. Ceramide-dependent signaling involves activation of extracellularly regulated kinases 1 and 2 (ERK1/2), downregulation of Period (Per)-aryl hydrocarbon receptor nuclear translocator (Arnt)-single-minded (Sim) kinase (PASK), activation of okadaic-acid-sensitive protein phosphatase 2A (PP2A) and stimulation of NADPH-oxidase with generation of superoxides and lipid peroxides. Ceramide reduces the activity of voltage gated potassium (Kv)-channels in insulin-secreting cells. The role of ceramide in β-cell survival and function may be therapeutically relevant, because ceramide formation can be suppressed by pharmacological inhibition of ceramide synthetase and/or sphingomyelinase.

Intensive research over the past 2 decades has implicated ceramide in the regulation of several cell responses. However, emerging evidence points to dramatic complexities in ceramide metabolism and structure that defy the prevailing unifying hypothesis on ceramide function that is based on the understanding of ceramide as a single entity. Here, we develop the concept that "ceramide" constitutes a family of closely related molecules, subject to metabolism by >28 enzymes and with >200 structurally distinct mammalian ceramides distinguished by specific structural modifications. These ceramides are synthesized in a combinatorial fashion with distinct enzymes responsible for the specific modifications. These multiple pathways of ceramide generation led to the hypothesis that individual ceramide molecular species are regulated by specific biochemical pathways in distinct subcellular compartments and execute distinct functions. In this minireview, we describe the "many ceramides" paradigm, along with the rationale, supporting evidence, and implications for our understanding of bioactive sphingolipids and approaches for unraveling these pathways.

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti-cancer treatment have been sought from natural resources. Here, we have investigated anti-proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells., Material and Methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate levels were analysed by HPLC., Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G(2) /M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21(waf1/cip1) was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine-1-phosphate in myriocin-treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells., Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21(waf1/cip1) , followed by inhibition of cyclin B1 and cdc2, resulting in G(2) /M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism-based therapy for this type of skin cancer., (© 2011 Blackwell Publishing Ltd.)