Sphinganine Research Articles

Sphingosine Promotes Fiber Early Elongation in Upland Cotton.

Sphingolipids play an important role in cotton fiber development, but the regulatory mechanism is largely unclear. We found that serine palmitoyltransferase (SPT) enzyme inhibitors, myriocin and sphingosine (dihydrosphingosine (DHS) and phytosphingosine (PHS)), affected early fiber elongation in cotton, and we performed a sphingolipidomic and transcriptomic analysis of control and PHS-treated fibers. Myriocin inhibited fiber elongation, while DHS and PHS promoted it in a dose-effect manner. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that contents of 22 sphingolipids in the PHS-treated fibers for 10 days were changed, of which the contents of 4 sphingolipids increased and 18 sphingolipids decreased. The transcriptome analysis identified 432 differentially expressed genes (238 up-regulated and 194 down-regulated) in the PHS-treated fibers. Among them, the phenylpropanoid biosynthesis pathway is the most significant enrichment. The expression levels of transcription factors such as MYB, ERF, LBD, and bHLH in the fibers also changed, and most of MYB and ERF were up-regulated. Auxin-related genes IAA, GH3 and BIG GRAIN 1 were up-regulated, while ABPs were down-regulated, and the contents of 3 auxin metabolites were decreased. Our results provide important sphingolipid metabolites and regulatory pathways that influence fiber elongation.

Aft1 Nuclear Localization and Transcriptional Response to Iron Starvation Rely upon TORC2/Ypk1 Signaling and Sphingolipid Biosynthesis.

Iron scarcity provokes a cellular response consisting of the strong expression of high-affinity systems to optimize iron uptake and mobilization. Aft1 is a primary transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Aft1 responds to iron deprivation by translocating from the cytoplasm to the nucleus. Here, we demonstrate that the AGC kinase Ypk1, as well as its upstream regulator TOR Complex 2 (TORC2), are required for proper Aft1 nuclear localization following iron deprivation. We exclude a role for TOR Complex 1 (TORC1) and its downstream effector Sch9, suggesting this response is specific for the TORC2 arm of the TOR pathway. Remarkably, we demonstrate that Aft1 nuclear localization and a robust transcriptional response to iron starvation also require biosynthesis of sphingolipids, including complex sphingolipids such as inositol phosphorylceramide (IPC) and upstream precursors, e.g., long-chain bases (LCBs) and ceramides. Furthermore, we observe the deficiency of Aft1 nuclear localization and impaired transcriptional response in the absence of iron when TORC2-Ypk1 is impaired is partially suppressed by exogenous addition of the LCB dihydrosphingosine (DHS). This latter result is consistent with prior studies linking sphingolipid biosynthesis to TORC2-Ypk1 signaling. Taken together, these results reveal a novel role for sphingolipids, controlled by TORC2-Ypk1, for proper localization and activity of Aft1 in response to iron scarcity.

Analysis of Sphingolipids in Pediatric Patients with Cholelithiasis-A Preliminary Study.

(1) Background: Disturbances in the sphingolipid profile are observed in many diseases. There are currently no data available on the evaluation of sphingolipids and ceramides in cholelithiasis in children. The aim of this study was to evaluate the concentrations of sphingolipids in the sera of pediatric patients with gallstones. We determined their relationship with anthropometric and biochemical parameters. (2) Methods: The concentrations of sphingolipids in serum samples were evaluated using a quantitative method, ultra-high-performance liquid chromatography–tandem mass spectrometry. (3) Results: The prospective study included 48 children and adolescents diagnosed with gallstones and 38 controls. Serum concentrations of total cholesterol (TC); sphinganine (SPA); ceramides—C14:0-Cer, C16:0-Cer, C18:1-Cer, C18:0-Cer, C20:0-Cer and C24:1-Cer; and lactosylceramides—C16:0-LacCer, C18:0-LacCer, C18:1-LacCer, C24:0-LacCer and C24:1-LacCer differed significantly between patients with cholelithiasis and without cholelithiasis. After adjusting for age, gender, obesity and TC and TG levels, we found the best differentiating sphingolipids for cholelithiasis in the form of decreased SPA, C14:0-Cer, C16:0-Cer, C24:1-LacCer and C24:0-LacCer concentration and increased C20:0-Cer, C24:1-Cer, C16:0-LacCer and C18:1-LacCer. The highest area under the curve (AUC), specificity and sensitivity were determined for C16:0-Cer with cholelithiasis diagnosis. (4) Conclusions: Our results suggest that serum sphingolipids may be potential biomarkers in pediatric patients with cholelithiasis.

Involvement of the mitochondrial retrograde pathway in dihydrosphingosine-induced cytotoxicity in budding yeast

Sphingoid long-chain bases are essential intermediates of ceramides and complex sphingolipids, and function in the regulation of various signal transduction systems. Previously, we found that, in budding yeast, intracellularly accumulated dihydrosphingosine (DHS) causes mitochondrial reactive-oxygen species (ROS)-mediated cytotoxicity, which is much stronger than phytosphingosine. In this study, we screened for suppressor mutations that confer resistance to DHS, and identified RTG2, which encodes upregulation of the mitochondrial retrograde signaling pathway (RTG pathway). Deletion of RTG3 encoding transcriptional factor for the RTG pathway suppressed the cytotoxicity of DHS, whereas deletion of MKS1 or point mutation of LST8, both of which cause increased activity of the RTG pathway, enhanced the cytotoxicity. Moreover, the deletion of RTG3 also suppressed the DHS-induced increases in ROS levels. Finally, it was found that the RTG pathway is activated on DHS treatment. These results suggested that the cytotoxicity of DHS is partially mediated through activation of the RTG pathway.

Serum C18:1-Cer as a Potential Biomarker for Early Detection of Gestational Diabetes.

We hypothesized that sphingolipids may be early biomarkers of gestational diabetes mellitus (GDM). Here, 520 women with normal fasting plasma glucose levels were recruited in the first trimester and tested with a 75 g oral glucose tolerance test in the 24th–28th week of pregnancy. Serum sphingolipids concentrations were measured in the first and the second trimester by ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/MS/MS) in 53 patients who were diagnosed with GDM, as well as 82 pregnant women with normal glucose tolerance (NGT) and 32 non-pregnant women. In the first trimester, pregnant women showed higher concentrations of C16:0, C18:1, C22:0, C24:1, and C24:0-Cer and lower levels of sphinganine (SPA) and sphingosine-1-phosphate (S1P) compared to non-pregnant women. During pregnancy, we observed significant changes in C16:0, C18:0, C18:1, and C24:1-Cer levels in the GDM group and C18:1 and C24:0-Cer in NGT. The GDM (pre-conversion) and NGT groups in the first trimester differed solely in the levels of C18:1-Cer (AUC = 0.702 p = 0.008), also considering glycemia. Thus, C18:1-Cer revealed its potential as a GDM biomarker. Sphingolipids are known to be a modulator of insulin resistance, and our results indicate that ceramide measurements in early pregnancy may help with GDM screening.

A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

Regulation of sphingolipid synthesis by the G1/S transcription factor Swi4

SBF (Swi4/Swi6 Binding Factor) complex is a crucial regulator of G1/S transition in Saccharomyces cerevisiae. Here, we show that SBF complex is required for myriocin resistance, an inhibitor of sphingolipid synthesis. This phenotype was not shared with MBF complex mutants nor with deletion of the Swi4p downstream targets, CLN1/CLN2. Based on data mining results, we selected putative Swi4p targets related to sphingolipid metabolism and studied their gene transcription as well as metabolite levels during progression of the cell cycle. Genes which encode key enzymes for the synthesis of long chain bases (LCBs) and ceramides were periodically transcribed during the mitotic cell cycle, having a peak at G1/S, and required SWI4 for full transcription at this stage. In addition, HPLC-MS/MS data indicated that swi4Δ cells have decreased levels of sphingolipids during progression of the cell cycle, particularly, dihydrosphingosine (DHS), C24-phytoceramides and C24-inositolphosphoryl ceramide (IPC) while it had increased levels of mannosylinositol phosphorylceramide (MIPC). Furthermore, we demonstrated that both inhibition of de novo sphingolipid synthesis by myriocin or SWI4 deletion caused partial arrest at the G2/M phase. Importantly, our lipidomic data demonstrated that the sphingolipid profile of WT cells treated with myriocin resembled that of swi4Δ cells, with lower levels of DHS, IPC and higher levels of MIPC. Taken together, these results show that SBF complex plays an essential role in the regulation of sphingolipid homeostasis, which reflects in the correct progression through the G2/M phase of the cell cycle.

Sphingosine Promotes Embryo Biomass in Upland Cotton: A Biochemical and Transcriptomic Analysis.

Sphingolipids are essential membrane components and signal molecules, but their regulatory role in cotton embryo growth is largely unclear. In this study, we evaluated the effects of treatment with the sphingolipid synthesis inhibitor fumonisin B1 (FB1), the serine palmityl transferase (SPT) inhibitor myriocin, the SPT sphingolipid product DHS (d18:0 dihydrosphingosine), and the post-hydroxylation DHS product PHS (t18:0 phytosphingosine) on embryo growth in culture, and performed comparative transcriptomic analysis on control and PHS-treated samples. We found that FB1 could inhibit cotton embryo development. At the five-day ovule/embryo developmental stage, PHS was the most abundant sphingolipid. An SPT enzyme inhibitor reduced the fresh weight of embryos, while PHS had the opposite effect. The transcriptomic analysis identified 2769 differentially expressed genes (1983 upregulated and 786 downregulated) in the PHS samples. A large number of transcription factors were highly upregulated, such as zinc finger, MYB, NAC, bHLH, WRKY, MADS, and GRF in PHS-treated samples compared to controls. The lipid metabolism and plant hormone (auxin, brassinosteroid, and zeatin) related genes were also altered. Our findings provide target metabolites and genes for cotton seed improvement.

BiP-overexpressing soybean plants display accelerated hypersensitivity response (HR) affecting the SA-dependent sphingolipid and flavonoid pathways

Biotic and abiotic environmental stresses have limited the increase in soybean productivity. Overexpression of the molecular chaperone BiP in transgenic plants has been associated with the response to osmotic stress and drought tolerance by maintaining cellular homeostasis and delaying hypersensitive cell death. Here, we evaluated the metabolic changes in response to the hypersensitivity response (HR) caused by the non-compatible bacteria Pseudomonas syringae pv. tomato in BiP-overexpressing plants. The HR-modified metabolic profiles in BiP-overexpressing plants were significantly distinct from the wild-type untransformed. The transgenic plants displayed a lower abundance of HR-responsive metabolites as amino acids, sugars, carboxylic acids and signal molecules, including p-aminobenzoic acid (PABA) and dihydrosphingosine (DHS), when compared to infected wild-type plants. In contrast, salicylic acid (SA) biosynthetic and signaling pathways were more stimulated in transgenic plants, and both pathogenesis-related genes (PRs) and transcriptional factors controlling the SA pathway were more induced in the BiP-overexpressing lines. Furthermore, the long-chain bases (LCBs) and ceramide biosynthetic pathways showed alterations in gene expression and metabolite abundance. Thus, as a protective pathway against pathogens, HR regulation by sphingolipids and SA may account at least in part by the enhanced resistance of transgenic plants. GmNAC32 transcriptional factor was more induced in the transgenic plants and it has also been reported to regulate flavonoid synthesis in response to SA. In fact, the BiP-overexpressing plants showed an increase in flavonoids, mainly prenylated isoflavones, as precursors for phytoalexins. Our results indicate that the BiP-mediated acceleration in the hypersensitive response may be a target for metabolic engineering of plant resistance against pathogens.

Impact of increasing levels of fumonisin on performance, liver toxicity, and tissue histopathology of finishing beef steers.

To address the gaps in current scientific knowledge, the objective of the present study was to investigate the impact of fumonisin exposure on feedlot cattle intake and performance. Fifty steers were received (day 0; 361 ± 6.4 kg), housed individually and fed once daily at 0800 hours. All steers were transitioned to a dry-rolled corn-based finishing diet from days 0 to 21 and then were fed the control finishing diet until day 50. Treatment diets were formulated to achieve ≤5 (CON), 15 (15PPM), 30 (30PPM), 60 (60PPM), or 90 ppm (90PPM) of total dietary fumonisin. Steers were fed the fumonisin treatment diets from day 50 until harvest on day 160; individual animal body weights (BW) were measured on days 0, 50, 100, 150, 159, and 160. Liver, kidney, and skeletal muscle tissue samples were collected at harvest for histopathological analyses, and liver samples were further analyzed for sphinganine (SA) and sphingosine (SO) concentration. Animal performance, carcass data, and liver enzyme concentration were analyzed using a mixed model; categorical data were analyzed via nonparametric models. Contrasts were used to test for linear and quadratic responses. Throughout the study, there was no effect of treatment (P > 0.60), or a linear response (P > 0.16) from increasing fumonisin levels, on BW or dry matter intake (DMI). However, CON tended to have a lower average daily gain (ADG) than the fumonisin treatments during the fumonisin treatment period (P = 0.10), and there was a positive linear response (P = 0.02) of ADG to fumonisin during the treatment period. There were no treatment differences in hot carcass weight, dressing percentage, marbling score, ribeye area, or yield grade. There were no effects of treatment on either liver abscesses (P = 0.95) or telangiectasis (P = 0.13). We observed a treatment difference for SA and SA:SO (P < 0.01), as well as a quadratic response (P < 0.02); both SA and SA:SO increased as dietary fumonisin increased. There were no observed differences between treatments for histopathology scores of kidney (P = 0.16), liver (P = 0.25), or skeletal muscle (P = 0.59) tissue. No adverse effects were observed in steers fed increasing dietary levels of fumonisin for 110 d prior to harvest. While elevated liver amino alcohol concentration did occur, negative effects on growth and carcass characters were not observed.

Repeated low-dose exposures to sarin disrupted the homeostasis of phospholipid and sphingolipid metabolism in guinea pig hippocampus

Repeated low-level exposure to sarin results to hippocampus dysfunction. Metabonomics involves a holistic analysis of a set of metabolites in an organism in the search for a relationship between these metabolites and physiological or pathological changes. The objective of the present study was to evaluate the effects of repeated exposure to low-level sarin on the metabonomics in hippocampus of a guinea pig model. Guinea pigs were divided randomly into control and sarin treated groups (n = 14). Guinea pigs in the control group received saline; while the sarin-treated group received 0.4×LD50 (16.8 μg/kg) sarin. Daily injections (a total of 14 days) were administered sc between the shoulder blades in a volume of 1.0 ml/kg body weight. At the end of the final injection, 6 animals in each group were chosen for Morris water maze test. The rest guinea pigs (n = 8 for each group) were sacrificed by decapitation, and hippocampus were dissected for analysis. Compared with the control-group, the escape latency in sarin-group was significantly (p < 0.05) longer while the crossing times were significantly decreased in the Morris water task (p < 0.05). Sarin inhibited activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in hippocampus. The AChE activity of hippocampus from sarin-treated groups is equivalent to 59.9 ± 6.4 %, and the NTE activity of hippocampus from sarin-groups is equivalent to 78.1 ± 8.3 % of that from control-group. Metabolites were identified and validated. A total of 14 variables were selected as potential biomarkers. Phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), Lysophosphatidylethanolamine (LysoPE or LPE)] and sphingolipids (SPs) [sphinganine (SA), phytosphingosine (PSO) and sphinganine-1-phosphate (SA1P)] were clearly modified. In conclusion, repeated low-dose exposures to sarin disrupted the homeostasis of phospholipid and sphingolipid metabolism in guinea pig hippocampus and may lead to a neuronal-specific function disorders. Identified metabolites such as SA1P need to be studied more deeply on their biological function that against sarin lesions. In future research, we should pay more attention to characterize the physiological roles of lipid metabolism enzymes as well as their involvement in pathologies induced by repeated low-level sarin exposure.

Effects of Fumonisin-Contaminated Corn on Growth Performance of 9 to 28 kg Nursery Pigs.

Fumonisin contamination in corn is an emerging issue in animal feed production. Fumonisin disrupts the metabolism of sphingolipids and reduces growth performance. This experiment was conducted to determine the effect of feeding fumonisin-contaminated corn on growth performance and sphinganine (SA) to sphingosine (SO) ratios of 9 to 28 kg pigs. A total of 350 pigs, were used with 5 pigs/pen and 14 pens/treatment. Dietary treatments contained fumonisin-contaminated corn (50 mg/kg of fumonisin B1 + B2) blended with low fumonisin corn (10 mg/kg of fumonisin B1 + B2) to provide dietary fumonisin concentrations of 7.2, 14.7, 21.9, 32.7, and 35.1 mg/kg. From day 0 to 28, increasing fumonisin concentration decreased (linear, p < 0.001) average daily gain, average daily feed intake (linear, p = 0.055), and gain:feed ratio (linear, p = 0.016). Although these response criteria tested linear, the greatest reduction in performance was in pigs fed with 32.7 and 35.1 mg/kg of fumonisin (B1 + B2). Increasing fumonisin concentration increased the serum SA:SO ratio (linear, p < 0.001) on day 14 and 28. In summary, for 9 to 28 kg nursery pigs, increasing fumonisin linearly decreased average daily gain and gain:feed ratio. However, despite the linear response, diets containing up to 21.9 mg/kg of fumonisin did not have as dramatic a decrease in growth performance as those fed more than 32.7 mg/kg. Further research is warranted to determine the effect of fumonisin concentrations between 21.9 and 32.7 mg/kg.

Mitochondrial reactive oxygen species-mediated cytotoxicity of intracellularly accumulated dihydrosphingosine in the yeast Saccharomycescerevisiae.

In eukaryotic cells, the content of sphingoid long-chain bases (LCBs) is generally much lower than that of complex sphingolipids and ceramides, and the quantitative balance of these metabolites in cells is tightly regulated. In the budding yeast Saccharomycescerevisiae, it has been demonstrated that exogenously added phytosphingosine (PHS) causes a strong growth defect in tryptophan auxotrophic cells, due to delayed uptake of tryptophan from the culture medium; however, the growth inhibitory effect of dihydrosphingosine (DHS) is less than that of PHS in tryptophan auxotrophic cells. Here, we found that, in tryptophan-prototrophic yeast cells, exogenously added DHS is much more toxic than PHS. Exogenously added DHS is converted to PHS, Cers, or LCB 1-phosphates through the action of sphingolipid C4-hydroxylase, Cer synthases, or LCB kinases, respectively; however, suppression of further metabolism of DHS in cells resulted in an increase in the growth inhibitory activity of exogenously added DHS, indicating that DHS itself is causative of the cytotoxicity. The cytotoxicity of DHS was not mediated by Pkh1/2, Sch9, and Ypk1/2 kinases, intracellular targets of LCBs. DHS treatment caused an increase in mitochondria-derived reactive oxygen species, and the cytotoxic effect of DHS was suppressed by depletion of mitochondrial DNA or antioxidant N-acetylcysteine, but enhanced by deletion of SOD1 and SOD2 encoding superoxide dismutases. Thus, collectively, these results indicated that intracellularly accumulated DHS has mitochondrial reactive oxygen species-mediated cytotoxic activity, which is much more potent than that of PHS.

Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg−1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance.

Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21

It is now well established that sphingoid Long Chain Bases (LCBs) are crucial mediators of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts the sphingolipid biosynthesis pathway by inhibiting the ceramide synthase leading to an increase in the amount of phytosphingosine (PHS) and dihydrosphingosine (DHS), the two major LCBs in Arabidopsis thaliana. To date, the signaling pathway involved in FB1-induced cell death remains largely uncharacterized. It is also well acknowledged that plant proteases such as papain-like cysteine protease are largely involved in plant immunity. Here, we show that the papain-like cysteine protease RD21 (responsive-to-desiccation-21) is activated in response to PHS and FB1 in Arabidopsis cultured cells and leaves, respectively. Using two allelic null mutants of RD21, and two different PCD bioassays, we demonstrate that the protein acts as a negative regulator of FB1-induced cell death in Arabidopsis.

Fatty acids and sphingolipids profile in the blood plasma of experienced divers in response to hyperbaric exposure

Introduction: Hyperbaric exposure mimics air-breathing scuba diving, which is reaching enormous popularity around the world. The diver’s body is subjected to a broad range of divergent effects exerted by, e.g.: an increased partial pressure of inert gases, microclotting, oxidative stress and/or production of gas bubbles. However, very little is known regarding the impact of hyperbaric exposure on plasma fatty acids content and composition, together with the body’s sphingolipids profile. Material and Methods: The aim of this study was to investigate the contents of major fatty acids present in the plasma as well as sphingolipids, namely: sphingosine (SPH); sphingosine1-phosphate (S1P); sphinganine (SPA); and ceramide (CER), after hyperbaric exposure corresponding to dives conducted to the depths of 30 and 60 meters of seawater. For the plasma lipids measurements, high-performance liquid chromatography together with gas-liquid chromatography were applied. Results: We demonstrated that hyperbaric exposure does not affect the content and composition of plasma fatty acids of experienced divers. Similarly, the amounts of major sphingolipids fractions were not influenced, as only the content of sphingosine-1-phosphate in the plasma was significantly decreased. Conclusions: Observed lack of significant changes in plasma lipid profile after hyperbaric exposure suggests that the procedure might be considered as secure. However, decreased sphingosine-1- phosphate content in the plasma might possibly exert some adverse effects.

The UPR Activator ATF6 Responds to Proteotoxic and Lipotoxic Stress by Distinct Mechanisms

The unfolded protein response (UPR) is induced byproteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.

Abstract P1-01-21: The levels of sphingosine-1-phosphate and its related gene expressions in breast cancer patients

Abstract Background: The bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression. We previously demonstrated that S1P is a crucial mediator of breast cancer-induced angiogenesis and lymphangiogenesis, and promote metastasis. Although increasing number of in vitro and in vivo experiments have revealed the importance of S1P in cancer progression, the data on the roles of S1P in human patients are very limited. The aim of this study is to reveal the clinical relevance of S1P in the interaction between cancer and the tumor microenvironment by examining the levels of the sphingolipids in patient breast cancer tissue samples. Material and Method: Breast cancer tissue, peri-tumor tissue, and normal breast tissue were collected from 20 breast cancer patients immediately after surgery that were conducted from November 2015 to February 2016 at Niigata University Medical and Dental Hospital. Sphingolipids were quantified by liquid chromatography–electrospray ionization tandem mass spectrometry. The expression level of each enzyme-encoding gene involved in S1P production was evaluated by retrieving RNA sequencing and gene expression quantification data from breast cancer tissues (n = 112) and paired normal breast tissues (n = 112) using the Genomics Data Commons (GDC) data portal of the The Cancer Genome Atlas (TCGA) cohort. Gene expression levels were derived using normalization methods provided in the DESeq2 package. Result: The levels of the sphingolipids sphingosine (Sph), dihydro-sphingosine (DHSph), S1P, and dihydro-S1P (DHS1P) were successfully determined in breast cancer, peri-tumor, and normal breast tissues from all of the 20 patients. As expected, a one-way ANOVA revealed that S1P levels were significantly different depending on the location (F(2,57) = 7.029, P = 0.002). The Tukey post hoc test revealed that S1P levels in tumors were significantly higher than those in normal breast tissue and peri-tumor tissue (P &amp;lt; 0.05). Similarly, Sph and DHSph levels in tumors were significantly higher than in normal breast tissue and peri-tumor tissue. Both SPHK1 and SPHK2 gene expression levels in breast cancer tissue were higher than those in normal breast tissue. Interestingly, expression of some of the S1P-related genes; S1PR3, ABCC1, SGPL1, and ORMDL2, were significantly increased in the breast cancer tissue compared to normal breast tissue. On the other hand, there was significantly decreased expression of the S1P-related genes S1PR1, S1PR2, ABCG2, SPNS2, SGPP1 and ORMDL3, in breast cancer tissue compared to normal breast tissue. Conclusion: We demonstrated that the major source of S1P is the tumor tissue, and not the peri-tumor tissue despite the fact that angiogenesis and lymphangiogenesis are occurring more in the peri-tumor area, which implicate that S1P may have further role inside the tumor. Our results indicated the complexity of S1P signaling in human cancer than expected based on the results in vivo experiments. Citation Format: Tsuchida J, Nagahashi M, Nakajima M, Takabe K, Wakai T. The levels of sphingosine-1-phosphate and its related gene expressions in breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-21.

Atherosclerotic dyslipidemia revealed by plasma lipidomics on ApoE−/− mice fed a high-fat diet

Background and aimsThe pathological process of atherosclerosis is closely related to lipid accumulation at arterial wall caused by lipoproteins transporting lipids through blood. In this study, we investigate the lipid composition in the plasma of apolipoprotein E deficient (ApoE−/−) mice fed a high-fat diet to reveal atherosclerosis-induced dyslipidemia. MethodsApoE−/− and corresponding wild-type C57BL/6J mice were used as the pathology model and control, respectively, and were fed a high-fat or normal diet. Lipidomics approach based on chromatography coupled with time of fight mass spectrometry (UPLC-Q/TOF-MS) was applied to profile lipid species. ResultsDiscrimination analysis revealed that 1 lysophosphatidylcholine (LPC) and 6 phosphatidylcholines (PCs) were identified to distinguish C57BL/6J mice fed a normal and high-fat diet. PC (16:0/18:1) and PC (18:0/18:1) were also extracted when the comparison was done between ApoE−/− and C57BL/6J mice, both fed a high-fat diet. Besides the 2 PCs, the other 4 PCs, 1 sphinganine (SP) and 3 sphingomyelins (SMs) were identified in the second comparing case, among which PC (16:0/16:0), PC (18:0/16:1), SM (d16:0/28:5), SM (d18:1/24:1) and SM (d18:1/16:0) showed obviously positive correlations with the plasma levels of TC and LDL-C. However, no significant relationship was observed between the differential lipids and TG or HDL-C. ConclusionsThis study reveals that SP, SMs and PCs are the particularly changed lipid species induced by atherosclerotic lesions in the ApoE-/- model, indicating a disturbance on sphingolipid and glycerophospholipid metabolism during the progression of atherosclerotic dyslipidemia.

Inositol Depletion Induced by Acute Treatment of the Bipolar Disorder Drug Valproate Increases Levels of Phytosphingosine

Bipolar disorder (BD) is a severe psychiatric illness affecting ∼1% of the world population. Valproate (VPA) and lithium, widely used for the treatment of BD, are not universally effective. These drugs have been shown to cause inositol depletion, but translating this observation to a specific therapeutic mechanism has been difficult, hampering the development of more effective therapies. We have shown previously in yeast that chronic VPA treatment induces the unfolded protein response due to increasing ceramide levels. To gain insight into the mechanisms activated during acute VPA treatment, we performed a genome-wide expression study in yeast treated with VPA for 30 min. We observed increased mRNA and protein levels of RSB1, which encodes an exporter of long chain bases dihydrosphingosine (DHS) and phytosphingosine (PHS), and further saw that VPA increased sensitivity of an rsb1Δ mutant to PHS, suggesting that VPA increases long chain base levels. Consistent with this, PHS levels were elevated in wild type and, to a greater extent, in rsb1Δ cells. Expression of ORM genes (negative regulators of PHS synthesis) and of fatty acid elongase genes FEN1 and SUR4 were decreased, and expression of YOR1 (exporter of PHS-1P) and DPL1 (lyase that degrades DHS-1P and PHS-1P) was increased. These effects were more pronounced in medium lacking inositol, and were mirrored by inositol starvation of an ino1Δ mutant. These findings provide a metabolic explanation as to how VPA-mediated inositol depletion causes increased synthesis of PHS and further support the therapeutic relevance of inositol depletion as a bipolar disorder treatment.