Phosphatidylcholine and ceramide derivatives from white rot fungus Microporus xanthropus PP17-20.

The undescribed phosphatidylcholine (1), along with twelve known compounds, was isolated from the cultures of white rot fungus Microporus xanthropus PP17-20. In this work the fungus was cultivated in Yeast-Malt extract medium to explore active compound production. The chemical structures were elucidated on the basis of spectroscopic and HRESIMS data. Several isolated compounds were evaluated for anti-proliferative activity against A549 and MCF-7 cancer cell lines.

The effect of normobaric hypoxia on acute exercise-induced changes in blood sphingoid base-1-phosphates metabolism in cyclists.

Extracellular sphingosine-1-phosphate (S1P) emerged as an important regulator of muscle function. We previously found that plasma S1P concentration is elevated in response to acute exercise and training. Interestingly, hypoxia, which is commonly utilized in training programs, induces a similar effect. Therefore, the aim of the current study was to determine the effect of normobaric hypoxia on exercise-induced changes in blood sphingolipid metabolism. Fifteen male competitive cyclists performed a graded cycling exercise until exhaustion (GE) and a simulated 30 km individual time trial (TT) in either normoxic or hypoxic (FiO2 = 16.5%) conditions. Blood samples were taken before the exercise, following its cessation, and after 30 min of recovery. We found that TT increased dihydrosphingosine-1-phosphate (dhS1P) concentration in plasma (both HDL- and albumin-bound) and blood cells, as well as the rate of dhS1P release from erythrocytes, regardless of oxygen availability. Plasma concentration of S1P was, however, reduced during the recovery phase, and this trend was augmented by hypoxia. On the other hand, GE in normoxia induced a selective increase in HDL-bound S1P. This effect disappeared when the exercise was performed in hypoxia, and it was associated with reduced S1P level in platelets and erythrocytes. We conclude that submaximal exercise elevates total plasma dhS1P concentration via increased availability of dihydrosphingosine resulting in enhanced dhS1P synthesis and release by blood cells. Maximal exercise, on the other hand, induces a selective increase in HDL-bound S1P, which is a consequence of mechanisms not related to blood cells. We also conclude that hypoxia reduces post-exercise plasma S1P concentration.

[Research on mechanism of hypolipidemic effect of Massa Medicata Fermentata based on metabolomics].

This paper aims to study the therapeutic effect of Massa Medicata Fermentata on hyperlipidemia model rats and investigate its mechanism of hypolipidemic effect with the help of non-targeted metabolomics. The mixed hyperlipidemia model rats were constructed by giving high-fat chow. After successful modeling, the rats were divided into the model group, pravastatin sodium group(4.4 mg·kg~(-1)), lipotropic group(0.1 g·kg~(-1)), high-dose group(2.4 g·kg~(-1)), medium-dose group(1.2 g·kg~(-1)), and low-dose group(0.6 g·kg~(-1)) of Massa Medicata Fermentata, and they were administered for four weeks once daily. An equal volume of ultrapure water was given to the blank group and model group. Serum lipid level and liver hematoxylin-eosin(HE) staining were used as indicators to estimate the intervention effect of Massa Medicata Fermentata on mixed hyperlipidemia, and the changes in metabolites in plasma of mixed hyperlipidemia model rats were analyzed by non-targeted metabolomics. The mechanism of the hypolipidemic effect of Massa Medicata Fermentata was analyzed through metabolite pathway enrichment. The results showed that compared with the model group, the Massa Medicata Fermentata administration group, especially the high-dose group, could significantly reduce the content of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P<0.05 or P<0.01), and liver HE staining revealed that the number of adipocytes in the high-dose group was reduced to some extent. The potential biomarkers obtained by non-targeted metabolomics screening included glycerol 3-phosphate, sphingomyelin, sphingosine 1-phosphate, and deoxyuridine, which were mainly involved in the sphingolipid metabolism process, glycerophospholipid metabolism process, glycerol ester metabolism pathway, and pyrimidine metabolism pathway, totaling four possible metabolic pathways related to lipid metabolism. This study provides a reference for an in-depth investigation of the hypolipidemic mechanism of Massa Medicata Fermentata, which is of great significance for further promoting the clinical application of Massa Medicata Fermentata and increasing the indications.

The Interplay between Bioactive Sphingolipids in the Psoriatic Skin and the Severity of the Disease.

Psoriasis is a complex chronic immunologically mediated disease that may involve skin, nails, and joints. It is characterized by hyperproliferation, deregulated differentiation, and impaired apoptosis of keratinocytes. Sphingolipids, namely ceramide, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate, are signal molecules that may regulate cell growth, immune reactions, and apoptosis. Fifteen patients with psoriasis and seventeen healthy persons were enrolled in the study. Skin samples were taken from psoriatic lesions and non-lesional areas. Tissue concentration of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate was measured by liquid chromatography. We assessed that all levels of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate were higher in lesioned psoriatic skin than in non-affected skin. The profile of bioactive lipids in the lesional skin of patients with psoriasis differed significantly from non-involved psoriatic skin and skin in healthy subjects.

Crosstalk between Serum and Skin Sphingolipids in Psoriasis.

Psoriasis is a chronic, complex, immunological disorder, which may lead to many different systemic complications. Sphingolipids, including ceramide, are bioactive lipids, which take part in the regulation of immune reactions, cell growth, and apoptosis. Twenty psoriatic patients and twenty-eight control subjects were included in the study. Skin (both lesional and non-lesional) and serum samples were collected from both the control group and the psoriatic patients. The levels of sphingosine (SFO), sphingosine-1-phosphate (S1P), sphingomyelin, sphinganine (SFA), sphinganine-1-phosphate (SFA1P), and ceramide (CER) were assessed in both tissue (t) and serum (s) samples using high-performance liquid chromatography (HPLC). We identified elevated serum levels of SFO, S1P, SFA, and SFA1P in psoriatic patients when compared to healthy individuals. As far as the lesional skin and serum of psoriatic patients are concerned, we demonstrated positive associations between CER_t and CER_s, SFA_t and CER_s, and SFO_t and CER_s. Additionally, we found negative correlations in the non-lesional skin and serum of psoriatic patients, including SFO_t vs. SFO_s, CER_t vs. SFA_s, CER_t vs. SFO_s, and SFO_t vs. SFA_s. Finally, we observed a positive correlation between S1P and SFA1P in both the serum samples of psoriatic patients and the serum samples of the control group. In this study, we did not observe any correlations between psoriasis area and severity index (PASI) scores and sphingolipid levels. In conclusion, our findings indicate an interplay between skin and serum lipids in psoriatic patients, which is not observed in healthy individuals.

Diverse Sphingolipid Profiles in Rectal and Colon Cancer.

Colorectal cancer is a heterogenous group of neoplasms showing a variety of clinical and pathological features depending on their anatomical location. Sphingolipids are involved in the formation and progression of cancers, and their changes are an important part of the abnormalities observed during carcinogenesis. Because the course of rectal and colonic cancer differs, the aim of the study was to assess whether the sphingolipid profile is also different in tumors of these two regions. Using a combination of ultra-high-performance liquid chromatography combined with triple quadrupole mass spectrometry, differences in the amounts of cellular sphingolipids were found in colorectal cancer. Sphingosine content was higher in rectal cancer than in adjacent healthy tissue, while the content of two ceramides (C18:0-Cer and C20:0-Cer) was lower. In colon cancer, a higher content of sphingosine, sphinganine, sphingosine-1-phosphate, and two ceramides (C14:0-Cer and C24:0-Cer) was found compared to healthy tissue, but there was no decrease in the amount of any of the assessed sphingolipids. In rectal cancer, the content of sphinganine and three ceramides (C16:0-Cer, C22:0-Cer, C24:0-Cer), as well as the entire pool of ceramides, was significantly lower compared to colon cancer. The S1P/Cer ratio in rectal cancer (S1P/C18:1-Cer, S1P/C20:0-Cer, S1P/C22:0-Cer, S1P/C24:1-Cer) and in colon cancer (S1P/C18:0-Cer, S1P/C18:1-Cer, S1P/C20:0-Cer) was higher than in adjacent healthy tissue and did not differ between the two sites (rectal cancer vs. colonic cancer). It seems that the development of colorectal cancer is accompanied by complex changes in the metabolism of sphingolipids, causing not only qualitative shifts in the ceramide pool of cancer tissue but also quantitative disturbances, depending on the location of the primary tumor.

Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron.

Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

UHPLC/MS-Based Serum Metabolomics Reveals the Mechanism of Radiation-Induced Thrombocytopenia in Mice.

Radiation-induced thrombocytopenia is a common and life-threatening side effect of ionizing radiation (IR) therapy. However, the underlying pathological mechanisms remain unclear. In the present study, irradiation was demonstrated to significantly reduce platelet levels, inhibit megakaryocyte differentiation, and promote the apoptosis of bone marrow (BM) cells. A metabolomics approach and a UHPLC-QTOF MS system were subsequently employed for the comprehensive analysis of serum metabolic profiles of normal and irradiated mice. A total of 66 metabolites were significantly altered, of which 56 were up-regulated and 10 were down-regulated in irradiated mice compared to normal mice on day 11 after irradiation. Pathway analysis revealed that disorders in glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, sphingolipid metabolism, inositol phosphate metabolism, and tryptophan metabolism were involved in radiation-induced thrombocytopenia. In addition, three important differential metabolites, namely L-tryptophan, LysoPC (17:0), and D-sphinganine, which were up-regulated in irradiated mice, significantly induced the apoptosis of K562 cells. L-tryptophan inhibited megakaryocyte differentiation of K562 cells. Finally, serum metabolomics was performed on day 30 (i.e., when the platelet levels in irradiated mice recovered to normal levels). The contents of L-tryptophan, LysoPC (17:0), and D-sphinganine in normal and irradiated mice did not significantly differ on day 30 after irradiation. In conclusion, radiation can cause metabolic disorders, which are highly correlated with the apoptosis of hematopoietic cells and inhibition of megakaryocyte differentiation, ultimately resulting in thrombocytopenia. Further, the metabolites, L-tryptophan, LysoPC (17:0), and D-sphinganine can serve as biomarkers for radiation-induced thrombocytopenia.

Dietary sphinganine is selectively assimilated by members of the mammalian gut microbiome.

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet-microbiome interactions. Here, we used a click chemistry-based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine [sphinganine alkyne (SAA)] into the murine gut microbial community (bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet-microbiome interactions.

Sphingosine-1-phosphate in acute exercise and training.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid found in all eukaryotic cells. Although it may function as an intracellular second messenger, most of its effects are induced extracellularly via activation of a family of five specific membrane receptors. Sphingosine-1-phosphate is enriched in plasma, where it is transported by high-density lipoprotein and albumin, as well as in erythrocytes and platelets which store and release large amounts of this sphingolipid. Sphingosine-1-phosphate regulates a host of cellular processes such as growth, proliferation, differentiation, migration, and apoptosis suppression. It was also shown to play an important role in skeletal muscle physiology and pathophysiology. In recent years, S1P metabolism in both muscle and blood was found to be modulated by exercise. In this review, we summarize the current knowledge on the effect of acute exercise and training on S1P metabolism, highlighting the role of this sphingolipid in skeletal muscle adaptation to physical effort.

Pharmacological inhibition of sphingosine-1-phosphate lyase partially reverses spatial memory impairment in streptozotocin-diabetic rats.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with strong neuroprotective properties that is important for normal excitability and synaptic transmission in the hippocampal neurons. Considering the above, the aim of the present study was to determine whether increasing brain S1P level is able to reverse spatial memory impairment in streptozotocin-diabetic rats. The experiment was carried out on diabetic (n = 22) and nondiabetic (n = 10) male Wistar rats. Diabetes was induced by a single injection of streptozotocin. Eleven weeks later, 11 diabetic animals received injections of THI (S1P lyase inhibitor) for seven days. During the last five days of the experiment spatial reference memory acquisition and retention were tested in the Morris water maze task. The animals were then anaesthetized and samples of the hippocampus, prefrontal cortex, striatum, and cerebellum were excised. The content of S1P and related sphingolipids was measured using a HPLC method. Diabetes induced a depletion of ceramide in the hippocampus and cerebellum that was associated with impaired spatial memory and learning. Administration of THI to the diabetic animals prevented ceramide depletion in the hippocampus and cerebellum, and induced an increase in S1P content in all examined brain structures. These effects were associated with an improvement in spatial memory. We conclude that pharmacological inhibition of S1P lyase partially reverses the impairment in spatial memory induced by chronic hyperglycemia, and that this effect may be related to the prevention of ceramide depletion in the hippocampus and cerebellum, the increase in brain S1P level, or both.

Identification and distribution of gene clusters required for synthesis of sphingolipid metabolism inhibitors in diverse species of the filamentous fungus Fusarium.

BACKGROUND: Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs. RESULTS: Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM. CONCLUSION: Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.

Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin-induced cell death and Alternaria stem canker in tomato.

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)-toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map-based cloning of Arabidopsis FB1-resistant mutant fbr41, then ectopically expressed in AAL-toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41-overexpressing tomato plants exhibited less severe cell death phenotype upon AAL-toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41-overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL-toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41-overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT-induced PCD and controlling Alternaria stem canker in tomato.

Altered Levels of Sphingosine, Sphinganine and Their Ceramides in Atopic Dermatitis Are Related to Skin Barrier Function, Disease Severity and Local Cytokine Milieu.

Dysfunctional skin barrier plays a key role in the pathophysiology of atopic dermatitis (AD), a common inflammatory skin disease. Altered composition of ceramides is regarded as a major cause of skin barrier dysfunction, however it is not clear whether these changes are intrinsic or initiated by inflammation and aberrant immune response in AD. This study investigated the levels of free sphingoid bases (SBs) sphingosine and sphinganine and their ceramides and glucosylceramide in the stratum corneum (SC) and related them to skin barrier function, disease severity and local cytokine milieu. Ceramides were measured in healthy skin, and lesional and non-lesional skin of AD patients by a novel method based on deacylation of ceramides which were subsequently determined as corresponding sphingoid bases by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytokine levels were determined by multiplex immunoassay. Atopic skin showed increased levels of most investigated markers, predominantly in lesional skin. The largest difference in respect to healthy skin was found for glucosylceramide with respective median values of 0.23 (IQR 0.18-0.61), 0.56 (IQR 0.32-0.76) and 19.32 (IQR 7.86-27.62) pmol/g protein for healthy, non-lesional and lesional skin. The levels of investigated ceramide markers were correlated with disease severity (scoring atopic dermatitis, SCORAD) and skin barrier function (trans-epidermal water loss, TEWL) and furthermore with cytokines involved in innate, Th-1, and Th-2 immune response. Interestingly, the strongest association with SCORAD was found for sphinganine/sphingosine ratio (r = -0.69, p < 0.001; non-lesional skin), emphasizing the importance of SBs in AD. The highest correlation with TEWL was found for glucosylceramide (r2 = 0.60, p < 0.001), which was investigated for the first time in AD. Findings that the changes in SBs and ceramide levels were predominant in lesional skin and their association with disease severity and cytokine levels suggest an immune-system driven effect. a novel analysis method demonstrates a robust and simple approach that might facilitate wider use of lipid biomarkers in the clinics e.g., to monitor (immune) therapy or dissect disease endotypes.

Validation of urinary sphingolipid metabolites as biomarker of effect for fumonisins exposure in Kenyan children.

Context: Fumonisins (FNs), a group of mycotoxins produced mainly by Fusarium species, are ubiquitous food contaminants, especially for maize. Fumonisin B1 (FB1) caused severe toxicities in farm animals, induced kidney and liver tumours in rodents and is associated with many human adverse health effects, including oesophageal cancer. International Agency for Research on Cancer (IARC) categorizes FB1 as a possible human carcinogen (Group 2B). Inhibition of ceramide synthesis and disruption of sphingolipids metabolism are well studied as the major mechanisms of FB1-induced toxicity. Increases in sphinganine (Sa) and decrease in sphingosine (So) levels and their ratio are validated biomarkers of FB1 effects. Methods: In this study, we measured urinary levels of Sa, So and Sa/So in 284 children aged 1-14 years who consume maize as a staple diet. Exfoliated cells from urine were processed and sphingolipids quantified by High Pressure Liquid Chromatography. Results and conclusions: Sa and So were detectable in 95.07% and 98.94% of samples, respectively. Creatinine adjusted mean levels and standard deviation of Sa, So and Sa/So ratio were 1.23 ± 2.18, 4.99 ± 8.3 and 0.296 ± 0.587 nM. These results further confirmed the findings in studies with human adults, i.e. urinary Sa, So levels and Sa/So ratio are good biomarkers to assess FNs exposure in children.

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.

Preanalytical Biases in the Measurement of Human Blood Sphingolipids.

Dysregulation of blood sphingolipids is an emerging topic in clinical science. The objective of this study was to determine preanalytical biases that typically occur in clinical and translational studies and that influence measured blood sphingolipid levels. Therefore, we collected blood samples from four healthy male volunteers to investigate the effect of storage conditions (time, temperature, long-term storage, freeze⁻thaw cycles), blood drawing (venous or arterial sampling, prolonged venous compression), and sample preparation (centrifugation, freezing) on sphingolipid levels measured by LC-MS/MS. Our data show that sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (SA1P) were upregulated in whole blood samples in a time- and temperature-dependent manner. Increased centrifugation at higher speeds led to lower amounts of S1P and SA1P. All other preanalytical biases did not significantly alter the amounts of S1P and SA1P. Further, in almost all settings, we did not detect differences in (dihydro)ceramide levels. In summary, besides time-, temperature-, and centrifugation-dependent changes in S1P and SA1P levels, sphingolipids in blood remained stable under practically relevant preanalytical conditions.

ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.

ORMDL proteins are believed to be negative regulators of serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single-nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and total SL levels. Ormdl3-knockout (Ormdl3-/-) and transgenic (Ormdl3Tg/wt) mice were generated to study the effect of ORMDL3 on total SL levels in plasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormdl3-/- mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Furthermore, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population-based cohort of 971 individuals. Total C18-long chain bases were not significantly altered in the plasma and tissues of Ormdl3-/- mice, whereas C18-sphinganine showed a small and significant increase in plasma, lung, and liver tissues. Mouse embryonic fibroblast cells from Ormdl3-/- mice did not show an altered SPT activity compared with Ormdl3+/- and Ormdl3+/+ mice. Overexpression or knockdown of ORMDL3 in HEK293 cells did not alter SPT activity; however, parallel knockdown of all 3 ORMDL isoforms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long-chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seem not to be directly associated with changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.-Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.

Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.