The sphingolipid inhibitor myriocin increases Candida auris susceptibility to amphotericin B.

BACKGROUND: The emergence of the pathogenic yeast Candida auris is of global concern due to its ability to cause hospital outbreaks and develop resistance against all antifungal drug classes. Based on published data for baker's yeast Saccharomyces cerevisiae, sphingolipid biosynthesis, which is essential for maintaining membrane fluidity and formation of lipid rafts, could offer a target for additive treatment. METHODS: We analysed the susceptibility of C. auris to myriocin, which is an inhibitor of the de novo synthesis of sphingolipids in eukaryotic cells in comparison to other Candida species. In addition, we combined sublethal concentrations of myriocin with the antifungal drugs amphotericin B and fluconazole in E-tests. Consequently, the combinatory effects of myriocin and amphotericin B were examined in broth microdilution assays. RESULTS: Myriocin-mediated inhibition of the sphingolipid biosynthesis affected the growth of C. auris. Sublethal myriocin concentrations increased fungal susceptibility to amphotericin B. Isolates which are phenotypically resistant (≥2 mg/L) to amphotericin B became susceptible in presence of myriocin. However, addition of myriocin had only limited effects onto the susceptibility of C. auris against fluconazole. CONCLUSIONS: Our results show that inhibition of de novo sphingolipid biosynthesis increases the susceptibility of C. auris to amphotericin B. This may potentially enhance antifungal treatment options fighting this often resistant yeast pathogen.

FTY720 increases paclitaxel efficacy in cisplatin-resistant oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma has high recurrence and cisplatin resistance. As cancer stem cells, autophagy, and sphingolipids have been appointed as associated with chemotherapy resistance, we tested combined treatments targeting autophagy and/or sphingolipid metabolism with paclitaxel using cisplatin-resistant oral squamous cell carcinoma cells. METHODS: Cisplatin-resistant oral squamous cell carcinoma cells were maintained under exposition to FTY720 and chloroquine combined with paclitaxel and submitted to viability, clonogenicity, and spheres formation assays. The xenograft tumor model using cisplatin-resistant CAL27 cells was adopted to examine the drug combinations' potential antitumoral efficacy. Using an animal model, sphingolipids profiles from plasma and tissue samples were obtained by liquid chromatography coupled to mass spectrometry to identify potential lipids associated with drug response. RESULTS AND DISCUSSION: Our results showed higher autophagic flux in cisplatin-resistant Ooral squamous cell carcinoma (CAL27 and SCC9) cells than in parental cells. The combinations of an autophagy inhibitor (chloroquine) or an autophagy inducer/sphingosine kinase 1 antagonist (FTY720) with paclitaxel (PTX) had a synergistic antitumor effect. Treated CisR cells lost clonogenicity and tumor sphere abilities and reduced proteins associated with proliferation, survival, and cancer stem cells. FTY720 plus PTX had higher antitumor efficacy than PTX against CAL27 CisR xenograft tumor formation. Additionally, increases in glucosylceramide, dehydroglucosylceramide, and sphingomyelin were presented in responsive tumors. CONCLUSION: FTY720 sensitizes cisplatin-resistant oral squamous cell carcinoma cells for paclitaxel.

A lipidomic approach to bisphenol F-induced non-alcoholic fatty liver disease-like changes: altered lipid components in a murine model.

Bisphenol A (BPA), a typical environmental endocrine disruptor, is an "obesogen" that can induce lipid accumulation in the liver. Highly similar in structure to BPA, bisphenol F (BPF) is becoming the dominant BPA substitute on the market, which attracts more and more attention due to its potential adverse effects. Recently, BPF exposure is found to cause non-alcoholic fatty liver disease (NAFLD)-like changes; however, the underlying toxic effects remain poorly understood. Therefore, in the current study, we focused on BPF-mediated lipid homeostasis, especially the alterations of lipid components and metabolism. In human serum, the BPF levels in healthy controls and NAFLD patients were assessed by ELISA, and BPF-induced disturbance of lipid metabolism was evaluated in mouse model via non-targeted lipomic methods with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. It suggested that BPF exposure was positively correlated with NAFLD severity and triglyceride level in patients. Based on the relationships, lipid metabolites were assessed in mouse livers between control and BPF-treated group, and it revealed that twenty-six lipid metabolites (including phospholipids, sphingolipids, and glycerides) were significantly changed in mouse livers. Phosphatidylcholine, phosphatidylethanolamine, and diglyceryl ester levels were lower than those in the control mice; hexose ceramide content in sphingolipids markedly increased in BPF-treated mouse livers. Noteworthily, the glycerophospholipid metabolic pathway was found to be the most pronounced in BPF-induced disturbance of lipid metabolism. Therefore, the current study, for the first time, is deciphering the BPF-induced lipid metabolic disturbance, which may provide novel intervention strategies for BPF-induced NAFLD-like changes.

Phytosterol-mediated glycerosomes combined with peppermint oil enhance transdermal delivery of lappaconitine by modulating the lipid composition of the stratum corneum.

Although the introduction of glycerosomes has enriched strategies for efficient transdermal drug delivery, the inclusion of cholesterol as a membrane stabilizer has limited their clinical application. The current study describes the development and optimization of a new type of glycerosome (S-glycerosome) that is formed in glycerol solution with ß-sitosterol as the stabilizer. Moreover, the transdermal permeation properties of lappaconitine (LA)-loaded S-glycerosomes and peppermint oil (PO)-mediated S-glycerosomes (PO-S-glycerosomes) are evaluated, and the lipid alterations in the stratum corneum are analyzed via lipidomics. The LA-loaded S-glycerosomes prepared by the preferred formulation from the uniform design have a mean size of 145.3 ± 7.81 nm and an encapsulation efficiency of 73.14 ± 0.35%. Moreover, the addition of PO positively impacts transdermal flux, peaking at 0.4% (w/v) PO. Tracing of the fluorescent probe P4 further revealed that PO-S-glycerosomes penetrate deeper into the skin than S-glycerosomes and conventional liposomes. Additionally, treatment with PO-S-glycerosomes alters the isoform type, number, and composition of sphingolipids, glycerophospholipids, glycerolipids, and fatty acids in the stratum corneum, with the most notable effect observed for ceramides, the main component of sphingolipids. Furthermore, the transdermal administration of LA-loaded PO-S-glycerosomes improved the treatment efficacy of xylene-induced inflammation in mice without skin irritation. Collectively, these findings demonstrate the feasibility of ß-sitosterol as a stabilizer in glycerosomes. Additionally, the inclusion of PO improves the transdermal permeation of S-glycerosomes, potentially by altering the stratum corneum lipids.

The Effect of Silencing the Genes Responsible for the Level of Sphingosine-1-phosphate on the Apoptosis of Colon Cancer Cells.

Sphingosine-1-phosphate (S1P) and ceramides (Cer) are engaged in key events of signal transduction, but their involvement in the pathogenesis of colorectal cancer is not conclusive. The aim of our study was to investigate how the modulation of sphingolipid metabolism through the silencing of the genes involved in the formation (SPHK1) and degradation (SGPL1) of sphingosine-1-phosphate would affect the sphingolipid profile and apoptosis of HCT-116 human colorectal cancer cells. Silencing of SPHK1 expression decreased S1P content in HCT-116 cells, which was accompanied by an elevation in sphingosine, C18:0-Cer, and C18:1-Cer, increase in the expression and activation of Caspase-3 and -9, and augmentation of apoptosis. Interestingly, silencing of SGLP1 expression increased cellular content of both the S1P and Cer (C16:0-; C18:0-; C18:1-; C20:0-; and C22:0-Cer), yet inhibited activation of Caspase-3 and upregulated protein expression of Cathepsin-D. The above findings suggest that modulation of the S1P level and S1P/Cer ratio regulates both cellular apoptosis and CRC metastasis through Cathepsin-D modulation. The cellular ratio of S1P/Cer seems to be a crucial component of the above mechanism.

Sphingosine 1-Phosphate Activates S1PR3 to Induce a Proinflammatory Phenotype in Human Myometrial Cells.

One of the common mechanisms responsible for obstetric complications, affecting millions of women every year, is abnormal uterine contractility. Despite the critical importance of this process for women's health, the mechanisms of uterine contraction regulation remain poorly understood. The initiation of uterine smooth muscle (myometrial) contraction is an inflammatory process, accompanied by upregulation of proinflammatory genes and cytokine release. In this study, we show that sphingolipid metabolism is activated during human labor and that sphingosine 1-phosphate (S1P), the main bioactive sphingolipid, may modify the myometrial proinflammatory phenotype. Our data in both primary and immortalized human myometrial cells show that exogenous S1P induces a proinflammatory gene signature and upregulates the expression of known inflammatory markers of parturition, such as IL8 and COX2. Using expression of IL8 as a readout for S1P activity in myometrial cells, we established that these S1P effects are mediated through the activation of S1P receptor 3 (S1PR3) and downstream activation of ERK1/2 pathways. Inhibition of S1PR3 in human myometrial cells attenuates upregulation of IL8, COX2, and JUNB both at the mRNA and protein levels. Furthermore, activation of S1PR3 with a receptor-specific agonist recapitulated the effects seen after treatment with exogenous S1P. Collectively, these results suggest a signaling pathway activated by S1P in human myometrium during parturition and propose new targets for development of novel therapeutics to alter uterine contractility during management of preterm labor or labor dystocia.

The natural occurrence, toxicity mechanisms and management strategies of Fumonisin B1：A review.

Fumonisin B1 (FB1) contaminates various crops, causing huge losses to agriculture and livestock worldwide. This review summarizes the occurrence regularity, toxicity, toxic mechanisms and management strategies of FB1. Specifically, FB1 contamination is particularly serious in developing countries, humid and hot regions. FB1 exposure can produce different toxic effects on the nervous system, respiratory system, digestive system and reproductive system. Furthermore, FB1 can also cause systemic immunotoxicity. The mechanism of toxic effects of FB1 is to interfere with the normal pathway of sphingolipid de novo biosynthesis by acting as a competitive inhibitor of ceramide synthase. Meanwhile, the toxic products of sphingolipid metabolic disorders can cause oxidative stress and apoptosis. FB1 also often causes feed contamination by mixing with other mycotoxins, and then exerts combined toxicity. For detection, lateral flow dipstick technology and enzyme linked immunosorbent assay are widely used in the detection of FB1 in commercial feeds, while mainstream detection methods such as high performance liquid chromatography and liquid chromatography-mass spectrometry are widely used in the laboratory theoretical study of FB1. For purification means of FB1, some natural plant extracts (such as Zingiber officinale and Litsea Cubeba essential oil) and their active compounds have been proved to inhibit the toxic effects of FB1 and protect livestock due to their antifungal and antioxidant effects. Natural plant extract has the advantages of high efficiency, low cost and no contamination residue. This review can provide information for comprehensive understanding of FB1, and provide reference for formulating reasonable treatment and management strategies in livestock production.

Targeted sphingolipid analysis in chickens suggests different mechanisms of fumonisin toxicity in kidney, lung, and brain.

Most of the toxic effects of fumonisins can be related to sphingolipid alteration, but there is little sphingolipidomic data in animals fed fumonisins in organs other than the liver. This study aimed to measure fumonisin B1 (FB1) in kidney, lung, and brain and determine its effects on sphingolipids. Thirty chickens divided into three groups received a diet containing 20.8 mg FB1+FB2/kg for 0, 4, or 9 days. FB1 increased in kidney from 1.7 to 5.6 nmol/kg and in lung from 0.5 to 1 nmol/kg at 4 and 9 days, respectively. No FB1 was detected in brain. In kidney, sphinganine increased, C14-C16 ceramides decreased, whereas C18-C26 ceramides increased. Most of the changes in dihydroceramides, dihydrodeoxyceramides, deoxyceramides sphingomyelins, dihydrosphingomyelins, and hexosylceramides paralleled those on ceramides. In lung, sphinganine was unaffected by fumonisins, whereas sphinganine-1-phosphate increased. Other major changes corresponded to decreases in glycosylceramides. In brain, sphinganine was unchanged, whereas deoxysphinganine, sphingosine, C14-C20 ceramides, and C14-C20 sphingomyelins increased. These results revealed that alterations in sphingolipids in kidney were close to those measured in liver and could correspond to inhibition of ceramide synthase 5 activity. In contrast, effects of fumonisins in lung and brain cannot be explained by inhibition of ceramide synthase.

Desaturation of the Sphingofungin Polyketide Tail Results in Increased Serine Palmitoyltransferase Inhibition.

Serine palmitoyltransferase catalyzes the first step of the sphingolipid biosynthesis. Recently, sphingolipid homeostasis has been connected to several human diseases, making serine palmitoyltransferases an interesting therapeutic target. Known and efficient serine palmitoyltransferase-inhibitors are sphingofungins, a group of natural products isolated from fungi. To further characterize newly isolated sphingofungins, we designed an easy to use colorimetric serine palmitoyltransferase activity assay using FadD, which can be performed in 96-well plates. Because sphingofungins exert antifungal activitiy as well, we compared the in vitro assay results with an in vivo growth assay using Saccharomyces cerevisiae. The reported experiments showed differences among the assayed sphingofungins, highlighting an increase of activity based on the saturation levels of the polyketide tail. IMPORTANCE Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed. A central role is played by the serine palmitoyltransferase, which catalyzes the initial and rate limiting step of sphingolipid de novo synthesis and, as such, the development of inhibitory compounds for this enzyme is of interest. Our work here established an alternative approach for determining the activity of serine palmitoyltransferase adding another tool for the validation of its inhibition. We also determined the effect of different modifications to sphingofungins on their inhibitory activity against serine palmitoyltransferase, revealing important differences on said activity against enzymes of bacterial and fungal origin.

Paraquat exposure induces Parkinsonism by altering lipid profile and evoking neuroinflammation in the midbrain.

Paraquat (PQ) is the most widely used herbicide in the world and a well-known potent neurotoxin for humans. PQ exposure has been linked to increase the risk of Parkinson's disease (PD). However, the mechanism underlying its neurotoxic effects in PD pathogenesis is unclear. In our present study, C57BL/6J mice treated with PQ manifested severe motor deficits indicated by the significant reductions in suspension score, latency to fall from rotarod, and grip strength at 8 weeks after PQ exposure. Pathological hallmarks of Parkinsonism in the midbrain such as dopaminergic neuron loss, increased α-synuclein protein, and dysregulated PD-related genes were observed. Non-targeted lipidome analysis demonstrated that PQ exposure alters lipid profile and abundance, increases pro-inflammatory lipids.27 significantly altered subclasses of lipids belonged to 6 different lipid categories. Glycerophospholipids, sphingolipids, and glycerides were the most abundant lipids. Abundance of pro-inflammatory lipids such as Cer, LPC, LPS, and LPI was significantly increased in the midbrain. mRNA expressions of genes regulating ceramide biosynthesis in the midbrain were markedly up-regulated. Moreover, PQ exposure increased serum pro-inflammatory cytokines and provoked neuroinflammation in the midbrain. Pro-inflammatory lipids and cytokines in the midbrain were positively correlated with motor deficits. PQ poisoning in humans significantly also elevated serum pro-inflammatory cytokines and induced an intense systemic inflammation. In summary, we presented initial investigations of PQ induced molecular events related to the PD pathogenesis, capturing aspects of disturbed lipid metabolism, neuroinflammation, impairment of dopaminergic neurons in the midbrain, and an intense systemic inflammation. These neurotoxic effects of PQ exposure may mechanistically contribute to the pathogenesis of PQ induced Parkinsonism. Results of this study also strongly support the hypothesis that ever-increasing prevalence of Parkinson's disease is etiologically linked to the health risk of exposure to neurotoxic environmental pollutants.

Alpha4 contributes to the dysfunction of the pancreatic beta cell under metabolic stress.

The current study examined the roles of Alpha4, a non-canonical subunit of protein phosphatase 2A, in the regulation of acute (insulin secretion) and chronic (cell dysfunction) effects of glucose in pancreatic beta cells. Alpha4 is expressed in human islets, rat islets and INS-1832/13 cells. Incubation of INS-1832/13 cells and rat islets with high glucose (HG) significantly increased the expression of Alpha4. C2-Ceramide, a biologically active sphingolipid, also increased the expression of Alpha4 in INS-1832/13 cells and rat islets. Subcellular distribution studies of Alpha4 in low glucose (LG) and HG exposed INS-1832/13 cells revealed that it is predominantly cytosolic, and its expression is significantly increased in the non-nuclear/cytosolic fractions in cells exposed to HG. siRNA-mediated knockdown of Alpha4 exerted minimal effects on glucose- or KCl-induced insulin secretion. siRNA-mediated deletion of Alpha4 significantly increased p38MAPK and JNK1/2 phosphorylation under LG conditions, comparable to the degree seen under HG conditions. Paradoxically, a significant potentiation of HG-induced p38MAPK and JNK2 phosphorylation was noted following Alpha4 deletion. HG-induced CHOP expression (ER stress marker) and caspase-3 activation were markedly attenuated in cells following Alpha4 knockdown. Deletion of Alpha4 in INS-1832/13 cells prevented HG-induced loss in the expression of Connexin36, a gap junction channel protein, which has been implicated in normal beta cell function. Lastly, depletion of endogenous Alpha4 significantly reduced HG-induced cell death in INS-1832/13 cells. Based on these findings we conclude that Alpha4 contributes to HG-induced metabolic dysfunction of the islet beta cell.

Methuosis Contributes to Jaspine-B-Induced Cell Death.

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

Triterpenoid saponins from Ilex pubescens promote blood circulation in blood stasis syndrome by regulating sphingolipid metabolism and the PI3K/AKT/eNOS signaling pathway.

BACKGROUND: Blood stasis syndrome (BSS) is a severe disorder involving disturbances in glycerophosphocholine metabolism. Ilex pubescens (IP) can regulate the levels of lipids, such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE); however, the main active constituent of IP and its corresponding mechanism in BSS treatment are still unclear. PURPOSE: To explore the mechanisms by which triterpenoid saponins of IP (IPTS) promote blood circulation using system pharmacology-based approaches. METHODS: Sprague-Dawley (SD) rat BSS model was prepared by oral administration of IPTS for 7 days followed by adrenaline hydrochloride injection before immersion in ice water. Coagulation parameters in plasma and thromboxane B2 (TXB2), endothelin (ET) and 6-keto-PGF1α in serum were measured. The possible influence on abdominal aortas was evaluated by histopathology assessment. Human vein endothelial cells (HUVECs) were incubated with ox-LDL, and the effects of IPTS on cell viability and LDH release were investigated. UPLC-QTOF-MS/MS was used for metabolic profile analysis of lipid-soluble components in rat plasma and intracellular metabolites in HUVECs. Network pharmacology was used to predict the relevant targets and model pathways of BSS and the main components of IPTS. Molecular docking, molecular dynamics (MD) simulation and biochemical assays were used to predict molecular interactions between the active components of IPTS and target proteins. RT-PCR was used to detect the mRNA level of target proteins. Western blotting and immunohistochemistry (IHC) were used to verify the mechanisms by which IPTS promotes blood circulation in BSS. RESULTS: IPTS improved blood biochemical function in the process of BSS and played a role in vascular protection and maintenance of the normal morphology of blood vessels. Furthermore, metabolite pathways involved in steroid biosynthesis and sphingolipid metabolism were significantly perturbed. Both metabolomics analysis and network pharmacology results showed that IPTS ameliorates vascular injury and that lipid accumulation may be mediated by PI3K/AKT signaling pathway activation. MD simulation and enzyme inhibitory activity results suggested that the main components of IPTS can form stable complexes with PI3K, AKT and eNOS and that the complexes have significant binding affinity. PI3K, AKT, p-AKT, and eNOS mRNA and protein levels were considerably elevated in the IPTS-treated group. Thus, IPTS protects the vasculature by regulating the PI3K/AKT signaling pathway, activating eNOS and increasing the release of NO. CONCLUSION: A possible mechanism by which IPTS prevents BSS is proposed: IPTS can promote blood circulation by modulating sphingolipid metabolism and activating the PI3K/AKT/eNOS signaling pathways.

Targeted Analysis of Sphingolipids in Turkeys Fed Fusariotoxins: First Evidence of Key Changes That Could Help Explain Their Relative Resistance to Fumonisin Toxicity.

The effects of fumonisins on sphingolipids in turkeys are unknown, except for the increased sphinganine to sphingosine ratio (Sa:So) used as a biomarker. Fumonisins fed at 20.2 mg/kg for 14 days were responsible for a 4.4 fold increase in the Sa:So ratio and a decrease of 33% and 36% in C14-C16 ceramides and C14-C16 sphingomyelins, respectively, whereas C18-C26 ceramides and C18-C26 sphingomyelins remained unaffected or were increased. Glucosyl- and lactosyl-ceramides paralleled the concentrations of ceramides. Fumonisins also increased dihydroceramides but had no effect on deoxysphinganine. A partial least squfares discriminant analysis revealed that all changes in sphingolipids were important in explaining the effect of fumonisins. Because deoxynivalenol and zearalenone are often found in feed, their effects on sphingolipids alone and in combination with fumonisins were investigated. Feeding 5.12 mg deoxynivalenol/kg reduced dihydroceramides in the liver. Zearalenone fed at 0.47 mg/kg had no effect on sphingolipids. When fusariotoxins were fed simultaneously, the effects on sphingolipids were similar to those observed in turkeys fed fumonisins alone. The concentration of fumonisin B1 in the liver of turkeys fed fumonisins was 0.06 µmol/kg. Changes in sphingolipid concentrations differed but were consistent with the IC50 of fumonisin B1 measured in mammals; these changes could explain the relative resistance of turkeys to fumonisins.

Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.

Sphingolipids are key signaling lipids and their dysregulation has been associated with various cellular processes. We have previously shown significant changes in sphingolipids in therapy-induced senescence, a state of cell cycle arrest as a response to chemotherapy, including the accumulation of ceramides, and provided evidence suggesting that ceramide processing is important for this process. Herein, we conducted a focused small molecule inhibitor screen targeting the sphingolipid pathway, which highlighted a new lipid regulator of therapy-induced senescence. Among the inhibitors tested, the inhibition of ceramide kinase by NVP-231 reduced the levels of senescent cells. Ceramide kinase knockdown exhibited similar effects, strongly supporting the involvement of ceramide kinase during this process. We showed that ceramide-1-phosphate was upregulated in therapy-induced senescence and that NVP-231 reduced ceramide-1-phosphate levels in different cell line models of therapy-induced senescence. Finally, ceramide-1-phosphate addition to NVP-231-treated cells reversed the effects of NVP-231 during senescence. Overall, our results identify a previously unknown lipid player in therapy-induced senescence and highlight a potential targetable enzyme to reduce the levels of therapy-induced senescent cells.

Bioactive lipids from the fruits of Solanum xanthocarpum and their anti-inflammatory activities.

Bioactive lipids widely found in daily consumed plants and animals are essential or beneficial to health and some of them are important physiological regulators in the human body. In our current investigation, 18 bioactive lipids (1-18), including 8 sphingolipids (1-8), 7 oxylipins (9-15), 3 phenolic lipids (16-18) were isolated from the fruits of Solanum xanthocarpum. And compounds 1, 9, 15, 16, and 18 were new lipids. In this study, homologues (4-8, 16, and 17) and configuration isomers (2 and 3) of bioactive lipids were separated, and NMR combined with MS/MS2 was an effective method to identify these compounds. These findings provided the reference for the separation and structural identification of bioactive lipids. The anti-inflammatory activities of all isolated lipids were evaluated by their inhibition of the NO release of LPS-induced RAW 264.7 cells. Aglycone components of sphingolipids, oxylipids with free carboxylic acid groups, phenylpropionic acid-fatty acid glyceride polymer exhibited significant anti-inflammatory activities. Further analysis by molecular docking revealed the interactions of bioactive compounds with the iNOS protein.

1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development.

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain.

The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.

Metabolism of HSAN1- and T2DM-associated 1-deoxy-sphingolipids inhibits the migration of fibroblasts.

Hereditary sensory neuropathy type 1 (HSAN1) is a rare axonopathy, characterized by a progressive loss of sensation (pain, temperature, and vibration), neuropathic pain, and wound healing defects. HSAN1 is caused by several missense mutations in the serine palmitoyltransferase long-chain base subunit 1 and serine palmitoyltransferase long-chain base subunit 2 of the enzyme serine palmitoyltransferase-the key enzyme for the synthesis of sphingolipids. The mutations change the substrate specificity of serine palmitoyltransferase, which then forms an atypical class of 1-deoxy-sphinglipids (1-deoxySLs). Similarly, patients with type 2 diabetes mellitus also present with elevated 1-deoxySLs and a comparable clinical phenotype. The effect of 1-deoxySLs on neuronal cells was investigated in detail, but their impact on other cell types remains elusive. Here, we investigated the consequences of externally added 1-deoxySLs on the migration of fibroblasts in a scratch assay as a simplified cellular wound-healing model. We showed that 1-deoxy-sphinganine (1-deoxySA) inhibits the migration of NIH-3T3 fibroblasts in a dose- and time-dependent manner. This was not seen for a non-native, L-threo stereoisomer. Supplemented 1-deoxySA was metabolized to 1-deoxy-(dihydro)ceramide and downstream to 1-deoxy-sphingosine. Inhibiting downstream metabolism by blocking N-acylation rescued the migration phenotype. In contrast, adding 1-deoxy-sphingosine had a lesser effect on cell migration but caused the massive formation of intracellular vacuoles. Further experiments showed that the effect on cell migration was primarily mediated by 1-deoxy-dihydroceramides rather than by the free base or 1-deoxyceramides. Based on these findings, we suggest that limiting the N-acylation of 1-deoxySA could be a therapeutic approach to improve cell migration and wound healing in patients with HSAN1 and type 2 diabetes mellitus.

Dietary Ceramide Prepared from Soy Sauce Lees Improves Skin Barrier Function in Hairless Mice.

Dietary sphingolipids such as glucosylceramide and sphingomyelin are known to improve the skin barrier function of damaged skin. In this study, we focused on free-ceramide prepared from soy sauce lees, which is a byproduct of soy sauce production. The effects of dietary soy sauce lees ceramide on the skin of normal mice were evaluated and compared with those of dietary maize glucosylceramide. We found that transepidermal water loss value was significantly suppressed by dietary supplementation with soy sauce lees ceramide as effectively as or more effectively than maize glucosylceramide. Although the content of total and each subclass of ceramide in the epidermis was not significantly altered by dietary sphingolipids, that of 12 types of ceramide molecules, which were not present in dietary sources, was significantly increased upon ingestion of maize glucosylceramide and showed a tendency to increase with soy sauce lees ceramide intake. In addition, the mRNA expression of ceramide synthase 4 and involucrin in the skin was downregulated by sphingolipids. This study, for the first time, demonstrated that dietary soy sauce lees ceramide enhances skin barrier function in normal hairless mice, although further studies are needed to clarify the molecular mechanism.