Search

Scope: Branched-chain amino acids, especially leucine, have been reported to play a role in regulating lipid metabolism. This study aims to examine the effects of leucine deprivation on hepatic lipid metabolism., Methods and Results: C57BL/6 mice are fed with a chow diet (control group, n = 8) or a leucine-free diet (-Leu group, n = 8) for 7 days. Histology, lipidomics, targeted metabolomics, and transcriptomics are performed to analyze the liver tissue. Compared to control group, -Leu group exhibits a notably reduced liver weight, accompanied by hepatic injury, and disorders of lipid metabolism. The level of sphingomyelin (SM) is significantly increased in the liver of -Leu group, while the glycerolipids (GL) level is significantly decreased. The expression of sphingomyelin synthase 1 (SGMS1) is upregulated by leucine deprivation in a time-dependent manner, leading to hepatic SM accumulation. Moreover, leucine deprivation results in hepatic GL loss via suppressing fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1) expression., Conclusion: The findings demonstrate that leucine deprivation results in abnormal lipid metabolism in the liver, mainly manifested as SM accumulation and GL loss. These results provide insights into the role of leucine in regulating lipid metabolism., (© 2023 Wiley-VCH GmbH.)

Neurotransmitter release from sympathetic terminals is a key avenue for heart regulation. Herein, presynaptic exocytotic activity was monitored in mice atrial tissue using a false fluorescent neurotransmitter FFN511, a substrate for monoamine transporters. FFN511 labeling had similarity with tyrosine hydroxylase immunostaining. High [K + ] o depolarization caused FFN511 release, which was augmented by reserpine, an inhibitor of neurotransmitter uptake. However, reserpine lost the ability to increase depolarization-induced FFN511 unloading after depletion of ready releasable pool with hyperosmotic sucrose. Cholesterol oxidase and sphingomyelinase modified atrial membranes, changing in opposite manner fluorescence of lipid ordering-sensitive probe. Plasmalemmal cholesterol oxidation increased FFN511 release upon K + -depolarization and more markedly potentiated FFN511 unloading in the presence of reserpine. Hydrolysis of plasmalemmal sphingomyelin profoundly enhanced the rate of FFN511 loss due to K + -depolarization, but completely prevented potentiating action of reserpine on FFN511 unloading. If cholesterol oxidase or sphingomyelinase got access to membranes of recycling synaptic vesicles, then the enzyme effects were suppressed. Hence, a fast neurotransmitter reuptake dependent on exocytosis of vesicles from ready releasable pool occurs during presynaptic activity. This reuptake can be enhanced or inhibited by plasmalemmal cholesterol oxidation or sphingomyelin hydrolysis, respectively. These modifications of plasmalemmal (but not vesicular) lipids increase the evoked neurotransmitter release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All authors consent to publish the article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Sphingomyelinase (SMase), a hydrolase of sphingomyelin (SM) enriched in the outer leaflet of the plasma membrane of mammalian cells, is closely associated with the onset and development of many diseases, but the specific mechanisms of SMase on the cell structure, function, and behavior are not yet fully understood due to the complexity of the cell structure. Artificial cells are minimal biological systems constructed from various molecular components designed to mimic cellular processes, behaviors, and structures, which are excellent models for studying biochemical reactions and dynamic changes in cell membranes. In this work, we presented an artificial cell model that mimics the lipid composition and content of the outer leaflet of mammalian plasma membranes for studying the effect of SMase on cell behavior. The results confirmed that the artificial cells can respond to SM degradation by producing ceramides that enrich and alter the membrane charge and permeability, thus inducing the budding and fission of the artificial cells. Thus, the artificial cells developed here provide a powerful tool to study the mechanism of action of cell membrane lipids on cell biological behavior, paving the way for further molecular mechanism studies.

Aims: Sphingomyelin is an abundant component of the presynaptic membrane and an organizer of lipid rafts. In several pathological conditions, sphingomyelin is hydrolyzed due to an upregulation and release of secretory sphingomyelinases (SMases). Herein, the effects of SMase on exocytotic neurotransmitter release were studied in the diaphragm neuromuscular junctions of mice., Main Methods: Microelectrode recordings of postsynaptic potentials and styryl (FM) dyes were used to estimate neuromuscular transmission. Membrane properties were assessed with fluorescent techniques., Key Findings: Application of SMase at a low concentration (0.01 U ml -1 ) led to a disruption of lipid-packing in the synaptic membranes. Neither spontaneous exocytosis nor evoked neurotransmitter release (in response to single stimuli) were affected by SMase treatment. However, SMase significantly increased neurotransmitter release and the rate of fluorescent FM-dye loss from the synaptic vesicles at 10, 20 and 70 Hz stimulation of the motor nerve. In addition, SMase treatment prevented a shift of the exocytotic mode from "full-collapse" fusion to "kiss-and-run" during high-frequency (70 Hz) activity. The potentiating effects of SMase on neurotransmitter release and FM-dye unloading were suppressed when synaptic vesicle membranes were also exposed to this enzyme (i.e., stimulation occurred during SMase treatment)., Significance: Thus, hydrolysis of the plasma membrane sphingomyelin can enhance mobilization of synaptic vesicles and facilitate full fusion mode of exocytosis, but SMase acting on vesicular membrane had a depressant effect on the neurotransmission. Partially, the effects of SMase can be related with the changes in synaptic membrane properties and intracellular signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All authors consent to publish the article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Scope: Milk fat globule membrane (MFGM) is an essential component of milk. Bovine MFGM (bMFGM) has been shown to support cognitive development and increase relative concentrations of serum phospholipids. This study investigates bioavailability of bMFGM components after oral administration in two preclinical models to explore whether dietary bMFGM induces parallel changes to plasma and brain lipidomes., Methods and Results: Transgenic APOE*3.Leiden mice (n = 18 per group) and Sprague-Dawley rats (n = 12 per group) are fed bMFGM-enriched (MFGM+) or Control diet, followed by phospholipid profile-determination in plasma, hippocampus, and prefrontal cortex tissue by targeted mass spectrometry. Multivariate analysis of lipidomic profiles demonstrates a separation between MFGM+ and Control plasma across rodents. In plasma, sphingomyelins contributed the most to the separation of lipid patterns among both models, where three sphingomyelins (d18:1/14:0, d18:1/23:0, d18:1/23:1[9Z]) are consistently higher in the circulation of MFGM+ groups. A similar trend is observed in rat prefrontal cortex, although no significant separation of the brain lipidome is demonstrated., Conclusion: bMFGM-enriched diet alters plasma phospholipid composition in rodents, predominantly increasing sphingomyelin levels in the systemic circulation with similar, but non-significant, trends in central brain regions. These changes may contribute to the beneficial effects of bMFGM on neurodevelopment during early life., (© 2022 Wiley-VCH GmbH.)

Sphingomyelinases (SMase), enzymes that catalyze the hydrolysis of sphingomyelin to ceramide, are important sensors for inflammatory cytokines and apoptotic signaling. Studies have provided evidence that increased SMase activity can contribute to retinal injury. In most tissues, two major SMases are responsible for stress-induced increases in ceramide: acid sphingomyelinase (ASMase) and Mg 2+ -dependent neutral sphingomyelinase (NSMase). The purposes of the current study were to determine the localization of SMases and their substrates in the retina and optic nerve head and to investigate the effects of ocular hypertension and ischemia on ASMase and NSMase activities. Tissue and cellular localization of ASMase and NSMase were determined by immunofluorescence imaging. Tissue localization of sphingomyelin in retinas was further determined by Matrix-Assisted Laser Desorption/Ionization mass spectrometry imaging. Tissue levels of sphingomyelins and ceramide were determined by liquid chromatography with tandem mass spectrometry. Sphingomyelinase activities under basal conditions and following acute ischemic and ocular hypotensive stress were measured using the Amplex Red Sphingomyelinase Assay Kit. Our data show that ASMase is in the optic nerve head and the retinal ganglion cell layer. NSMase is in the optic nerve head, photoreceptor and retinal ganglion cell layers. Both ASMase and NSMase were identified in human induced pluripotent stem cell-derived retinal ganglion cells and optic nerve head astrocytes. The retina and optic nerve head each exhibited unique distribution of sphingomyelins with the abundance of very long chain species being higher in the optic nerve head than in the retina. Basal activities for ASMase in retinas and optic nerve heads were 54.98 ± 2.5 and 95.6 ± 19.5 mU/mg protein, respectively. Ocular ischemia significantly increased ASMase activity to 86.2 ± 15.3 mU/mg protein in retinas (P = 0.03) but not in optic nerve heads (81.1 ± 15.3 mU/mg protein). Ocular hypertension significantly increased ASMase activity to 121.6 ± 7.3 mU/mg protein in retinas (P < 0.001) and 267.0 ± 66.3 mU/mg protein in optic nerve heads (P = 0.03). Basal activities for NSMase in retinas and optic nerve heads were 12.3 ± 2.1 and 37.9 ± 8.7 mU/mg protein, respectively. No significant change in NSMase activity was measured following ocular ischemia or hypertension. Our results provide evidence that both ASMase and NSMase are expressed in retinas and optic nerve heads; however, basal ASMase activity is significantly higher than NSMase activity in retinas and optic nerve heads. In addition, only ASMase activity was significantly increased in ocular ischemia or hypertension. These data support a role for ASMase-mediated sphingolipid metabolism in the development of retinal ischemic and hypertensive injuries., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Patients with inflammatory bowel diseases tend to show alteration of lipid profiles. It remains unknown whether dietary intake with specific lipids, such as phosphatidylcholine (PC) and sphingomyelin (SM), have distinguishable effects against IBD. Here, a preclinical study using dextran sulphate sodium (DSS)-induced colitis mice model was applied to explore/compare the effects by PC, and SM. Results showed that PC treatment (p.o., 30 mg/kg b.w., 15 d) exerted higher inhibitory activity than the same dosage of SM supplementation on colonic tissue lesions and pro-inflammatory cytokines expressions induced by DSS. Integrative analysis of the metabolome and microbiome indicated that PC and SM supplementation could modulate endogenous tryptophan metabolism, arginine and proline metabolism, purine metabolism, bile secretion, as well as vitamin digestion and absorption, closely correlated with their regulation on the abundance of Lactobacillus, Faecalibacterium, Dubosiella, Turicibacter, and Parasutterella communities in the gut. Based on these data, PC is a more promising candidate for preventing colitis than SM. Our findings provided a scientific foundation for further clinical research to screen more efficient dietary intervention strategy for colitis prevention., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Niemann-Pick type A disease (NPA) is a rare lysosomal storage disorder caused by mutations in the gene coding for the lysosomal enzyme acid sphingomyelinase (ASM). ASM deficiency leads to the consequent accumulation of its uncatabolized substrate, the sphingolipid sphingomyelin (SM), causing severe progressive brain disease. To study the effect of the aberrant lysosomal accumulation of SM on cell homeostasis, we loaded skin fibroblasts derived from a NPA patient with exogenous SM to mimic the levels of accumulation characteristic of the pathological neurons. In SM-loaded NPA fibroblasts, we found the blockage of the autophagy flux and the impairment of the mitochondrial compartment paralleled by the altered transcription of several genes, mainly belonging to the electron transport chain machinery and to the cholesterol biosynthesis pathway. In addition, SM loading induces the nuclear translocation of the transcription factor EB that promotes the lysosomal biogenesis and exocytosis. Interestingly, we obtained similar biochemical findings in the brain of the NPA mouse model lacking ASM (ASMKO mouse) at the neurodegenerative stage. Our work provides a new in vitro model to study NPA etiopathology and suggests the existence of a pathogenic lysosome-plasma membrane axis that with an impairment in the mitochondrial activity is responsible for the cell death., (© 2022. The Author(s).)

Reactive oxygen species (ROS) generated by ultraviolet (UV) exposure cause skin barrier dysfunction, which leads to dry skin. In this study, the skin moisturizing effect of sphingomyelin-containing milk phospholipids in UV-induced hairless mice was evaluated. Hairless mice were irradiated with UVB for eight weeks, and milk phospholipids (50, 100, and 150 mg/kg) were administered daily. Milk phospholipids suppressed UV-induced increase in erythema and skin thickness, decreased transepidermal water loss, and increased skin moisture. Milk phospholipids increased the expression of filaggrin, involucrin, and aquaporin3 (AQP3), which are skin moisture-related factors. Additionally, hyaluronic acid (HA) content in the skin tissue was maintained by regulating the expression of HA synthesis- and degradation-related enzymes. Milk phospholipids alleviated UV-induced decrease in the expression of the antioxidant enzymes superoxidase dismutase1 and 2, catalase, and glutathione peroxidase1. Moreover, ROS levels were reduced by regulating heme oxygenase-1 (HO-1), an ROS regulator, through milk phospholipid-mediated activation of nuclear factor erythroid-2-related factor 2 (Nrf2). Collectively, sphingomyelin-containing milk phospholipids contributed to moisturizing the skin by maintaining HA content and reducing ROS levels in UVB-irradiated hairless mice, thereby, minimizing damage to the skin barrier caused by photoaging.

Sphingomyelin nanosystems have already shown to be promising carriers for efficient delivery of anticancer drugs. For further application in the treatment of pancreatic tumor, the investigation on relevant in vitro models able to reproduce its physio-pathological complexity is mandatory. Accordingly, a 3D heterotype spheroid model of pancreatic tumor has been herein constructed to investigate the potential of bare and polyethylene glycol-modified lipid nanosystems in terms of their ability to penetrate the tumor mass and deliver drugs. Regardless of their surface properties, the lipid nanosystems successfully diffused through the spheroid without inducing toxicity, showing a clear safety profile. Loading of the bare nanosystems with a lipid prodrug of gemcitabine was used to evaluate their therapeutic potential. While the nanosystems were more effective than the free drug on 2D cell monocultures, this advantage, despite their efficient penetration capacity, was lost in the 3D tumor model. The latter, being able to mimic the tumor and its microenvironment, was capable to provide a more realistic information on the cell sensitivity to treatments. These results highlight the importance of using appropriate 3D tumor models as tools for proper in vitro evaluation of nanomedicine efficacy and their timely optimisation, so as to identify the best candidates for later in vivo evaluation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Senescent cells accumulation can contribute to the development of several age-related diseases, including cancer. Targeting and eliminating senescence cells, would allow the development of new therapeutic approaches for the treatment of different diseases. The 4N1Ks peptide, a 10 amino acid peptide derived from TSP1 protein, combines both features by targeting the CD47 receptor present in the surface of senescent cells and demonstrating senolytic activity, thereby representing a new strategy to take into account. Nonetheless, peptide drugs are known for their biopharmaceutical issues, such as low short half-life and tendency to aggregate, which reduces their bioavailability and limits their therapeutic potential. In order to overcome this problem, herein we propose the use of biodegradable and biocompatible sphingomyelin nanosystems (SNs), decorated with this peptide for the targeting of senescent cells. In order to efficiently associate the 4N1Ks peptide to the nanosystems while exposing it on their surface for an effective targeting of senescent cells, the 4N1Ks peptide was chemically conjugated to a PEGylated hydrophobic chain. The resulting SNs-4N1Ks (SNs-Ks), were extensively characterized for their physicochemical properties, by dynamic light scattering, multiple-angle dynamic light scattering, nanoparticle tracking analysis and atomic force microscopy. The SNs-Ks demonstrated suitable features in terms of size (∼100 nm), association efficiency (87.2 ± 6.9%) and stability in different biorelevant media. Cell toxicity experiments in MCF7 cancer cells indicated an improved cytotoxic effect of SNs-Ks, decreasing cancer cells capacity to form colonies, with respect to free peptide, and an improved hemocompatibility. Lastly, senescence escape preliminary experiments demonstrated the improvement of SNs-Ks senolytic activity of in chemotherapy-induced senescence model of breast cancer cells. Therefore, these results demonstrate for the first time the potential of the combination of SNs with 4N1Ks peptide for the development of innovative senolytic therapies to battle cancer., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Doxorubicin (DOX) is one of the most efficient antitumor drugs employed in numerous cancer therapies. Its incorporation into lipid-based nanocarriers, such as liposomes, improves the drug targeting into tumor cells and reduces drug side effects. The carriers' lipid composition is expected to affect the interactions of DOX and its partitioning into liposomal membranes. To get a rational insight into this aspect and determine promising lipid compositions, we use numerical simulations, which provide unique information on DOX-membrane interactions at the atomic level of resolution. In particular, we combine classical molecular dynamics simulations and free energy calculations to elucidate the mechanism of penetration of a protonated Doxorubicin molecule (DOX + ) into potential liposome membranes, here modeled as lipid bilayers based on mixtures of phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol lipid molecules, of different compositions and lipid phases. Moreover, we analyze DOX + partitioning into relevant regions of SM-based lipid bilayer systems using a combination of free energy methods. Our results show that DOX + penetration and partitioning are facilitated into less tightly packed SM-based membranes and are dependent on lipid composition. This work paves the way to further investigations of optimal formulations for lipid-based carriers, such as those associated with pH-responsive membranes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

Host cell lipids play a pivotal role in the pathogenesis of respiratory virus infection. However, a direct comparison of the lipidomic profile of influenza virus and rhinovirus infections is lacking. In this study, we first compared the lipid profile of influenza virus and rhinovirus infection in a bronchial epithelial cell line. Most lipid features were downregulated for both influenza virus and rhinovirus, especially for the sphingomyelin features. Pathway analysis showed that sphingolipid metabolism was the most perturbed pathway. Functional study showed that bacterial sphingomyelinase suppressed influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, but promoted rhinovirus replication. These findings suggest that sphingomyelin pathway can be a potential target for antiviral therapy, but should be carefully evaluated as it has opposite effects on different respiratory viruses. Furthermore, the differential effect of sphingomyelinase on rhinovirus and influenza virus may explain the interference between rhinovirus and influenza virus infection.

Determining mechanisms that naturally protect species from developing cancer is critical in order to prevent and treat cancer. Here, we describe a novel cancer-suppressing mechanism, via the secretion of bioactive factors by mammary cells, that is present in domesticated mammals with a low mammary cancer incidence. Specifically, these bioactive factors induced triple-negative breast cancer cell (TNBC) death in vitro and reduced tumorigenicity in a xenograft TNBC mouse model in vivo. RNA deep sequencing showed significant downregulation of genes associated with breast cancer progression in secretome-cultured TNBC cells. Further in-depth multi-omics analysis identified sphingomyelins as key secreted factors, and their role was confirmed via inhibition of the sphingomyelin signaling pathway. We speculate that secreted sphingomyelins in the mammary gland of mammals with a naturally low incidence of mammary cancer mediate the elimination of cancer cells. This study contributes to the growing list of protective mechanisms identified in cancer-proof species.

The structures of bioactive polar lipids (PLs) of Irish ale with potent antithrombotic and cardioprotective properties were elucidated. Ale PL was fractionated by preparative thin layer chromatography (TLC) into subclasses, and their antithrombotic effect was assessed against human platelet aggregation induced by the pro-inflammatory mediator, platelet-activating factor (PAF). The fatty acid content and the overall structures of ale PL were elucidated by liquid chromatography mass spectrometry (LC-MS). Phosphatidylcholines (PC) and molecules of the sphingomyelin (SM) family exhibited the strongest anti-PAF effects, followed by phosphatidylethanolamines (PE). PC contained higher amounts of omega-3 polyunsaturated fatty acids (n-3 PUFA) and thus the lowest n-6/n-3 ratio. Bioactive diacyl and alkyl-acyl PC and PE molecules bearing n-3 PUFA at their sn -2 position, especially docosahexaenoic acid (DHA) and α-linolenic acid (ALA) but mostly oleic acid (OA), were identified in both PC and PE subclasses. Eicosapentaenoic acid (EPA) was present only in bioactive PC molecules and not in PE, explaining the lower anti-PAF effects of PE. Bioactive sphingolipid and glycolipid molecules with reported anti-inflammatory and anti-tumour properties, such as specific ceramides and glucosylcerebrosides with sphingosine, phytosphingosine and dihydrosphingosine bases but also specific monogalactodiglycerides and SM species bearing ALA at their sn -2 position, were identified in the SM subclass, providing a rational for its strong bioactivities against the PAF pathway. Further studies are required on the health benefits of bioactive PL from beer and brewery by-products.

Milk sphingomyelin (SM), a polar lipid (PL) component of milk fat globule membranes, is protective against dyslipidemia. However, it is unclear whether ingestion of milk PLs protect against atherosclerosis. To determine this, male LDLr -/- mice (age 6 weeks) were fed ad libitum either a high-fat, added-cholesterol diet (CTL; 45% kcal from fat, 0.2% cholesterol by weight; n=15) or the same diet supplemented with 1% milk PL (1% MPL; n=15) or 2% milk PL (2% MPL; n=15) added by weight from butter serum. After 14 weeks on diets, mice fed 2% MPL had significantly lower serum cholesterol (-51%) compared to CTL (P<.01), with dose-dependent effects in lowering VLDL- and LDL-cholesterol. Mice fed 2% MPL displayed lower inflammatory markers in the serum, liver, adipose and aorta. Notably, milk PLs reduced atherosclerosis development in both the thoracic aorta and the aortic root, with 2% MPL-fed mice having significantly lower neutral lipid plaque size by 59% (P<.01) and 71% (P<.02) compared to CTL, respectively. Additionally, the 2% MPL-fed mice had greater relative abundance of Bacteroidetes, Actinobacteria and Bifidobacterium, and lower Firmicutes in cecal feces compared to CTL. Milk PL feeding resulted in significantly different microbial communities as demonstrated by altered beta diversity indices. In summary, 2% MPL strongly reduced atherogenic lipoprotein cholesterol, modulated gut microbiota, lowered inflammation and attenuated atherosclerosis development. Thus, milk PL content may be important to consider when choosing dairy products as foods for cardiovascular disease prevention., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Free radical driven lipid peroxidation is a chain reaction which can lead to oxidative degradation of biological membranes. Propagation vs. termination rates of peroxidation in biological membranes are determined by a variety of factors including fatty acyl chain composition, presence of antioxidants, as well as biophysical properties of mono- or bilayers. Sphingomyelins (SMs), a class of sphingophospholipids, were previously described to inhibit lipid oxidation most probably via the formation of H-bond network within membranes. To address the "antioxidant" potential of SMs, we performed LC-MS/MS analysis of model SM/glycerophosphatidylcholine (PC) liposomes with different SM fraction after induction of radical driven lipid peroxidation. Increasing SM fraction led to a strong suppression of lipid peroxidation. Electrochemical oxidation of non-liposomal SMs eliminated the observed effect, indicating the importance of membrane structure for inhibition of peroxidation propagation. High resolution MS analysis of lipid peroxidation products (LPPs) observed in in vitro oxidized SM/PC liposomes allowed to identify and relatively quantify SM- and PC-derived LPPs. Moreover, mapping quantified LPPs to the known pathways of lipid peroxidation allowed to demonstrate significant decrease in mono-hydroxy(epoxy) LPPs relative to mono-keto derivatives in SM-rich liposomes. The results presented here illustrate an important property of SMs in biological membranes, acting as "biophysical antioxidant". Furthermore, a ratio between mono-keto/mono-hydroxy(epoxy) oxidized species can be used as a marker of lipid peroxidation propagation in the presence of different antioxidants.

This study aimed to determine the metabolites associated with ventricular septal defect (VSD) and the underlying mechanisms. Blood samples and thymus tissues were collected from VSD patients to perform LC-MS-based metabolomics assay and generate iPS cell-derived cardiomyocytes, respectively. VSD rat model was used in vivo study. RT-PCR, western blotting, immunohistochemistry, luciferase activity assay, GFP-LC3 adenovirus and GFP and RFP tfLC3 assay, and transmission electron microscopy were performed to investigate the underlying mechanisms. The metabolites uric acid (UA) and sphingomyelin (SM) increased in the serum of VSD patients, along with enhanced autophagy. The combination of UA and SM treatment could promote autophagy and inhibit EGFR and AKT3 expressions. Overexpression of EGFR and AKT3 suppressed autophagy in UA and SM-treated cardiomyocytes, respectively. Also, lncRNA MEG3 knockdown and overexpression could enhance and inhibit autophagy in UA and SM-treated cardiomyocytes, respectively, through targeting miR-7-5p. Moreover, miR-7-5p mimics and inhibitors promoted and inhibited autophagy in UA and SM-treated cardiomyocytes, respectively, via target EGFR. In VSD rat model, upregulation of MEG3 could reverse high level of autophagy and decrease serum UA and SM. In conclusion, UA and SM are essential VSD-associated metabolic biomarkers and MEG3/miR-7-5p/EGFR axis is critical to the regulation of autophagy in cardiomyocytes.

In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection., (Copyright © 2019 Hollmann, Wiese, Dennstädt, Fink, Schneider-Schaulies and Beyersdorf.)

Western-style diets have been linked with dyslipidemia and inflammation, two well-known risk factors associated with cardiovascular disease (CVD). Dietary sphingomyelin (SM) has been reported to modulate gut microbiota, and lower serum lipids and inflammation in mice on Western-style diets. However, few studies have examined if nutritionally-relevant intake of dietary SM can impact atherosclerosis progression. Thus, the aim of this study was to determine if incorporating 0.1% (w/w) egg SM (ESM) (equivalent to ~750 mg/day in humans) into a high-fat (45% kcal), cholesterol-enriched diet (HFD) could prevent atheroprogression in apoE -/- mice ( n = 15/group). We found that mice fed with the ESM-rich diet had significantly lower epididymal fat mass (-46%) and tended to have higher spleen weights (+15%). There were no significant differences in serum lipids between groups. However, ESM-fed mice had significantly lower alanine aminotransferase (ALT) activity. Additionally, ESM-fed mice displayed significantly less aortic root lipid accumulation (-31%) compared to controls. This improvement in atherosclerosis was paired with over a two-fold reduction in circulating serum amyloid A (SAA) in ESM-fed mice. Finally, there was also a modulation of the gut microbiota with ESM supplementation. ESM may have the potential to prevent atherosclerosis, however further research in the clinical setting is warranted.

Sphingomyelins and glycerophospholipids are structurally related phospholipids. Nevertheless, glycerophospholipids analogs are known as antitumor agents while sphingomyelin analogs were reported as cytoprotective agents. Herein, we have addressed the development of 3-deoxysphingomyelin analogs as cytotoxic agents possessing modified sphingobases. Thus, pyrrolidine-based 3-deoxysphingomyelin analogs were synthesized and evaluated against a panel of cell lines representing four major types of cancers. Compounds 3d, 4d and 6d elicited better GI 50 values than the FDA approved drug miltefosine. Investigation of their impact on Akt phosphorylation as a possible mechanism for the antiproliferative activity of this class of compounds revealed that these compounds might elicit a concentration-dependent mechanism via inhibition of Akt phosphorylation at the lower concentration. Molecular docking predicted their binding modes to Akt to involve polar head binding to the Pleckstrin homology domain and hydrophobic tail extension into a hydrophobic pocket connecting the Pleckstrin homology domain and the kinase domain. As a whole, the described work suggests compounds 3d, 4d and 6d as promising pyrrolidine-based 3-deoxysphingomyelin analogs for development of novel cancer therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Milk polar lipids (MPL) are specifically rich in milk sphingomyelin (MSM) which represents 24% of MPL. Beneficial effects of MPL or MSM have been reported on lipid metabolism, but information on gut physiology is scarce. Here we assessed whether MPL and MSM can impact tight junction expression. Human epithelial intestinal Caco-2/TC7 cells were incubated with mixed lipid micelles devoid of MSM (Control) or with 0.2 or 0.4 mM of MSM via pure MSM or via total MPL. C57Bl/6 mice received 5 or 10 mg of MSM via MSM or via MPL (oral gavage); small intestinal segments were collected after 4 h. Impacts on tight junction and cytokine expressions were assessed by qPCR; IL-8 and IL-8 murine homologs (Cxcl1, Cxcl2) were analyzed. In vitro, MSM increased tight junction expression (Occludin, ZO-1) vs Control, unlike MPL. However, no differences were observed in permeability assays (FITC-dextran, Lucifer yellow). MSM increased the secretion and gene expression of IL-8 but not of other inflammatory cytokines. Moreover, cell incubation with IL-8 induced an overexpression of tight junction proteins. In mice, mRNA level of Cxcl1 and Cxcl2 in the ileum were increased after gavage with MSM vs NaCl but not with MPL. Altogether, these results suggest a specific action of MSM on intestinal tight junction expression, possibly mediated by IL-8. Our study provides clues to shed light on the beneficial effects of MPL on intestinal functions and supports the need for further mechanistic exploration of the direct vs indirect effects of MSM and IL-8 on the gut barrier., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Scope: Enhanced adiposity and metabolic inflammation are major features of obesity associated with altered gut microbiota and intestinal barrier. How these metabolic outcomes can be impacted by milk polar lipids (MPL), naturally containing 25% of sphingomyelin, is investigated in mice fed a mixed high-fat (HF) diet ., Methods and Results: Male C57Bl/6 mice receive a HF-diet devoid of MPL (21% fat, mainly palm oil, in chow), or supplemented with 1.1% or 1.6% of MPL (HF-MPL1; HF-MPL2) via a total-lipid extract from butterserum concentrate for 8 weeks. HF-MPL2 mice gain less weight versus HF (p < 0.01). Diets do not impact plasma markers of inflammation but in the liver, HF-MPL2 tends to decrease hepatic gene expression of macrophage marker F4/80 versus HF-MPL1 (p = 0.06). Colonic crypt depth is the maximum in HF-MPL2 (p < 0.05). In cecal microbiota, HF-MPL1 increases Bifidobacterium animalis versus HF (p < 0.05). HF-MPL2 decreases Lactobacillus reuteri (p < 0.05), which correlates negatively with the fecal loss of milk sphingomyelin-specific fatty acids (p < 0.05)., Conclusion: In mice fed a mixed HF diet, MPL can limit HF-induced body weight gain and modulate gut physiology and the abundance in microbiota of bacteria of metabolic interest. This supports further exploration of how residual unabsorbed lipids reaching the colon can impact HF-induced metabolic disorders., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Context: Skeletal muscle endocannabinoids and sphingolipids (particularly sphingomyelins) are inversely associated with sleeping energy expenditure (SLEEP) in humans. The endocannabinoid system may increase sphingolipid synthesis via cannabinoid receptor-1., Objective: To investigate in human skeletal muscle whether endocannabinoids are responsible for the effect of sphingomyelins on SLEEP., Design: Muscle endocannabinoid [anandamide (AEA), 2-arachidonoylglycerol (2-AG)], endocannabinoid congeners [oleoylethanolamide (OEA), palmitoylethanolamide (PEA)], and sphingomyelin content were measured with liquid chromatography/mass spectrometry. SLEEP was assessed in a whole-room indirect calorimeter. Mediation analyses tested whether the inverse associations between sphingomyelins and SLEEP depended on endocannabinoids and endocannabinoid-related OEA and PEA., Setting: Inpatient study., Participants: Fifty-three Native Americans who are overweight., Main Outcome Measure: SLEEP., Results: AEA (r = 0.45, P = 0.001), 2-AG (r = 0.47, P = 0.0004), OEA (r = 0.27, P = 0.05), and PEA (r = 0.53, P < 0.0001) concentrations were associated with the total sphingomyelin content. AEA, OEA, and PEA correlated with specific sphingomyelins (SM18:1/23:0, SM18:1/23:1, and SM18:1/26:1) previously reported to be determinants of SLEEP in Native Americans (all r > 0.31, all P < 0.03). Up to half of the negative effect of these specific sphingomyelins on SLEEP was accounted for by AEA (all P < 0.04), rendering the direct effect by sphingomyelins per se on SLEEP negligible (P > 0.05)., Conclusions: In skeletal muscle, AEA is responsible for the sphingomyelin effect on SLEEP, indicating that endocannabinoids and sphingomyelins may jointly reduce human whole-body energy metabolism.

Deep profiling of glycosphingolipids and sphingomyelins in wild Cordyceps was carried out by using offline chromatographic enrichment followed by ultrahigh performance liquid chromatography-ultrahigh definition-quadrupole time-of-flight mass spectrometry (UHPLC-UHD-Q-TOF-MS). A total of 119 glycosphingolipids (72 new ones) and 87 sphingomyelins (43 new ones) were identified from wild Cordyceps on the basis of the accurate mass and MS/MS fragmentations, isotope patterns, sphingolipid (SPL) database matching, confirmation by SPL standards, and the reversed-phase liquid chromatographic retention rule. This study is the most comprehensive report on the identification of glycosphingolipids and sphingomyelins from fungus. A subsequent lipopolysaccharide-induced mouse splenic lymphocyte proliferation assay showed that the Cordyceps glycosphingolipid fraction exhibits higher immunosuppressive activity compared to that of Cordyceps sphingomyelins. Our findings provided insight into the chemical diversity of sphingolipids in Cordyceps and chemical evidence for the therapeutic application of wild Cordyceps.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), typified by the pulse-field type USA300, is an emerging endemic pathogen that is spreading rapidly among healthy people. CA-MRSA causes skin and soft tissue infections, life-threatening necrotizing pneumonia and sepsis, and is remarkably resistant to many antibiotics. Here we show that engineered liposomes composed of naturally occurring sphingomyelin were able to sequester cytolytic toxins secreted by USA300 and prevent necrosis of human erythrocytes, peripheral blood mononuclear cells and bronchial epithelial cells. Mass spectrometric analysis revealed the capture by liposomes of phenol-soluble modulins, α-hemolysin and other toxins. Sphingomyelin liposomes prevented hemolysis induced by pure phenol-soluble modulin-α3, one of the main cytolytic components in the USA300 secretome. In contrast, sphingomyelin liposomes harboring a high cholesterol content (66 mol/%) were unable to protect human cells from phenol-soluble modulin-α3-induced lysis, however these liposomes efficiently sequestered the potent staphylococcal toxin α-hemolysin. In a murine cutaneous abscess model, a single dose of either type of liposomes was sufficient to significantly decrease tissue dermonecrosis. Our results provide further insights into the promising potential of tailored liposomal therapy in the battle against infectious diseases., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Phospholipids have been shown to modulate intestinal cholesterol absorption in cells and animals, a process that is regulated by several transporter proteins. Of these proteins, Niemann-Pick C1-Like 1 (NPC1L1) is a major contributor to this process. The mechanism by which phospholipids modulate cholesterol absorption remains unknown. Here, we evaluate the effects of egg-yolk phospholipids on cholesterol absorption and transport in human colon carcinoma cell line (Caco-2 cells) and on the expression of NPC1L1 and others proteins associated with cholesterol absorption (ABCG5, ABCG8, ABCA1, ACAT2, MTP, CAV-1, ANX-2). The roles of SREBP-1 and SREBP-2 in this process were also investigated. The results show that egg-yolk sphingomyelin (CerPCho) and phosphatidylcholine (PtdCho) inhibit cholesterol transport in the Caco-2 monolayer in a dose-dependent manner. These might be due to the decrease of the cholesterol solubility in micelles as well as to the increases in the micellar sizes and the bile acid-binding capacity. Furthermore, the treatments with egg-yolk CerPCho or PtdCho at 1.2 mmol/L reduced the expression levels of NPC1L1 protein to 21 or 22%, respectively, and its mRNA to 9 or 31% of that in the control group (p < 0.05). Moreover, there was a general inhibitory effect of egg-yolk PtdCho and CerPCho on the mRNA levels of SREBP-1, and SREBP-2. These results suggest that the inhibitory effect of egg-yolk CerPCho and PtdCho on cholesterol transport might be due to their interference with the physicochemical properties of micelles and their regulations on the expression of the NPC1L1 gene., (© 2018 AOCS.)

Lysenin, which is an earthworm toxin, strongly binds to sphingomyelin (SM). Lysenin oligomerizes on SM-rich domains and can induce cell death by forming pores in the membrane. In this review, the assembly of lysenin on SM-containing membranes is discussed mostly on the basis of the information gained by atomic force microscopy (AFM). AFM data show that lysenin assembles into a hexagonal close packed (hcp) structure by rapid reorganization of its oligomers on an SM/cholesterol membrane. In case of a phase-separated membrane of SM, lysenin induces phase mixing as a result of pore formation in SM-rich domains, and consequently its hcp assembly covers the entire membrane. Besides the lytic action, lysenin is important as an SM marker and its pore has the potential to be used as a biosensor in the future. These points are also highlighted in this review., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation., Methods: Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA., Results: Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation., Conclusions: Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Western-type diets can induce obesity and related conditions such as dyslipidemia, insulin resistance and hepatic steatosis. We evaluated the effects of milk sphingomyelin (SM) and egg SM on diet-induced obesity, the development of hepatic steatosis and adipose inflammation in C57BL/6J mice fed a high-fat, cholesterol-enriched diet for 10 weeks. Mice were fed a low-fat diet (10% kcal from fat) (n=10), a high-fat diet (60% kcal from fat) (HFD, n=14) or a high-fat diet modified to contain either 0.1% (w/w) milk SM (n=14) or 0.1% (w/w) egg SM (n=14). After 10 weeks, egg SM ameliorated weight gain, hypercholesterolemia and hyperglycemia induced by HFD. Both egg SM and milk SM attenuated hepatic steatosis development, with significantly lower hepatic triglycerides (TGs) and cholesterol relative to HFD. This reduction in hepatic steatosis was stronger with egg SM supplementation relative to milk SM. Reductions in hepatic TGs observed with dietary SM were associated with lower hepatic mRNA expression of PPARγ-related genes: Scd1 and Pparg2 in both SM groups, and Cd36 and Fabp4 with egg SM. Egg SM and, to a lesser extent, milk SM reduced inflammation and markers of macrophage infiltration in adipose tissue. Egg SM also reduced skeletal muscle TG content compared to HFD. Overall, the current study provides evidence of dietary SM improving metabolic complications associated with diet-induced obesity in mice. Further research is warranted to understand the differences in bioactivity observed between egg and milk SM., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters., Design: Experimental study., Setting: University-affiliated tertiary hospital., Patient(s): A total of 20 normospermic fertile men., Intervention(s): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC)., Main Outcome Measure(s): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS)., Result(s): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC., Conclusion(s): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

β-Amyloid (Aβ) oligomers are neurotoxic and implicated in Alzheimer's disease. Neuronal plasma membranes may mediate formation of Aβ oligomers in vivo. Membrane components sphingomyelin and GM1 have been shown to promote aggregation of Aβ; however, these studies were performed under extreme, non-physiological conditions. We demonstrate that physiological levels of GM1 , organized in nanodomains do not seed oligomerization of Aβ40 monomers. We show that sphingomyelin triggers oligomerization of Aβ40 and that GM1 is counteractive thus preventing oligomerization. We propose a molecular explanation that is supported by all-atom molecular dynamics simulations. The preventive role of GM1 in the oligomerization of Aβ40 suggests that decreasing levels of GM1 in the brain, for example, due to aging, could reduce protection against Aβ oligomerization and contribute to the onset of Alzheimer's disease., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Streptococcus pneumoniae causes several human diseases, including pneumonia and meningitis, in which pathology is associated with an excessive inflammatory response. A major inducer of this response is the cholesterol dependent pneumococcal toxin, pneumolysin. Here, we measured the amount of inflammatory cytokine CXCL8 (interleukin (IL)-8) by ELISA released by human nasopharyngeal epithelial (Detroit 562) cells as inflammatory response to a 24 h exposure to different pneumococcal strains., Results: We found pneumolysin to be the major factor influencing the CXCL8 response. Cholesterol and sphingomyelin-containing liposomes designed to sequester pneumolysin were highly effective at reducing CXCL8 levels from epithelial cells exposed to different clinical pneumococcal isolates. These liposomes also reduced CXCL8 response from epithelial cells exposed to pneumolysin knock-out mutants of S. pneumoniae indicating that they also reduce the CXCL8-inducing effect of an unidentified pneumococcal virulence factor, in addition to pneumolysin., Conclusion: The results indicate the potential of liposomes in attenuating excessive inflammation as a future adjunctive treatment of pneumococcal diseases.

Cancer poses a significant threat to human health worldwide, and many therapies have been used for its palliative and curative treatments. Vincristine has been extensively used in chemotherapy. However, there are two major challenges concerning its applications in various tumors: (1) Vincristine's antitumor mechanism is cell-cycle-specific, and the duration of its exposure to tumor cells can significantly affect its antitumor activity and (2) Vincristine is widely bio-distributed and can be rapidly eliminated. One solution to these challenges is the encapsulation of vincristine into liposomes. Vincristine can be loaded into conventional liposomes, but it quickly leak out owing to its high membrane permeability. Numerous approaches have been attempted to overcome this problem. Vincristine has been loaded into PEGylated liposomes to prolong circulation time and improve tumor accumulation. These liposomes indeed prolong circulation time, but the payout characteristic of vincristine is severer, resulting in a compromised outcome rather than a better efficacy compared to conventional sphingomyelin (SM)/cholesterol (Chol) liposomes. In 2012, the USA Food and Drug Administration (FDA) approved SM/Chol liposomal vincristine (Marqibo®) for commercial use. In this review, we mainly focus on the drug's rapid leakage problem and the potentially relevant solutions that can be applied during the development of liposomal vincristine and the reason for conventional liposomal vincristine rather than PEGylated liposomes has access to the market.

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Sphingomyelin (SM), an essential phospholipid for the skin, is contained largely in the milk fat globule membrane surrounding milk fat, concentrated fractions of which are also generated concurrently during the manufacture of dairy products. Such an SM-containing milk phospholipid concentrate (SM-MPC) is useful for investigating the benefits of dietary SM. Here, we examined the effect of consuming SM-MPC on the condition of skin in a double-blind, placebo-controlled, randomized trial. Ninety-six healthy subjects aged 20 to 39 yr with low skin hydration were randomly assigned to 3 groups: a high-SM group supplemented with SM-MPC at a dose equivalent to 10 mg/d of SM, a low-SM group supplemented with SM-MPC equivalent to 5 mg/d of SM, and a placebo group fed a vehicle composed of olive oil and beeswax. During daily supplementation for 12 wk, parameters related to the condition of skin were evaluated at baseline and every 3 wk. Skin hydration at the heel was significantly increased at wk 9 and 12 in the low-SM group compared with the placebo group. Skin elasticity in the region below the eye was significantly increased at wk 9 in the high-SM group versus placebo. Questionnaire-based subjective perceptions of skin conditions were significantly improved for facial skin moisture at wk 3 and 12, and in the wrinkle around the eyes at wk 9 and 12 in the high-SM group versus placebo. Our results indicate that constant and long-term supplementation with SM-MPC is capable of improving the general condition of skin., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.

Introduction: Periapical infections secondary to pulpal necrosis are associated with bacterial contamination of the pulp. Porphyromonas endodontalis, a gram-negative organism, is considered to be a pulpal pathogen. P. gingivalis is phylogenetically related to P. endodontalis and synthesizes several classes of novel complex lipids that possess biological activity, including the capacity to promote osteoclastogenesis and osteoclast activation. The purpose of this study was to extract and characterize constituent lipids of P. endodontalis and evaluate their capacity to promote proinflammatory secretory responses in the macrophage cell line, RAW 264.7, as well as their capacity to promote osteoclastogenesis and inhibit osteoblast activity., Methods: Constituent lipids of both organisms were fractionated by high-performance liquid chromatography and were structurally characterized using electrospray mass spectrometry or electrospray-mass spectrometry/mass spectrometry. The virulence potential of P. endodontalis lipids was then compared with known biologically active lipids isolated from P. gingivalis., Results: P. endodontalis total lipids were shown to promote tumor necrosis factor alpha secretion from RAW 264.7 cells, and the serine lipid fraction appeared to account for the majority of this effect. P. endodontalis lipid preparations also increased osteoclast formation from RAW 264.7 cells, but osteoblast differentiation in culture was inhibited and appeared to be dependent on Toll-like receptor 2 expression., Conclusions: These effects underscore the importance of P. endodontalis lipids in promoting inflammatory and bone cell activation processes that could lead to periapical pathology., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of (125)I-lysenin* binding to erythrocytes upon SM depletion by SMase. The (125)I-lysenin* binding isotherm indicated saturation at 3.5 × 10(6) molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub-micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Scope: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis., Methods and Results: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis., Conclusion: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The arachidonic acid (AA) cascade is regulated mainly by the actions of two rate-limiting enzymes, phospholipase A₂ (PLA₂) and inducible cyclooxygenase-2 (COX-2). PLA₂ acts to generate AA, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to prostaglandin production. Amongst more than 30 members of the PLA₂ family, cytosolic PLA₂α (cPLA₂α, group IVA) plays a major role in releasing AA from cellular membranes. Sphingolipids are a novel class of bioactive lipids that play key roles in the regulation of several cellular processes including growth, differentiation, inflammatory responses, and apoptosis. Recent studies implicated a regulatory function of sphingolipids in prostaglandin production. Whereas ceramide-1-phosphate and lactosylceramide activate cPLA₂α directly, sphingosine-1-phosphate induces COX-2 expression. Sphingomyelin has been shown to inhibit the activity of cPLA₂α. In addition, several sphingolipid analogs including a therapeutic agent currently used clinically are also reported to be inhibitors of cPLA₂α. This review explores the role of sphingolipids in the regulation of cPLA₂α and COX-2.

Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4°C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55-67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain., (© 2013. Published by Elsevier B.V. All rights reserved.)

Background: Diets enriched with sphingolipids may improve blood lipid profiles. Studies in animals have shown reductions in cholesterol absorption and alterations in blood lipids after treatment with sphingomyelin (SM). However, minimal information exists on effect of SM on cholesterol absorption and metabolism in humans. The objective was to assess the effect of SM consumption on serum lipid concentrations and cholesterol metabolism in healthy humans., Methods: Ten healthy adult males and females completed a randomized crossover study. Subjects consumed controlled diets with or without 1 g/day SM for 14 days separated by at least 4 week washout period. Serum lipid profile and markers of cholesterol metabolism including cholesterol absorption and synthesis were analyzed., Results: Serum triglycerides, total, LDL- and VLDL- cholesterol were not affected while HDL cholesterol concentrations were increased (p = 0.043) by SM diet consumption. No change in cholesterol absorption and cholesterol fractional synthesis rate was observed with supplementation of SM compared to control. Intraluminal cholesterol solubilization was also not affected by consumption of SM enriched diet., Conclusions: In humans, 1 g/day of dietary SM does not alter the blood lipid profile except for an increased HDL-cholesterol concentration and has no effect on cholesterol absorption, synthesis and intraluminal solubilization compared to control., Trial Registration: Clinicaltrials.gov # NCT00328211.

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.

Controlling intestinal lipid absorption is an important strategy for maintaining lipid homeostasis. Accumulation of lipids in the liver is a major risk factor for metabolic syndrome and nonalcoholic fatty liver disease. It is well-known that sphingomyelin (SM) can inhibit intestinal cholesterol absorption. It is, however, unclear if dietary SM also lowers liver lipid levels. In the present study (i) the effect of pure dietary egg SM on hepatic lipid metabolism and intestinal cholesterol absorption was measured with [(14)C]cholesterol and [(3)H]sitostanol in male C57BL/6 mice fed a high-fat (HF) diet with or without 0.6% wt/wt SM for 18 days; and (ii) hepatic lipid levels and gene expression were determined in mice given a HF diet with or without egg SM (0.3, 0.6 or 1.2% wt/wt) for 4 weeks. Mice supplemented with SM (0.6% wt/wt) had significantly increased fecal lipid and cholesterol output and reduced hepatic [(14)C]cholesterol levels after 18 days. Relative to HF-fed mice, SM-supplemented HF-fed mice had significantly lower intestinal cholesterol absorption (-30%). Liver weight was significantly lower in the 1.2% wt/wt SM-supplemented mice (-18%). Total liver lipid (mg/organ) was significantly reduced in the SM-supplemented mice (-33% and -40% in 0.6% wt/wt and 1.2% wt/wt SM, respectively), as were triglyceride and cholesterol levels. The reduction in liver triglycerides was due to inactivation of the LXR-SREBP-1c pathway. In conclusion, dietary egg SM has pronounced hepatic lipid-lowering properties in mice maintained on an obesogenic diet.

The bovine milk fat globule membrane (MFGM) contains several antimicrobial components with proven efficacy in vitro, but in vivo evidence is scarce. The present study was performed to determine the efficacy of the bovine MFGM in vivo. Rats were fed diets based on bovine skimmed milk powder (low in MFGM) or bovine sweet buttermilk powder (high in MFGM). After dietary adaptation, rats were orally infected with Salmonella enteritidis or Listeria monocytogenes. Whereas sweet buttermilk powder did not protect rats against infection with S. enteritidis, it protected against L. monocytogenes, as shown by a lower colonisation and translocation of this pathogen. Protection coincided with higher listericidal capacity of gastric and caecal contents. The digestion products of phosphoglycerides and sphingomyelin are bactericidal in vitro. To study their role, rats were fed diets containing either 0·1 % phosphatidylcholine or sphingomyelin, or a control diet. After dietary adaptation, rats were infected with L. monocytogenes. Since Listeria colonisation was not affected by these diets, phosphoglycerides and sphingomyelin are not involved in the protective effect of sweet buttermilk. Additional in vitro experiments were performed to further explore the mechanism of the beneficial effects of sweet buttermilk. Inhibition of the adherence of L. monocytogenes to the intestinal mucosa is the most likely explanation, since sweet buttermilk powder inhibited the binding of L. monocytogenes in both a haemagglutination assay and a Caco-2 cell adherence assay. In conclusion, sweet buttermilk powder, which is rich in MFGM, protects against L. monocytogenes infection in rats, probably by preventing adherence of this pathogen to the intestinal mucosa.