We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

The prefrontal cortex is essential for executive functions such as decision-making and planning. There is also accumulating evidence that it is important for the modulation of pain. In this study, we investigated a possible role of prefrontal cortical calcium-independent phospholipase A2 (iPLA2) in antinociception induced by the norepinephrine reuptake inhibitor (NRI) and tetracyclic (tricyclic) antidepressant, maprotiline. Intraperitoneal injections of maprotiline increased iPLA2 mRNA and protein expression in the prefrontal cortex. This treatment also reduced grooming responses to von-Frey hair stimulation of the face after facial carrageenan injection, indicating decreased sensitivity to pain. The antinociceptive effect of maprotiline was abrogated by iPLA2 antisense oligonucleotide injection to the prefrontal cortex, indicating a role of this enzyme in antinociception. In contrast, injection of iPLA2 antisense oligonucleotide to the somatosensory cortex did not reduce the antinociceptive effect of maprotiline. Lipidomic analysis of the prefrontal cortex showed decrease in phosphatidylcholine species, but increase in lysophosphatidylcholine species, indicating increased PLA2 activity, and release of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) after maprotiline treatment. Differences in sphingomyelin/ceramide were also detected. These changes were not observed in maprotiline-treated mice that received iPLA2 antisense oligonucleotide to the prefrontal cortex. Metabolites of DHA and EPA may help to strengthen a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate a role of prefrontal cortical iPLA2 and its enzymatic products in the antinociceptive effect of maprotiline.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation.

SignificanceWith obesity on the rise, there is a growing appreciation for intracellular lipid droplet (LD) regulation. Here, we show how saturated fatty acids (SFAs) reduce fat storage-inducing transmembrane protein 2 (FIT2)-facilitated, pancreatic β cell LD biogenesis, which in turn induces β cell dysfunction and death, leading to diabetes. This mechanism involves direct acylation of FIT2 cysteine residues, which then marks the FIT2 protein for endoplasmic reticulum (ER)-associated degradation. Loss of β cell FIT2 and LDs reduces insulin secretion, increases intracellular ceramides, stimulates ER stress, and exacerbates diet-induced diabetes in mice. While palmitate and stearate degrade FIT2, unsaturated fatty acids such as palmitoleate and oleate do not, results of which extend to nutrition and diabetes.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.

Quantitative MS of human plasma lipids is a promising technology for translation into clinical applications. Current MS-based lipidomic methods rely on either direct infusion (DI) or chromatographic lipid separation methods (including reversed phase and hydrophilic interaction LC). However, the use of lipid markers in laboratory medicine is limited by the lack of reference values, largely because of considerable differences in the concentrations measured by different laboratories worldwide. These inconsistencies can be explained by the use of different sample preparation protocols, method-specific calibration procedures, and other experimental and data-reporting parameters, even when using identical starting materials. Here, we systematically investigated the roles of some of these variables in multiple approaches to lipid analysis of plasma samples from healthy adults by considering: 1) different sample introduction methods (separation vs. DI methods); 2) different MS instruments; and 3) between-laboratory differences in comparable analytical platforms. Each of these experimental variables resulted in different quantitative results, even with the inclusion of isotope-labeled internal standards for individual lipid classes. We demonstrated that appropriate normalization to commonly available reference samples (i.e., "shared references") can largely correct for these systematic method-specific quantitative biases. Thus, to harmonize data in the field of lipidomics, in-house long-term references should be complemented by a commonly available shared reference sample, such as NIST SRM 1950, in the case of human plasma.

White matter lesions (WML) are thought to contribute to vascular cognitive impairment in elderly patients. Growing evidence show that failure of myelin formation arising from the disruption of oligodendrocyte progenitor cell (OPC) differentiation is a cause of chronic vascular white matter damage. The sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) signaling pathway regulates oligodendroglia differentiation and function, and is known to be altered in hypoxia. In this study, we measured SphK, S1P as well as markers of WML, hypoxia and OPC (NG2) in a mouse bilateral carotid artery stenosis (BCAS) model of chronic cerebral hypoperfusion. Our results indicated that BCAS induced hypoxia inducible factor (HIF)-1α, Sphk2, S1P, and NG2 up-regulation together with accumulation of WML. In contrast, BCAS mice treated with the SphK inhibitor, SKI-II, showed partial reversal of SphK2, S1P and NG2 elevation and amelioration of WML. In an in vitro model of hypoxia, SKI-II reversed the suppression of OPC differentiation. Our study suggests a mechanism for hypoperfusion-associated WML involving HIF-1α-SphK2-S1P-mediated disruption of OPC differentiation, and proposes the SphK signaling pathway as a potential therapeutic target for white matter disease.

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.

Lipids comprise an exceptionally diverse class of bioactive macromolecules. While quantitatively abundant lipid species serve fundamental roles in cell structure and energy metabolism, thousands of structurally-distinct, quantitatively minor species may serve as important regulators of cellular processes. Historically, a complete understanding of the biological roles of these lipids has been limited by a lack of sensitive, discriminating analytical techniques. The class of sphingolipids alone, for example, is known to consist of over 600 different confirmed species, but is likely to include tens of thousands of metabolites with potential biological significance. Advances in mass spectrometry (MS) have improved the throughput and discrimination of lipid analysis, allowing for the determination of detailed lipid profiles in large cohorts of clinical samples. Databases emerging from these studies will provide a rich resource for the identification of novel biomarkers and for the discovery of potential drug targets, analogous to that of existing genomics databases. In this review, we will provide an overview of the field of sphingolipidomics, and will discuss some of the challenges and considerations facing the generation of robust lipidomics databases.

Meibomian gland (MG) dysfunction is the leading cause of evaporative dry eye and it leads to inflammation of the ocular surface. Eicosanoids may be involved in inflammation of dry eye. This study aimed to profile tear eicosanoid levels in healthy individuals and those with MG dysfunction, and to examine if these levels are associated with clinical factors and expressibility of MG. Forty participants with MG dysfunction and 30 healthy controls were recruited in this study. Clinical signs of MG dysfunction were assessed, and tear lactoferrin concentration was evaluated. Tear eicosanoids were extracted from Schirmer's strips and analyzed using mass spectrometry. We were able to quantify 38 tear eicosanoids and levels were increased in older individuals. In participants with MG dysfunction, higher 5-HETE, LTB4, 18-HEPE, 12-HEPE and 14-HDoHE were associated with poorer MG expressibility. The eicosanoids PGF2α, 18-HEPE, 20-HDoHE and 17-HDoHE were elevated with increased corneal staining; higher 5-HETE, LTB4 were associated with lower tear lactoferrin levels. The receiver-operating-characteristics analysis shows higher levels of 5-HETE, LTB4 and 18-HEPE were able to predict poor expressibility of MGs. In conclusion, tear eicosanoid levels are age-dependent and specific eicosanoids may be indicators of clinical obstruction of MG or the severity of ocular surface damage.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biological conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets.

Cell state transitions control the functional behavior of cancer cells. Epithelial-to-mesenchymal transition (EMT) confers cancer stem cell-like properties, enhanced tumorigenicity and drug resistance to tumor cells, while mesenchymal-epithelial transition (MET) reverses these phenotypes. Using high-throughput chemical library screens, retinoids are found to be potent promoters of MET that inhibit tumorigenicity in basal-like breast cancer. Cell state transitions are defined by reprogramming of lipid metabolism. Retinoids bind cognate nuclear receptors, which target lipid metabolism genes, thereby redirecting fatty acids for β-oxidation in the mesenchymal cell state towards lipid storage in the epithelial cell state. Disruptions of key metabolic enzymes mediating this flux inhibit MET. Conversely, perturbations to fatty acid oxidation (FAO) rechannel fatty acid flux and promote a more epithelial cell phenotype, blocking EMT-driven breast cancer metastasis in animal models. FAO impinges on the epigenetic control of EMT through acetyl-CoA-dependent regulation of histone acetylation on EMT genes, thus determining cell states.

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer in equilibrium, although it is unclear whether these species translocate to the PM via the same or different mechanisms. We therefore investigated the structural determinants involved to better understand how translocation might potentially be targeted for therapeutic intervention. We report here that monomeric WT mouse SK1 (GFP-mSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol 12-myristate 13-acetate, whereas the dimer translocates to the PM in response to sphingosine-1-phosphate; thus, the equilibrium between the monomer and dimer is sensitive to cellular stimulus. In addition, carbachol and phorbol 12-myristate 13-acetate induced translocation of monomeric GFP-mSK1 to lamellipodia, whereas sphingosine-1-phosphate induced translocation of dimeric GFP-mSK1 to filopodia, suggesting that SK1 regulates different cell biological processes dependent on dimerization. GFP-mSK1 mutants designed to modulate dimerization confirmed this difference in localization. Regulation by the C-terminal tail of SK1 was investigated using GFP-mSK1 truncations. Removal of the last five amino acids (PPEEP) prevented translocation of the enzyme to the PM, whereas removal of the last ten amino acids restored translocation. This suggests that the penultimate five amino acids (SRRGP) function as a translocation brake, which can be released by sequestration of the PPEEP sequence. We propose that these determinants alter the arrangement of N-terminal and C-terminal domains in SK1, leading to unique surfaces that promote differential translocation to the PM.

Acute kidney injury (AKI) is a global public health concern with high mortality and morbidity. In ischemic-reperfusion injury (IRI), a main cause of AKI, the brush border membrane of S3 proximal tubules (PT) is lost to the tubular lumen. How injured tubules reconstitute lost membrane lipids during renal recovery is not known. Here, we identified Mfsd2a, a sodium-dependent lysophosphatidylcholine (LPC) transporter, to be expressed specifically in the basolateral membrane of S3 PT. Using an in vivo activity probe for Mfsd2a, transport activity was found to be specific to the S3 PT. Mice with haploinsufficiency of Mfsd2a exhibited delayed recovery of renal function after acute IRI, with depressed urine osmolality and elevated levels of histological markers of damage, fibrosis, and inflammation, findings corroborated by transcriptomic analysis. Lipidomics revealed a deficiency in docosahexaenoic acid (DHA) containing phospholipids in Mfsd2a haploinsufficiency. Treatment of Mfsd2a haploinsufficient mice with LPC-DHA improved renal function and reduced markers of injury, fibrosis, and inflammation. Additionally, LPC-DHA treatment restored S3 brush border membrane architecture and normalized DHA-containing phospholipid content. These findings indicate that Mfsd2a-mediated transport of LPC-DHA is limiting for renal recovery after AKI and suggest that LPC-DHA could be a promising dietary supplement for improving recovery following AKI.

Untargeted lipidomics has been increasingly adopted for hypothesis generation in a biological context or discovery of disease biomarkers. Most of the current liquid chromatography mass spectrometry (LC-MS) based untargeted methodologies utilize a data dependent acquisition (DDA) approach in pooled samples for identification and MS-only acquisition for semi-quantification in individual samples. In this study, we present for the first time an untargeted lipidomic workflow that makes use of the newly implemented Quadrupole Resolved All-Ions (Q-RAI) acquisition function on the Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer to acquire MS2 spectra in data independent acquisition (DIA) mode. This is followed by data processing and analysis on MetaboKit, a software enabling DDA-based spectral library construction and extraction of MS1 and MS2 peak areas, for reproducible identification and quantification of lipids in DIA analysis. This workflow was tested on lipid extracts from human plasma and showed quantification at MS1 and MS2 levels comparable to multiple reaction monitoring (MRM) targeted analysis of the same samples. Analysis of serum from Ceramide Synthase 2 (CerS2) null mice using the Q-RAI DIA workflow identified 88 lipid species significantly different between CerS2 null and wild type mice, including well-characterized changes previously associated with this phenotype. Our results show the Q-RAI DIA as a reliable option to perform simultaneous identification and reproducible relative quantification of lipids in exploratory biological studies.

Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death.

Oral squamous cell carcinoma (OSCC) is a lethal disease with a 5-year mortality rate of around 50%. Molecular targeted therapies are not in routine use and novel therapeutic targets are required. Our previous microarray data indicated sphingosine 1-phosphate (S1P) metabolism and signalling was deregulated in OSCC. In this study, we have investigated the contribution of S1P signalling to the pathogenesis of OSCC. We show that the expression of the two major enzymes that regulate S1P levels were altered in OSCC: SPHK1 was significantly upregulated in OSCC tissues compared to normal oral mucosa and low levels of SGPL1 mRNA correlated with a worse overall survival. In in vitro studies, S1P enhanced the migration/invasion of OSCC cells and attenuated cisplatin-induced death. We also demonstrate that S1P receptor expression is deregulated in primary OSCCs and that S1PR2 is over-expressed in a subset of tumours, which in part mediates S1P-induced migration of OSCC cells. Lastly, we demonstrate that FTY720 induced significantly more apoptosis in OSCC cells compared to non-malignant cells and that FTY720 acted synergistically with cisplatin to induce cell death. Taken together, our data show that S1P signalling promotes tumour aggressiveness in OSCC and identify S1P signalling as a potential therapeutic target.

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.