Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Deep Characterisation of the sn-Isomer Lipidome Using High-Throughput Data-Independent Acquisition and Ozone-Induced Dissociation.

In recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn-isomers have resolved only a limited number of species. We report a workflow based on ozone-induced dissociation for untargeted characterisation of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20 min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified as compared to previous reports and reveals that sn-isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra-long monounsaturated acyl chains at the sn-1 position.

Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface.

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Lipidomic Characterization of Whey Concentrates Rich in Milk Fat Globule Membranes and Extracellular Vesicles.

Lipids from milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for cognitive development and human health. Milk-derived whey concentrates rich in these lipids are therefore used as ingredients in infant formulas to mimic human milk and in medical nutrition products to improve the metabolic fitness of adults and elderly people. In spite of this, there is no consensus resource detailing the multitude of lipid molecules in whey concentrates. To bridge this knowledge gap, we report a comprehensive and quantitative lipidomic resource of different whey concentrates. In-depth lipidomic analysis of acid, sweet, and buttermilk whey concentrates identified 5714 lipid molecules belonging to 23 lipid classes. The data show that the buttermilk whey concentrate has the highest level of fat globule-derived triacylglycerols and that the acid and sweet whey concentrates have the highest proportions of MFGM- and EV-derived membrane lipids. Interestingly, the acid whey concentrate has a higher level of cholesterol whereas sweet whey concentrate has higher levels of lactosylceramides. Altogether, we report a detailed lipid molecular compendium of whey concentrates and lay the groundwork for using in-depth lipidomic technology to profile the nutritional value of milk products and functional foods containing dairy-based concentrates.

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA-imaging technology where high-resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap-Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA-imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA-imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA-imaging on the Orbitrap Elite. Altogether, our work shows that DDA-imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.

Brain lipidomics and neurodevelopmental outcomes in intrauterine growth restricted piglets fed dairy or vegetable fat diets.

Breast milk has neurodevelopmental advantages compared to infant formula, especially in low-birth-weight infants, which may in part relate to the fat source. This study compared neurodevelopmental outcomes in three-day-old normal birth weight (NBW) and intrauterine growth restricted (IUGR) piglets fed a formula diet with either vegetable oil (VEG) or bovine milk fat sources (MILK) for three weeks in a 2 × 2 factorial design. Behavioural tests, lipidomics, MRI and RNA sequencing analyses of plasma and brain tissue were conducted. The absolute levels of 82% and 11% of lipid molecules were different between dietary groups in plasma and hippocampus, respectively. Of the lipid molecules with differential abundance in the hippocampus, the majority were upregulated in MILK versus VEG, and they mainly belonged to the group of glycerophospholipids. Lower absolute brain weights, absolute grey and white matter volumes and behaviour and motor function scores, and higher relative total brain weights were present in IUGR compared to NBW with minor influence of diet. Cognitive function and cerebellar gene expression profiles were similar for dietary and weight groups, and overall only minor interactive effects between diet and birth weight were observed. Overall, we show that the dietary fat source influences the plasma and to a lesser degree the hippocampal lipidome and is unable to improve on IUGR-induced brain structural and functional impairments.

Silencing of ceramide synthase 2 in hepatocytes modulates plasma ceramide biomarkers predictive of cardiovascular death.

Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders.

Molecular species selectivity of lipid transport creates a mitochondrial sink for di-unsaturated phospholipids.

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.

Adipose MDM2 regulates systemic insulin sensitivity.

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

Adipocyte-like signature in ovarian cancer minimal residual disease identifies metabolic vulnerabilities of tumor-initiating cells.

Similar to tumor-initiating cells (TICs), minimal residual disease (MRD) is capable of reinitiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multiregion transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells showed the same phenotype and were dependent on fatty acid oxidation (FAO) for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD.

Dairy-Derived Emulsifiers in Infant Formula Show Marginal Effects on the Plasma Lipid Profile and Brain Structure in Preterm Piglets Relative to Soy Lecithin.

Breastfed infants have higher intestinal lipid absorption and neurodevelopmental outcomes compared to formula-fed infants, which may relate to a different surface layer structure of fat globules in infant formula. This study investigated if dairy-derived emulsifiers increased lipid absorption and neurodevelopment relative to soy lecithin in newborn preterm piglets. Piglets received a formula diet containing soy lecithin (SL) or whey protein concentrate enriched in extracellular vesicles (WPC-A-EV) or phospholipids (WPC-PL) for 19 days. Both WPC-A-EV and WPC-PL emulsions, but not the intact diets, increased in vitro lipolysis compared to SL. The main differences of plasma lipidomics analysis were increased levels of some sphingolipids, and lipid molecules with odd-chain (17:1, 19:1, 19:3) as well as mono- and polyunsaturated fatty acyl chains (16:1, 20:1, 20:3) in the WPC-A-EV and WPC-PL groups and increased 18:2 fatty acyls in the SL group. Indirect monitoring of intestinal triacylglycerol absorption showed no differences between groups. Diffusor tensor imaging measurements of mean diffusivity in the hippocampus were lower for WPC-A-EV and WPC-PL groups compared to SL indicating improved hippocampal maturation. No differences in hippocampal lipid composition or short-term memory were observed between groups. In conclusion, emulsification of fat globules in infant formula with dairy-derived emulsifiers altered the plasma lipid profile and hippocampal tissue diffusivity but had limited effects on other absorptive and learning abilities relative to SL in preterm piglets.

Lipid molecular timeline profiling reveals diurnal crosstalk between the liver and circulation.

Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.

Accurate quantification of lipid species affected by isobaric overlap in Fourier-transform mass spectrometry.

Lipidomics data require consideration of ions with near-identical masses, which comprises among others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DBs) mainly because of the natural abundance of 13C-atoms. High-resolution mass spectrometry, such as Fourier-transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow injection analysis-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well-described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction because of peak interference. The described method was validated including intraday and interday precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis.

Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures.

A comprehensive and standardized system to report lipid structures analyzed by MS is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e., annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labeled compounds in metabolic labeling experiments or as internal standards. This update on lipid classification, nomenclature, and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

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.

Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.

Phosphoproteomic Analysis across the Yeast Life Cycle Reveals Control of Fatty Acyl Chain Length by Phosphorylation of the Fatty Acid Synthase Complex.

The ability to remodel lipid metabolism under changing conditions is pivotal for cellular functionality and homeostasis. Here, we characterize the regulatory landscape of phosphorylation-based signaling events across the life cycle of Saccharomyces cerevisiae and determine its impact on the regulation of lipid metabolism. Our data show that 50 lipid metabolic proteins are differentially phosphorylated as cells transit between different physiological states. To identify functional phosphosites, we devised a strategy where multiple phosphosites are simultaneously mutated into phosphomimetic or phosphodeficient alleles and mutants are phenotyped by in-depth lipidomics flux analysis. This uncovers functional phosphosites in the phosphatidate cytidylyltransferase Cds1, the phosphatidylserine synthase Cho1, and Fas2, the α-subunit of the fatty acid synthase (FAS) complex. Furthermore, we show that the fatty acyl chain length produced by FAS is governed by phosphorylation. Overall, our work demonstrates a vital role for phosphoregulation of lipid metabolism and provides a resource to investigate its molecular underpinnings.

LipidCreator workbench to probe the lipidomic landscape.

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.