Global Lipidomic Analysis Research Articles

Delta-5 elongase knockout reduces docosahexaenoic acid and lipid synthesis and increases heat sensitivity in a diatom.

Recent global marine lipidomic analysis reveals a strong relationship between ocean temperature and phytoplanktonic abundance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for human nutrition and primarily sourced from phytoplankton in marine food webs. In phytoplanktonic organisms, EPA may play a major role in regulating the phase transition temperature of membranes, while the function of DHA remains unexplored. In the oleaginous diatom Phaeodactylum tricornutum, DHA is distributed mainly on extraplastidial phospholipids, which is very different from the EPA enriched in thylakoid lipids. Here, CRISPR/Cas9-mediated knockout of delta-5 elongase (ptELO5a), which encodes a delta-5 elongase (ELO5) catalyzing the elongation of EPA to synthesize DHA, led to a substantial interruption of DHA synthesis in P. tricornutum. The ptELO5a mutants showed some alterations in transcriptome and glycerolipidomes, including membrane lipids and triacylglycerols under normal temperature (22°C), and were more sensitive to elevated temperature (28°C) than wild type. We conclude that PtELO5a-mediated synthesis of small amounts of DHA has indispensable functions in regulating membrane lipids, indirectly contributing to storage lipid accumulation, and maintaining thermomorphogenesis in P. tricornutum. This study also highlights the significance of DHA synthesis and lipid composition for environmental adaptation of P. tricornutum.

Comprehensive untargeted lipidomic analysis of sea buckthorn using UHPLC-HR-AM/MS/MS combined with principal component analysis

Sea buckthorn is an important ecological and economic plant which has multiple bioactivities. The fruits and seeds of sea buckthorn are rich in oil. However, there are few studies on the differences of lipid profiles of sea buckthorn varieties. Herein, the lipidomic fingerprints of sea buckthorn was established. First, a mixture solvent of methanol and chloroform (2:1, v/v) was selected to extract the lipid of the flesh and seed of sea buckthorn. Then, global lipidomic analysis of different varieties of sea buckthorn was conducted. A total of 16 lipid classes and 112 lipid molecular species were determined. Several molecular species, such as PE (phosphatidylethanolamine) 18:1/18:3, PE18:0/18:1, PE18:0/18:2, etc. were selected as the potential biomarkers to classify the samples. Our study provides a scientific basis for quality control of sea buckthorn and promotes the development of sea buckthorn oil.

Global serum metabolomic and lipidomic analyses reveal lipid perturbations and potential biomarkers of the colorectal cancer by adenoma-carcinoma sequence

Global serum metabolomic and lipidomic analyses reveal lipid perturbations and potential biomarkers of the colorectal cancer by adenoma-carcinoma sequence

Serine Racemase mediates subventricular zone neurogenesis via fatty acid metabolism.

The adult subventricular zone (SVZ) is a neurogenic niche that continuously produces newborn neurons. Here we show that serine racemase (SR), an enzyme that catalyzes the racemization of L-serine to D-serine and vice versa, affects neurogenesis in the adult SVZ by controlling de novo fatty acid synthesis. Germline and conditional deletion of SR (nestin precursor cells) leads to diminished neurogenesis in the SVZ. Nestin-cre+ mice showed reduced expression of fatty acid synthase and its substrate malonyl-CoA, which are involved in de novo fatty acid synthesis. Global lipidomic analyses revealed significant alterations in different lipid subclasses in nestin-cre+ mice. Decrease in fatty acid synthesis was mediated by phospho Acetyl-CoA Carboxylase that was AMP-activated protein kinase independent. Both L- and D-serine supplementation rescued defects in SVZ neurogenesis, proliferation, and levels of malonyl-CoA invitro. Our work shows that SR affects adult neurogenesis in the SVZ via lipid metabolism.

Lipidomic Profiling of PFOA-Exposed Mouse Liver by Multi-Modal Mass Spectrometry Analysis.

Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated chemical classified as a persistent organic pollutant. PFOA has been linked to many toxic effects, including liver injury. Many studies report that PFOA exposure alters serum and hepatic lipid metabolism. However, lipidomic pathways altered by PFOA exposure are largely unknown and only a few lipid classes, mostly triacylglycerol (TG), are usually considered in lipid analysis. Here, we performed a global lipidomic analysis on the liver of PFOA-exposed (high-dose and short-duration) and control mice by combining three mass spectrometry (MS) techniques: liquid chromatography with tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Among all hepatic lipids identified by LC-MS/MS analysis, more than 350 were statistically impacted (increased or decreased levels) after PFOA exposure, as confirmed by multi-variate data analysis. The levels of many lipid species from different lipid classes, most notably phosphatidylethanolamine (PE), phosphatidylcholine (PC), and TG, were significantly altered. Subsequent lipidomic analysis highlights the pathways significantly impacted by PFOA exposure, with the glycerophospholipid metabolism being the most impacted, and the changes in the lipidome network, which connects all the lipid species together. MALDI-MSI displays the heterogeneous distribution of the affected lipids and PFOA, revealing different areas of lipid expression linked to PFOA localization. TOF-SIMS localizes PFOA at the cellular level, supporting MALDI-MSI results. This multi-modal MS analysis unveils the lipidomic impact of PFOA in the mouse liver after high-dose and short-term exposure and opens new opportunities in toxicology.

Tuberculostearic Acid Controls Mycobacterial Membrane Compartmentalization.

The intracellular membrane domain (IMD) is a laterally discrete region of the mycobacterial plasma membrane, enriched in the subpolar region of the rod-shaped cell. Here, we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. The putative gene cfa showed the most significant effect on recovery from membrane compartment disruption by dibucaine. Enzymatic analysis of Cfa and lipidomic analysis of a cfa deletion mutant (Δcfa) demonstrated that Cfa is an essential methyltransferase for the synthesis of major membrane phospholipids containing a C19:0 monomethyl-branched stearic acid, also known as tuberculostearic acid (TBSA). TBSA has been intensively studied due to its abundant and genus-specific production in mycobacteria, but its biosynthetic enzymes had remained elusive. Cfa catalyzed the S-adenosyl-l-methionine-dependent methyltransferase reaction using oleic acid-containing lipid as a substrate, and Δcfa accumulated C18:1 oleic acid, suggesting that Cfa commits oleic acid to TBSA biosynthesis, likely contributing directly to lateral membrane partitioning. Consistent with this model, Δcfa displayed delayed restoration of subpolar IMD and delayed outgrowth after bacteriostatic dibucaine treatment. These results reveal the physiological significance of TBSA in controlling lateral membrane partitioning in mycobacteria. IMPORTANCE As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis. It was discovered in 1934, and yet the enzymes that mediate the biosynthesis of this fatty acid and the functions of this unusual fatty acid in cells have remained elusive. Through a genome-wide transposon sequencing screen, enzyme assay, and global lipidomic analysis, we show that Cfa is the long-sought enzyme that is specifically involved in the first step of generating tuberculostearic acid. By characterizing a cfa deletion mutant, we further demonstrate that tuberculostearic acid actively regulates lateral membrane heterogeneity in mycobacteria. These findings indicate the role of branched fatty acids in controlling the functions of the plasma membrane, a critical barrier for the pathogen to survive in its human host.

Lipidomic changes in the liver of beagle dogs associated with Toxocara canis infection.

A global lipidomic analysis using liquid chromatography–tandem mass spectrometry was performed on the liver of beagle dogs infected with Toxocara canis to profile hepatic lipid species at 12 h post-infection (hpi), 24 hpi, and 36 days post-infection (dpi). This analysis identified six categories and 42 subclasses of lipids, including 173, 64, and 116 differentially abundant lipid species at 12 hpi, 24 hpi, and 36 dpi, respectively. Many of the identified lysophospholipids, such as lysophosphatidylglycerol, lysophosphatidylserine, and lysophosphatidylcholine, may contribute to the migration and development of T. canis during the early infection stage. Pathway analysis revealed significant alterations of several immune-inflammatory pathways, such as the B-cell receptor signaling pathway, the NF-kappa B signaling pathway, and the C-type lectin receptor signaling pathway at 12 and 24 hpi. These findings demonstrate the value of lipidomic profiling in revealing the extent of changes in the composition and abundance of hepatic lipidome caused by T. canis infection and their relevance to the pathophysiology of toxocariasis in beagle dogs.

Key signaling networks are dysregulated in patients with the adipose tissue disorder, lipedema

ObjectivesLipedema, a poorly understood chronic disease of adipose hyper-deposition, is often mistaken for obesity and causes significant impairment to mobility and quality-of-life. To identify molecular mechanisms underpinning lipedema, we employed comprehensive omics-based comparative analyses of whole tissue, adipocyte precursors (adipose-derived stem cells (ADSCs)), and adipocytes from patients with or without lipedema.MethodsWe compared whole-tissues, ADSCs, and adipocytes from body mass index–matched lipedema (n = 14) and unaffected (n = 10) patients using comprehensive global lipidomic and metabolomic analyses, transcriptional profiling, and functional assays.ResultsTranscriptional profiling revealed >4400 significant differences in lipedema tissue, with altered levels of mRNAs involved in critical signaling and cell function-regulating pathways (e.g., lipid metabolism and cell-cycle/proliferation). Functional assays showed accelerated ADSC proliferation and differentiation in lipedema. Profiling lipedema adipocytes revealed >900 changes in lipid composition and >600 differentially altered metabolites. Transcriptional profiling of lipedema ADSCs and non-lipedema ADSCs revealed significant differential expression of >3400 genes including some involved in extracellular matrix and cell-cycle/proliferation signaling pathways. One upregulated gene in lipedema ADSCs, Bub1, encodes a cell-cycle regulator, central to the kinetochore complex, which regulates several histone proteins involved in cell proliferation. Downstream signaling analysis of lipedema ADSCs demonstrated enhanced activation of histone H2A, a key cell proliferation driver and Bub1 target. Critically, hyperproliferation exhibited by lipedema ADSCs was inhibited by the small molecule Bub1 inhibitor 2OH-BNPP1 and by CRISPR/Cas9-mediated Bub1 gene depletion.ConclusionWe found significant differences in gene expression, and lipid and metabolite profiles, in tissue, ADSCs, and adipocytes from lipedema patients compared to non-affected controls. Functional assays demonstrated that dysregulated Bub1 signaling drives increased proliferation of lipedema ADSCs, suggesting a potential mechanism for enhanced adipogenesis in lipedema. Importantly, our characterization of signaling networks driving lipedema identifies potential molecular targets, including Bub1, for novel lipedema therapeutics.

Simvastatin Blocks Reinstatement of Cocaine-induced Conditioned Place Preference in Male Mice with Brain Lipidome Remodeling.

Drug-associated reward memories are conducive to intense craving and often trigger relapse. Simvastatin has been shown to regulate lipids that are involved in memory formation but its influence on other cognitive processes is elusive. Here, we used a mass spectrometry-based lipidomic method to evaluate the impact of simvastatin on the mouse brain in a cocaine-induced reinstatement paradigm. We found that simvastatin blocked the reinstatement of cocaine-induced conditioned place preference (CPP) without affecting CPP acquisition. Specifically, only simvastatin administered during extinction prevented cocaine-primed reinstatement. Global lipidome analysis showed that the nucleus accumbens was the region with the greatest degree of change caused by simvastatin. The metabolism of fatty-acids, phospholipids, and triacylglycerol was profoundly affected. Simvastatin reversed most of the effects on phospholipids induced by cocaine. The correlation matrix showed that cocaine and simvastatin significantly reshaped the lipid metabolic pathways in specific brain regions. Furthermore, simvastatin almost reversed all changes in the fatty acyl profile and unsaturation caused by cocaine. In summary, pre-extinction treatment with simvastatin facilitates cocaine extinction and prevents cocaine relapse with brain lipidome remodeling.

SCD2-mediated monounsaturated fatty acid metabolism regulates cGAS-STING-dependent type I IFN responses in CD4+ T cells

Host lipid metabolism and viral responses are intimately connected. However, the process by which the acquired immune systems adapts lipid metabolism to meet demands, and whether or not the metabolic rewiring confers a selective advantage to host immunity, remains unclear. Here we show that viral infection attenuates the expression of genes related to lipid metabolism in murine CD4+ T cells, which in turn increases the expression of antiviral genes. Inhibition of the fatty acid synthesis pathway substantially increases the basal expression of antiviral genes via the spontaneous production of type I interferon (IFN). Using a combination of CRISPR/Cas9-mediated genome editing technology and a global lipidomics analysis, we found that the decrease in monounsaturated fatty acid caused by genetic deletion of Scd2 in mice was crucial for the induction of an antiviral response through activation of the cGAS-STING pathway. These findings demonstrate the important relationship between fatty acid biosynthesis and type I IFN responses that enhances the antiviral response.

Characterizing a role for NuA4 in the regulation of ergosterol in yeast

NuA4 is an essential lysine acetyltransferase (KAT) in yeast and is homologous to Tip60, a disease relevant protein complex in mammalian cells. As our lab and others have recently identified a novel role for NuA4 in lipid regulation, we performed a global lipidomic analysis. We determined that two mutants of NuA4, a temperature sensitive mutant of the catalytic subunit (esa1-ts) and deletion of the non-essential scaffolding subunit (eaf1Δ), have an increase in the relative abundance of ergosteryl esters, the yeast equivalent to cholesteryl esters. Following this discovery, we have also found that the NuA4 mutants have altered sensitivity to many compounds which target ergosterol synthesis and ergosterol in the membrane. This suggests that ergosterol regulation in NuA4 mutants may be altered. We are working to measure ergosterol directly in these mutants as well as to determine how NuA4 may affect ergosterol regulation in the cell. Specifically, we are using the deletion mutant array, a collection of yeast strains with each gene deleted, to look for genes that reverse the sensitivity of NuA4 mutants to ergosterol targeting compounds such as Amphotericin B and Nystatin. Overall, this study aims to characterize changes in ergosterol regulation in yeast NuA4 mutants. Given the exceptional conservation of KAT function across eukaryotes, this may provide insight into understanding the biological roles of Tip60 in lipid regulation and the cellular consequences of its deregulation in disease.

Disaccahrides-Based Cryo-Formulant Effect on Modulating Phospho/Mitochondrial Lipids and Biological Profiles of Human Leukaemia Cells.

The use of novel cryo-additive agents to increase cell viability post-cryopreservation is paramount to improve future cell based-therapy treatments. We aimed to establish the Human Leukemia (HL-60) cells lipidomic and biological patterns when cryo-preserved in DMSO alone and with 300 µM Nigerose (Nig), 200 µM Salidroside (Sal) or a combination of Nig (150 µM) and Sal (100 µM). HL-60 cells were pre-incubated with Nig/Sal prior, during and post cryopreservation, and subjected to global lipidomic analysis. Malondialdeyhde (MDA), released lactate dehydrogenase (LDH) and reactive oxygen scavenger (ROS) measurements were also carried out to evaluate levels of lipid peroxidation and cytotoxicity. Cryopreserving HL-60 cells in DMSO with Nig and Sal provided optimal protection against unsaturated fatty acid oxidation. Post-thaw, cellular phospholipids and mitochondrial cardiolipins were increased by Nig/Sal as the ratio of unsaturated to saturated fatty acids 2.08 +/- 0.03 and 0.95 +/- 0.09 folds respectively in comparison to cells cryopreserved in DMSO alone (0.49 +/- 0.05 and 0.86 +/- 0.10 folds). HL-60 lipid peroxidation levels in the presence of DMSO + Nig and Sal combined were significantly reduced relative to pre-cryopreservation levels (10.91 +/- 2.13 nmole) compared to DMSO (17.1 +/- 3.96 nmole). DMSO + Nig/Sal combined also significantly reduced cell cytotoxicity post-thaw (0.0128 +/- 0.00182 mU/mL) in comparison to DMSO (0.0164 +/- 0.00126 mU/mL). The combination of Nig/Sal also reduced significantly ROS levels to the levels of prior cryopreservation of HL-60. Overall, the establishment of the cryopreserved HL-60 cells lipidomic and the corresponding biological profiles showed an improved cryo-formulation in the presence of DMSO with the Nig/Sal combination by protecting the, mitochondrial inner membrane, unsaturated fatty acid components (i. e. Cardiolipins) and total phospholipids.

Aging selectively dampens oscillation of lipid abundance in white and brown adipose tissue

Lipid metabolism is under the control of the circadian system and circadian dysregulation has been linked to obesity and dyslipidemia. These factors and outcomes have also been associated to, or affected by, the process of aging. Here, we investigated whether murine white (WAT) and brown (BAT) adipose tissue lipids exhibit rhythmicity and if this is affected by aging. To this end, we have measured the 24 h lipid profiles of WAT and BAT using a global lipidomics analysis of > 1100 lipids. We observed rhythmicity in nearly all lipid classes including glycerolipids, glycerophospholipids, sterol lipids and sphingolipids. Overall, ~ 22% of the analyzed lipids were considered rhythmic in WAT and BAT. Despite a general accumulation of lipids upon aging the fraction of oscillating lipids decreased in both tissues to 14% and 18%, respectively. Diurnal profiles of lipids in BAT appeared to depend on the lipid acyl chain length and this specific regulation was lost in aged mice. Our study revealed how aging affects the rhythmicity of lipid metabolism and could contribute to the quest for targets that improve diurnal lipid homeostasis to maintain cardiometabolic health during aging.

Comprehensive and High-Coverage Lipidomic Analysis of Oilseeds Based on Ultrahigh-Performance Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry.

Oilseeds are an important source of dietary lipids, and a comprehensive analysis of oilseed lipids is of great significance to human health, while information about the global lipidomes in oilseeds was limited. Herein, an ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry method for comprehensive lipidomic profiling of oilseeds was established and applied. First, the lipid extraction efficiency and lipid coverage of four different lipid extraction methods were compared. The optimized methyl tert-butyl ether extraction method was superior to isopropanol, Bligh-Dyer, and Folch extraction methods, in terms of the operation simplicity, lipid coverage, and number of identified lipids. Then, global lipidomic analysis of soybean, sesame, peanut, and rapeseed was conducted. A total of 764 lipid molecules, including 260 triacylglycerols, 54 diacylglycerols, 313 glycerophospholipids, 36 saccharolipids, 35 ceramides, 30 free fatty acids, 21 fatty esters, and 15 sphingomyelins were identified and quantified. The compositions and contents of lipids significantly varied among different oilseeds. Our results provided a theoretical basis for the selection and breeding of varieties of oilseed as well as deep processing of oilseed for the edible oil industry.

Global metabolomic and lipidomic analysis reveals the potential mechanisms of hemolysis effect of Ophiopogonin D and Ophiopogonin D' in vivo

BackgroundOPD and OPD' are the two main active components of Ophiopogon japonicas in Shenmai injection (SMI). Being isomers of each other, they are supposed to have similar pharmacological activities, but the actual situation is complicated. The difference of hemolytic behavior between OPD and OPD' in vivo and in vitro was discovered and reported by our group for the first time. In vitro, only OPD' showed hemolysis reaction, while in vivo, both OPD and OPD' caused hemolysis. In vitro, the primary cause of hemolysis has been confirmed to be related to the difference between physical and chemical properties of OPD and OPD'. In vivo, although there is a possible explanation for this phenomenon, the one is that OPD is bio-transformed into OPD' or its analogues in vivo, the other one is that both OPD and OPD' were metabolized into more activated forms for hemolysis. However, the mechanism of hemolysis in vivo is still unclear, especially the existing literature are still difficult to explain why OPD shows the inconsistent hemolysis behavior in vivo and in vitro. Therefore, the study of hemolysis of OPD and OPD' in vivo is of great practical significance in response to the increase of adverse events of SMI.MethodsAiming at the hemolysis in vivo, this manuscript adopted untargeted metabolomics and lipidomics technology to preliminarily explore the changes of plasma metabolites and lipids of OPD- and OPD'-treated rats. Metabolomics and lipidomics analyses were performed on ultra-high performance liquid chromatography (UPLC) system tandem with different mass spectrometers (MS) and different columns respectively. Multivariate statistical approaches such as principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA) were applied to screen the differential metabolites and lipids.ResultsBoth OPD and OPD' groups experienced hemolysis, Changes in endogenous differential metabolites and differential lipids, enrichment of differential metabolic pathways, and correlation analysis of differential metabolites and lipids all indicated that the causes of hemolysis by OPD and OPD' were closely related to the interference of phospholipid metabolism.ConclusionsThis study provided a comprehensive description of metabolomics and lipidomics changes between OPD- and OPD'-treated rats, it would add to the knowledge base of the field, which also provided scientific guidance for the subsequent mechanism research. However, the underlying mechanism require further research.

Study on the interaction mechanism of phospholipid imbalance and endoplasmic reticulum protein secretion imbalance in Aspergillus niger

As the largest membrane organelle, the endoplasmic reticulum (ER) is the main location for protein preliminary processing and phospholipid synthesis. Phospholipid bilayer is the main component of the ER, so it plays an intuitively important role in the steady state of protein synthesis in the ER. Despite of their importance, relationship between phospholipid homeostasis and protein processing in Aspergillus niger remains poorly understood. In this study, phosphatidyl ethanolamine (PE)/phosphatidyl choline (PC) and phosphatidyl acid (PA) metabolic mutants and ER protein processing mutants were established by knockout the key genes in phospholipid synthesis or UPR effector hacA. Based on global transcriptome and lipidome analysis, the relationship between the phospholipids imbalance and ER protein secretory imbalance was revealed as followed: The cells compensate for the damage caused by ER protein secretory deficiency or phospholipid deficiency from enhancing the protein processing and the synthesis of phospholipids at the transcription level, therefore phospholipid deficiency (Δopi3) and continuous activation of UPR (hacAi) have a synergistic effect in promoting protein secretion and phospholipid biosynthesis. At the same time, the metabolic deficiencies of phospholipid homeostasis and the processing deficiencies of ER protein will also cause cells sensitive to oxidative stress, cell wall inhibition and DNA damage.

Alterations of the Gut Microbiome Composition and Lipid Metabolic Profile in Radiation Enteritis.

Radiation enteritis (RE) is a common complication in cancer patients receiving radiotherapy. Although studies have shown the changes of this disease at clinical, pathological and other levels, the dynamic characteristics of local microbiome and metabolomics are hitherto unknown. We aimed to examine the multi-omics features of the gut microecosystem, determining the functional correlation between microbiome and lipid metabolites during RE activity. By delivering single high-dose irradiation, a RE mouse model was established. High-throughput 16S rDNA sequencing and global lipidomics analysis were performed to examine microbial and lipidomic profile changes in the gut microecosystem. Spearman correlation analysis was used to determine the functional correlation between bacteria and metabolites. Clinical samples were collected to validate the above observations. During RE activity, the intestinal inflammation of the mice was confirmed by typical signs, symptoms, imaging findings and pathological evidences. 16S datasets revealed that localized irradiation dramatically altered the gut microbial composition, resulting in a decrease ratio of Bacteroidetes to Firmicutes. Lipidomics analysis indicated the remarkable lipidomic profile changes in enteric epithelial barrier, determining that glycerophospholipids metabolism was correlated to RE progression with the highest relevance. Spearman correlation analysis identified that five bacteria-metabolite pairs showed the most significant functional correlation in RE, including Alistipes-PC(36:0e), Bacteroides-DG(18:0/20:4), Dubosiella-PC(35:2), Eggerthellaceae-PC(35:6), and Escherichia-Shigella-TG(18:2/18:2/20:4). These observations were partly confirmed in human specimens. Our study provided a comprehensive description of microbiota dysbiosis and lipid metabolic disorders in RE, suggesting strategies to change local microecosystem to relieve radiation injury and maintain homeostasis.

Human adipocyte differentiation and composition of disease-relevant lipids are regulated by miR-221-3p

MicroRNA-221-3p (miR-221-3p) is associated with both metabolic diseases and cancers. However, its role in terminal adipocyte differentiation and lipid metabolism are uncharacterized. miR-221-3p or its inhibitor was transfected into differentiating or mature human adipocytes. Triglyceride (TG) content and adipogenic gene expression were monitored, global lipidome analysis was carried out, and mechanisms underlying the effects of miR-221-3p were investigated. Finally, cross-talk between miR-221-3p expressing adipocytes and MCF-7 breast carcinoma (BC) cells was studied, and miR-221-3p expression in tumor-proximal adipose biopsies from BC patients analyzed. miR-221-3p overexpression inhibited terminal differentiation of adipocytes, as judged from reduced TG storage and gene expression of the adipogenic markers SCD1, GLUT4, FAS, DGAT1/2, AP2, ATGL and AdipoQ, whereas the miR-221-3p inhibitor increased TG storage. Knockdown of the predicted miR-221-3p target, 14-3-3γ, had similar antiadipogenic effects as miR-221-3p overexpression, indicating it as a potential mediator of mir-221-3p function. Importantly, miR-221-3p overexpression inhibited de novo lipogenesis but increased the concentrations of ceramides and sphingomyelins, while reducing diacylglycerols, concomitant with suppression of sphingomyelin phosphodiesterase, ATP citrate lyase, and acid ceramidase. miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive BC. Conditioned medium of miR-221-3p overexpressing adipocytes stimulated the invasion and proliferation of BC cells, while medium of the BC cells enhanced miR-221-3p expression in adipocytes. Elevated miR-221-3p impairs adipocyte lipid storage and differentiation, and modifies their ceramide, sphingomyelin, and diacylglycerol content. These alterations are relevant for metabolic diseases but may also affect cancer progression.

Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

Development of a NanoLC-MS workflow for high-sensitivity global lipidomic analysis

Lipidomics focuses on the comprehensive analysis of lipids and their interactions with other molecules. Many biological samples are available in small volumes or limited amounts and display very complex lipid compositions; thus, highly sensitive lipidomic analysis methods are often needed. NanoLC-MS offers extremely high sensitivity, although it is known to be technically more challenging than the conventional LC-MS approach, requiring greater care and maintenance. This work describes the development and optimization of a nanoLC-MS method for routine analysis of the lipidome of small volumes of biological samples. We focused on achieving robust conditions for high sensitivity analysis of complex samples. The nanoLC method, mass spectrometry conditions and sample preparation by liquid-liquid extraction of lipids were fully optimized using serum samples and deuterated lipid standards, including an evaluation of contamination sources. The performance of the method was assessed through the analysis of human and pig sera, as well as cerebrospinal fluid samples from pigs. This method allowed the detection of 9900 to 12,200 features by employing only 1.0–2.5 μL of serum samples and identification of 5842 lipids from 36 subclasses within a 50-min gradient. The method can be easily adapted to other types of biological samples where only limited volumes are available.