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Abstract In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

Abstract The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.

Abstract Inflammation is an important factor in Alzheimer’s disease (AD). An NMR measurement in plasma, glycoprotein acetyls (GlycA), captures the overall level of protein production and glycosylation implicated in systemic inflammation. With its additional advantage of reducing biological variability, GlycA might be useful in monitoring the relationship between peripheral inflammation and brain changes relevant to AD. However, the associations between GlycA and these brain changes have not been fully evaluated. Here, we performed Spearman’s correlation analyses to evaluate these associations cross-sectionally and determined whether GlycA can inform AD-relevant longitudinal measurements among participants in the Alzheimer’s Disease Neuroimaging Initiative (n = 1506), with additional linear models and stratification analyses to evaluate the influences of sex or diagnosis status and confirm findings from Spearman’s correlation analyses. We found that GlycA was elevated in AD patients compared to cognitively normal participants. GlycA correlated negatively with multiple concurrent regional brain volumes in females diagnosed with late mild cognitive impairment (LMCI) or AD. Baseline GlycA level was associated with executive function decline at 3–9 year follow-up in participants diagnosed with LMCI at baseline, with similar but not identical trends observed in the future decline of memory and entorhinal cortex volume. Results here indicated that GlycA is an inflammatory biomarker relevant to AD pathogenesis and that the stage of LMCI might be relevant to inflammation-related intervention.

The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.

Summary: Background: Metabolic ageing biomarkers may capture the age-related shifts in metabolism, offering a precise representation of an individual’s overall metabolic health. Methods: Utilising comprehensive lipidomic datasets from two large independent population cohorts in Australia (n = 14,833, including 6630 males, 8203 females), we employed different machine learning models, to predict age, and calculated metabolic age scores (mAge). Furthermore, we defined the difference between mAge and age, termed mAgeΔ, which allow us to identify individuals sharing similar age but differing in their metabolic health status. Findings: Upon stratification of the population into quintiles by mAgeΔ, we observed that participants in the top quintile group (Q5) were more likely to have cardiovascular disease (OR = 2.13, 95% CI = 1.62–2.83), had a 2.01-fold increased risk of 12-year incident cardiovascular events (HR = 2.01, 95% CI = 1.45–2.57), and a 1.56-fold increased risk of 17-year all-cause mortality (HR = 1.56, 95% CI = 1.34–1.79), relative to the individuals in the bottom quintile group (Q1). Survival analysis further revealed that men in the Q5 group faced the challenge of reaching a median survival rate due to cardiovascular events more than six years earlier and reaching a median survival rate due to all-cause mortality more than four years earlier than men in the Q1 group. Interpretation: Our findings demonstrate that the mAge score captures age-related metabolic changes, predicts health outcomes, and has the potential to identify individuals at increased risk of metabolic diseases. Funding: The specific funding of this article is provided in the acknowledgements section.

Summary: Background: Decreased levels of circulating ethanolamine plasmalogens [PE(P)], and a concurrent increase in phosphatidylethanolamine (PE) are consistently reported in various cardiometabolic conditions. Here we devised, a plasmalogen score (Pls Score) that mirrors a metabolic signal that encompasses the levels of PE(P) and PE and captures the natural variation in circulating plasmalogens and perturbations in their metabolism associated with disease, diet, and lifestyle. Methods: We utilised, plasma lipidomes from the Australian Obesity, Diabetes and Lifestyle study (AusDiab; n = 10,339, 55% women) a nationwide cohort, to devise the Pls Score and validated this in the Busselton Health Study (BHS; n = 4,492, 56% women, serum lipidome) and in a placebo-controlled crossover trial involving Shark Liver Oil (SLO) supplementation (n = 10, 100% men). We examined the association of the Pls Score with cardiometabolic risk factors, type 2 diabetes mellitus (T2DM), cardiovascular disease and all-cause mortality (over 17 years). Findings: In a model, adjusted for age, sex and BMI, individuals in the top quintile of the Pls Score (Q5) relative to Q1 had an OR of 0.31 (95% CI 0.21–0.43), 0.39 (95% CI 0.25–0.61) and 0.42 (95% CI 0.30–0.57) for prevalent T2DM, incident T2DM and prevalent cardiovascular disease respectively, and a 34% lower mortality risk (HR = 0.66; 95% CI 0.56–0.78). Significant associations between diet and lifestyle habits and Pls Score exist and these were validated through dietary supplementation of SLO that resulted in a marked change in the Pls Score. Interpretation: The Pls Score as a measure that captures the natural variation in circulating plasmalogens, was not only inversely related to cardiometabolic risk and all-cause mortality but also associate with diet and lifestyle. Our results support the potential utility of the Pls Score as a biomarker for metabolic health and its responsiveness to dietary interventions. Further research is warranted to explore the underlying mechanisms and optimise the practical implementation of the Pls Score in clinical and population settings. Funding: National Health and Medical Research Council (NHMRC grant 233200), National Health and Medical Research Council of Australia (Project grant APP1101320), Health Promotion Foundation of Western Australia, and National Health and Medical Research Council of Australia Senior Research Fellowship (#1042095).

Abstract Recent advancements in plasma lipidomic profiling methodology have significantly increased specificity and accuracy of lipid measurements. This evolution, driven by improved chromatographic and mass spectrometric resolution of newer platforms, has made it challenging to align datasets created at different times, or on different platforms. Here we present a framework for harmonising such plasma lipidomic datasets with different levels of granularity in their lipid measurements. Our method utilises elastic-net prediction models, constructed from high-resolution lipidomics reference datasets, to predict unmeasured lipid species in lower-resolution studies. The approach involves (1) constructing composite lipid measures in the reference dataset that map to less resolved lipids in the target dataset, (2) addressing discrepancies between aligned lipid species, (3) generating prediction models, (4) assessing their transferability into the targe dataset, and (5) evaluating their prediction accuracy. To demonstrate our approach, we used the AusDiab population-based cohort (747 lipid species) as the reference to impute unmeasured lipid species into the LIPID study (342 lipid species). Furthermore, we compared measured and imputed lipids in terms of parameter estimation and predictive performance, and validated imputations in an independent study. Our method for harmonising plasma lipidomic datasets will facilitate model validation and data integration efforts.

An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues, however the mechanisms by which epidermal interactions shape processing of noxious inputs is still poorly understood. Here, we identify a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites, but not at other sites in the epidermis. From a genetic screen we identified miR-14 as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. We found that miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, we found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.

Abstract Obesity is a risk factor for type 2 diabetes and cardiovascular disease. However, a substantial proportion of patients with these conditions have a seemingly normal body mass index (BMI). Conversely, not all obese individuals present with metabolic disorders giving rise to the concept of “metabolically healthy obese”. We use lipidomic-based models for BMI to calculate a metabolic BMI score (mBMI) as a measure of metabolic dysregulation associated with obesity. Using the difference between mBMI and BMI (mBMIΔ), we identify individuals with a similar BMI but differing in their metabolic health and disease risk profiles. Exercise and diet associate with mBMIΔ suggesting the ability to modify mBMI with lifestyle intervention. Our findings show that, the mBMI score captures information on metabolic dysregulation that is independent of the measured BMI and so provides an opportunity to assess metabolic health to identify “at risk” individuals for targeted intervention and monitoring.

Complementary multi-omics approach, in silico metabolic flux, and neuroimaging analyses link the sphingolipid pathway to Alzheimer’s disease pathogenesis with therapeutic targets validated using behavioral assessments in amyloidogenic APP/PS1 mice.

Summary: SORL1 is implicated in the pathogenesis of Alzheimer’s disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor β (TGF-β)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

BackgroundBreastfed infants have lower disease risk compared to formula-fed infants, however, the mechanisms behind this protection are unknown. Human milk has a complex lipidome which may have many critical roles in health and disease risk. However, human milk lipidomics is challenging, and research is still required to fully understand the lipidome and to interpret and translate findings. This study aimed to address key human milk lipidome knowledge gaps and discuss possible implications for early life health.MethodsHuman milk samples from two birth cohorts, the Barwon Infant Study (n = 312) and University of Western Australia birth cohort (n = 342), were analysed using four liquid chromatography-mass spectrometry (LC–MS) methods (lipidome, triacylglycerol, total fatty acid, alkylglycerol). Bovine, goat, and soy-based infant formula, and bovine and goat milk were analysed for comparison. Composition was explored as concentrations, relative abundance, and infant lipid intake. Statistical analyses included principal component analysis, mixed effects modelling, and correlation, with false discovery rate correction, to explore human milk lipidome longitudinal trends and inter and intra-individual variation, differences between sample types, lipid intakes, and correlations between infant plasma and human milk lipids.ResultsLipidomics analysis identified 979 lipids. The human milk lipidome was distinct from that of infant formula and animal milk. Ether lipids were of particular interest, as they were significantly higher, in concentration and relative abundance, in human milk than in formula and animal milk, if present in the latter samples at all. Many ether lipids were highest in colostrum, and some changed significantly through lactation. Significant correlations were identified between human milk and infant circulating lipids (40% of which were ether lipids), and specific ether lipid intake by exclusively breastfed infants was 200-fold higher than that of an exclusively formula-fed infant.ConclusionThere are marked differences between the lipidomes of human milk, infant formula, and animal milk, with notable distinctions between ether lipids that are reflected in the infant plasma lipidome. These findings have potential implications for early life health, and may reveal why breast and formula-fed infants are not afforded the same protections. Comprehensive lipidomics studies with outcomes are required to understand the impacts on infant health and tailor translation.

Abstract Background Lipids play a vital role in health and disease, but changes to their circulating levels and the link with obesity remain poorly characterized in expecting mothers and their offspring in early childhood. Methods LC-MS/MS-based quantitation of 480 lipid species was performed on 2491 plasma samples collected at 4 time points in the mother-offspring Asian cohort GUSTO (Growing Up in Singapore Towards healthy Outcomes). These 4 time points constituted samples collected from mothers at 26–28 weeks of gestation (n=752) and 4–5 years postpartum (n=650), and their offspring at birth (n=751) and 6 years of age (n=338). Linear regression models were used to identify the pregnancy and developmental age-specific variations in the plasma lipidomic profiles, and their association with obesity risk. An independent birth cohort (n=1935), the Barwon Infant Study (BIS), comprising mother-offspring dyads of Caucasian origin was used for validation. Results Levels of 36% of the profiled lipids were significantly higher (absolute fold change > 1.5 and Padj < 0.05) in antenatal maternal circulation as compared to the postnatal phase, with phosphatidylethanolamine levels changing the most. Compared to antenatal maternal lipids, cord blood showed lower concentrations of most lipid species (79%) except lysophospholipids and acylcarnitines. Changes in lipid concentrations from birth to 6 years of age were much higher in magnitude (log2FC=−2.10 to 6.25) than the changes observed between a 6-year-old child and an adult (postnatal mother) (log2FC=−0.68 to 1.18). Associations of cord blood lipidomic profiles with birth weight displayed distinct trends compared to the lipidomic profiles associated with child BMI at 6 years. Comparison of the results between the child and adult BMI identified similarities in association with consistent trends (R 2=0.75). However, large number of lipids were associated with BMI in adults (67%) compared to the children (29%). Pre-pregnancy BMI was specifically associated with decrease in the levels of phospholipids, sphingomyelin, and several triacylglycerol species in pregnancy. Conclusions In summary, our study provides a detailed landscape of the in utero lipid environment provided by the gestating mother to the growing fetus, and the magnitude of changes in plasma lipidomic profiles from birth to early childhood. We identified the effects of adiposity on the circulating lipid levels in pregnant and non-pregnant women as well as offspring at birth and at 6 years of age. Additionally, the pediatric vs maternal overlap of the circulating lipid phenotype of obesity risk provides intergenerational insights and early opportunities to track and intervene the onset of metabolic adversities. Clinical trial registration This birth cohort is a prospective observational study, which was registered on 1 July 2010 under the identifier NCT01174875 .

Dysregulation of lipid metabolism is associated with coronary artery disease (CAD). Here, the authors perform GWAS of the serum lipidome to identify variants associated with lipid species that are putatively in the mechanistic pathway to CAD.

Abstract Background Both changes in circulating lipids represented by a validated poor prognostic 3-lipid signature (3LS) and somatic tumour genetic aberrations are individually associated with worse clinical outcomes in men with metastatic castration-resistant prostate cancer (mCRPC). A key question is how the lipid environment and the cancer genome are interrelated in order to exploit this therapeutically. We assessed the association between the poor prognostic 3-lipid signature (3LS), somatic genetic aberrations and clinical outcomes in mCRPC. Methods We performed plasma lipidomic analysis and cell-free DNA (cfDNA) sequencing on 106 men with mCRPC commencing docetaxel, cabazitaxel, abiraterone or enzalutamide (discovery cohort) and 94 men with mCRPC commencing docetaxel (validation cohort). Differences in lipid levels between men ± somatic genetic aberrations were assessed with t-tests. Associations between the 3LS and genetic aberrations with overall survival (OS) were examined using Kaplan-Meier methods and Cox proportional hazard models. Results The 3LS was associated with shorter OS in the discovery (hazard ratio [HR] 2.15, 95% confidence interval [CI] 1.4-3.3, p < 0.001) and validation cohorts (HR 2.32, 95% CI 1.59–3.38, p < 0.001). Elevated plasma sphingolipids were associated with AR, TP53, RB1 and PI3K aberrations (p < 0.05). Men with both the 3LS and aberrations in AR, TP53, RB1 or PI3K had shorter OS than men with neither in both cohorts (p ≤ 0.001). The presence of 3LS and/or genetic aberration was independently associated with shorter OS for men with AR, TP53, RB1 and PI3K aberrations (p < 0.02). Furthermore, aggressive-variant prostate cancer (AVPC), defined as 2 or more aberrations in TP53, RB1 and/or PTEN, was associated with elevated sphingolipids. The combination of AVPC and 3LS predicted for a median survival of ~12 months. The relatively small sample size of the cohorts limits clinical applicability and warrants future studies. Conclusions Elevated circulating sphingolipids were associated with AR, TP53, RB1, PI3K and AVPC aberrations in mCRPC, and the combination of lipid and genetic abnormalities conferred a worse prognosis. These findings suggest that certain genotypes in mCRPC may benefit from metabolic therapies.

Abstract Objectives The leading cause of mortality in patients with rheumatoid arthritis is atherosclerotic cardiovascular disease (CVD). We have shown that murine arthritis impairs atherosclerotic lesion regression, because of cellular cholesterol efflux defects in haematopoietic stem and progenitor cells (HSPCs), causing monocytosis and impaired atherosclerotic regression. Therefore, we hypothesised that improving cholesterol efflux using a Liver X Receptor (LXR) agonist would improve cholesterol efflux and improve atherosclerotic lesion regression in arthritis. Methods Ldlr−/− mice were fed a western‐type diet for 14 weeks to initiate atherogenesis, then switched to a chow diet to induce lesion regression and divided into three groups; (1) control, (2) K/BxN serum transfer inflammatory arthritis (K/BxN) or (3) K/BxN arthritis and LXR agonist T0901317 daily for 2 weeks. Results LXR activation during murine inflammatory arthritis completely restored atherosclerotic lesion regression in arthritic mice, evidenced by reduced lesion size, macrophage abundance and lipid content. Mechanistically, serum from arthritic mice promoted foam cell formation, demonstrated by increased cellular lipid accumulation in macrophages and paralleled by a reduction in mRNA of the cholesterol efflux transporters Abca1, Abcg1 and Apoe. T0901317 reduced lipid loading and increased Abca1 and Abcg1 expression in macrophages exposed to arthritic serum and increased ABCA1 levels in atherosclerotic lesions of arthritic mice. Moreover, arthritic clinical score was also attenuated with T0901317. Conclusion Taken together, we show that the LXR agonist T0901317 rescues impaired atherosclerotic lesion regression in murine arthritis because of enhanced cholesterol efflux transporter expression and reduced foam cell development in atherosclerotic lesions.

Exercise training can be therapeutic but how mitochondria respond remains unclear. Here, the authors use multiple omics techniques to reveal a complex network of non-stoichiometric mitochondrial adaptations that are prioritized or deprioritised during different phases of exercise training.

Objectives: Dysregulation of cholesterol metabolism in the liver and hematopoietic stem and progenitor cells (HSPCs) promotes atherosclerosis development. Previously, it has been shown that HMG-CoA-Reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, can be phosphorylated and inactivated by the metabolic stress sensor AMP-activated protein kinase (AMPK). However, the physiological significance of AMPK regulation of HMGCR to atherogenesis has yet to be elucidated. The aim of this study was to determine the role of AMPK/HMGCR axis in the development of atherosclerosis. Methods: We have generated a novel atherosclerotic-prone mouse model with defects in the AMPK regulation of HMGCR (Apoe−/−/Hmgcr KI mice). Atherosclerotic lesion size, plaque composition, immune cell and lipid profiles were assessed in Apoe−/− and Apoe−/−/Hmgcr KI mice. Results: In this study, we showed that both male and female atherosclerotic-prone mice with a disruption of HMGCR regulation by AMPK (Apoe−/−/Hmgcr KI mice) display increased aortic lesion size concomitant with an increase in plaque-associated macrophages and lipid accumulation. Consistent with this, Apoe−/−/Hmgcr KI mice exhibited an increase in total circulating cholesterol and atherogenic monocytes, Ly6-Chi subset. Mechanistically, increased circulating atherogenic monocytes in Apoe−/−/Hmgcr KI mice was associated with enhanced egress of bone marrow HSPCs and extramedullary myelopoiesis, driven by a combination of elevated circulating 27-hydroxycholesterol and intracellular cholesterol in HSPCs. Conclusions: Our results uncovered a novel signalling pathway involving AMPK-HMGCR axis in the regulation of cholesterol homeostasis in HSPCs, and that inhibition of this regulatory mechanism accelerates the development and progression of atherosclerosis. These findings provide a molecular basis to support the use of AMPK activators that currently undergoing Phase II clinical trial such as O–3O4 and PXL 770 for reducing atherosclerotic cardiovascular disease risks.

IntroductionCardiovascular disease (CVD) is the leading cause of mortality worldwide and is the leading cause of death in the US. Lipid dysregulation is a well-known precursor to metabolic diseases, including CVD. There is a growing body of literature that suggests MRI-derived epicardial fat volume, or epicardial adipose tissue (EAT) volume, is linked to the development of coronary artery disease. Interestingly, epicardial fat is also actively involved in lipid and energy homeostasis, with epicardial adipose tissue having a greater capacity for release and uptake of free fatty acids. However, there is a scarcity of knowledge on the influence of plasma lipids on EAT volume.AimThe focus of this study is on the identification of novel lipidomic species associated with CMRI-derived measures of epicardial fat in Mexican American individuals.MethodsWe performed lipidomic profiling on 200 Mexican American individuals. High-throughput mass spectrometry enabled rapid capture of precise lipidomic profiles, providing measures of 799 unique species from circulating plasma samples. Because of our extended pedigree design, we utilized a standard quantitative genetic linear mixed model analysis to determine whether lipids were correlated with EAT by formally testing for association between each lipid species and the CMRI epicardial fat phenotype.ResultsAfter correction for multiple testing using the FDR approach, we identified 135 lipid species showing significant association with epicardial fat. Of those, 131 lipid species were positively correlated with EAT, where increased circulating lipid levels were correlated with increased epicardial fat. Interestingly, the top 10 lipid species associated with an increased epicardial fat volume were from the deoxyceramide (Cer(m)) and triacylglycerol (TG) families. Deoxyceramides are atypical and neurotoxic sphingolipids. Triacylglycerols are an abundant lipid class and comprise the bulk of storage fat in tissues. Pathologically elevated TG and Cer(m) levels are related to CVD risk and, in our study, to EAT volume.ConclusionOur results indicate that specific lipid abnormalities such as enriched saturated triacylglycerols and the presence of toxic ceramides Cer(m) in plasma of our individuals could precede CVD with increased EAT volume.

Abstract While maternal mental health strongly influences neurodevelopment and health in the offspring, little is known about the determinants of inter-individual variation in the mental health of mothers. Likewise, the in utero biological pathways by which variation in maternal mental health affects offspring development remain to be defined. Previous studies implicate lipids, consistent with a known influence on cognitive and emotional function, but the relevance for maternal mental health and offspring neurodevelopment is unclear. This study characterizes the placental and circulatory lipids in antenatal depression, as well as socio-emotional outcomes in the offspring. Targeted liquid chromatography-mass spectrometry covering 470 lipid species was performed on placenta from 186 women with low (n = 70) or high (n = 116) levels of antenatal depressive symptoms assessed using the Edinburgh Postnatal Depression Scale at 26 weeks’ gestation. Child socio-emotional outcomes were assessed from the Child Behavior Check List (CBCL) at 48 months. Seventeen placental lipid species showed an inverse association with antenatal EPDS scores. Specifically, lower levels of phospholipids containing LC-PUFAs: omega-3 docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-6 arachidonic acid (AA) were significantly associated with depressive symptoms. Additional measurement of LC-PUFA in antenatal plasma samples at mid-gestation confirmed the reduced circulation of these specific fatty acids in mothers. Reduced concentration of the placental phospholipids also predicted poorer socio-emotional outcomes in the offspring. This study provides new insights into the role of the materno-fetal lipid cross-talk as a mechanism linking maternal mental health to that of the offspring. These findings show the potential utility of nutritional approaches among pregnant women with depressive symptoms to reduce offspring risk for later socio-emotional problems.

Background: There is mounting evidence that in utero and early life exposures may predispose an individual to metabolic disorders in later life; and dysregulation of lipid metabolism is critical in such outcomes. However, there is limited knowledge about lipid metabolism and factors causing lipid dysregulation in early life that could result in adverse health outcomes in later life. We studied the effect of antenatal factors such as gestational age, birth weight, and mode of birth on lipid metabolism at birth; changes in the circulating lipidome in the first 4 years of life and the effect of breastfeeding in the first year of life. From this study, we aim to generate a framework for deeper understanding into factors effecting lipid metabolism in early life, to provide early interventions for those at risk of developing metabolic disorders including cardiovascular diseases. Methods: We performed comprehensive lipid profiling of 1074 mother-child dyads in the Barwon Infant Study (BIS), a population-based pre-birth cohort and measured 776 distinct lipid features across 39 lipid classes using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). We measured lipids in 1032 maternal serum samples at 28 weeks’ gestation, 893 cord serum samples at birth, 793, 735, and 511 plasma samples at 6, 12 months, and 4 years, respectively. Cord serum was enriched with long chain poly-unsaturated fatty acids (LC-PUFAs), and corresponding cholesteryl esters relative to the maternal serum. We performed regression analyses to investigate the associations of cord serum lipid species with antenatal factors: gestational age, birth weight, mode of birth and duration of labour. Results: The lipidome differed between mother and newborn and changed markedly with increasing child’s age. Alkenylphosphatidylethanolamine species containing LC-PUFAs increased with child’s age, whereas the corresponding lysophospholipids and triglycerides decreased. Majority of the cord serum lipids were strongly associated with gestational age and birth weight, with most lipids showing opposing associations. Each mode of birth showed an independent association with cord serum lipids. Breastfeeding had a significant impact on the plasma lipidome in the first year of life, with up to 17-fold increases in a few species of alkyldiaclylglycerols at 6 months of age. Conclusions: This study sheds light on lipid metabolism in infancy and early childhood and provide a framework to define the relationship between lipid metabolism and health outcomes in early childhood. Funding: This work was supported by the A*STAR-NHMRC joint call funding (1711624031).

The onset and pathology of Alzheimer’s disease (AD) is associated with changes to lipid metabolism. Here, the authors analysed 569 lipids from 32 classes and subclasses in two independent patient cohorts to identify key lipid pathways to link the plasma lipidome with AD and the future onset of AD.

CVD is the leading cause of death worldwide, and genetic investigations into the human lipidome may provide insight into CVD risk. The aim of this study was to estimate the heritability of circulating lipid species and their genetic correlation with CVD traits. Targeted lipidomic profiling was performed on 4,492 participants from the Busselton Family Heart Study to quantify the major fatty acids of 596 lipid species from 33 classes. We estimated narrow-sense heritabilities of lipid species/classes and their genetic correlations with eight CVD traits: BMI, HDL-C, LDL-C, triglycerides, total cholesterol, waist-hip ratio, systolic blood pressure, and diastolic blood pressure. We report heritabilities and genetic correlations of new lipid species/subclasses, including acylcarnitine (AC), ubiquinone, sulfatide, and oxidized cholesteryl esters. Over 99% of lipid species were significantly heritable (h2: 0.06–0.50) and all lipid classes were significantly heritable (h2: 0.14–0.50). The monohexosylceramide and AC classes had the highest median heritabilities (h2 = 0.43). The largest genetic correlation was between clinical triglycerides and total diacylglycerol (rg = 0.88). We observed novel positive genetic correlations between clinical triglycerides and phosphatidylglycerol species (rg: 0.64–0.82), and HDL-C and alkenylphosphatidylcholine species (rg: 0.45–0.74). Overall, 51% of the 4,768 lipid species-CVD trait genetic correlations were statistically significant after correction for multiple comparisons. This is the largest lipidomic study to address the heritability of lipids and their genetic correlation with CVD traits. Future work includes identifying putative causal genetic variants for lipid species and CVD using genome-wide SNP and whole-genome sequencing data.

Sex and the APOE ε4 genotype are important risk factors for late-onset Alzheimer’s disease. In the current study, the authors investigate how sex and APOE ε4 genotype modify the association between Alzheimer’s disease biomarkers and metabolites in serum.

Background: Intrinsic resistance to androgen receptor signalling inhibitors (ARSI) occurs in 20–30% of men with metastatic castration-resistant prostate cancer (mCRPC). Ceramide metabolism may have a role in ARSI resistance. Our study's aim is to investigate the association of the ceramide-sphingosine-1-phosphate (ceramide-S1P) signalling axis with ARSI resistance in mCRPC. Methods: Lipidomic analysis (∼700 lipids) was performed on plasma collected from 132 men with mCRPC, before commencing enzalutamide or abiraterone. AR gene aberrations in 77 of these men were identified by deep sequencing of circulating tumour DNA. Associations between circulating lipids, radiological progression-free survival (rPFS) and overall survival (OS) were examined by Cox regression. Inhibition of ceramide-S1P signalling with sphingosine kinase (SPHK) inhibitors (PF-543 and ABC294640) on enzalutamide efficacy was investigated with in vitro assays, and transcriptomic and lipidomic analyses of prostate cancer (PC) cell lines (LNCaP, C42B, 22Rv1). Findings: Men with elevated circulating ceramide levels had shorter rPFS (HR=2·3, 95% CI=1·5–3·6, p = 0·0004) and shorter OS (HR=2·3, 95% CI=1·4–36, p = 0·0005). The combined presence of an AR aberration with elevated ceramide levels conferred a worse prognosis than the presence of only one or none of these characteristics (median rPFS time = 3·9 vs 8·3 vs 17·7 months; median OS time = 8·9 vs 19·8 vs 34·4 months). SPHK inhibitors enhanced enzalutamide efficacy in PC cell lines. Transcriptomic and lipidomic analyses indicated that enzalutamide combined with SPHK inhibition enhanced PC cell death by SREBP-induced lipotoxicity. Interpretation: Ceramide-S1P signalling promotes ARSI resistance, which can be reversed with SPHK inhibitors. Funding: None.

The sphingolipid de novo synthesis pathway, encompassing the sphingolipids, the enzymes and the cell membrane receptors, are being investigated for their role in diseases and as potential therapeutic targets. The intermediate sphingolipids such as dihydrosphingosine (dhSph) and sphingosine (Sph) have not been investigated due to them being thought of as precursors to other more active lipids such as ceramide (Cer) and sphingosine 1 phosphate (S1P). Here we investigated their effects in terms of collagen synthesis in primary rat neonatal cardiac fibroblasts (NCFs). Our results in NCFs showed that both dhSph and Sph did not induce collagen synthesis, whilst dhSph reduced collagen synthesis induced by transforming growth factor β (TGFβ). The mechanisms of these inhibitory effects were associated with the increased activation of the de novo synthesis pathway that led to increased dihydrosphingosine 1 phosphate (dhS1P). Subsequently, through a negative feedback mechanism that may involve substrate-enzyme receptor interactions, S1P receptor 1 expression (S1PR1) was reduced.

Dysregulation of lipid metabolism plays a major role in the etiology and sequelae of inflammatory disorders, cardiometabolic and neurological diseases, and several forms of cancer. Recent advances in lipidomic methodology allow comprehensive lipidomic profiling of clinically relevant biological samples, enabling researchers to associate lipid species and metabolic pathways with disease onset and progression. The resulting data serve not only to advance our fundamental knowledge of the underlying disease process but also to develop risk assessment models to assist in the diagnosis and management of disease. Currently, clinical applications of in-depth lipidomic profiling are largely limited to the use of research-based protocols in the analysis of population or clinical sample sets. However, we foresee the development of purpose-built clinical platforms designed for continuous operation and clinical integration—assisting health care providers with disease risk assessment, diagnosis, and monitoring. Herein, we review the current state of clinical lipidomics, including the use of research-based techniques and platforms in the analysis of clinical samples as well as assays already available to clinicians. With a primary focus on MS-based strategies, we examine instrumentation, analysis techniques, statistical models, prospective design of clinical platforms, and the possible pathways toward implementation of clinical lipidomics.

[This corrects the article DOI: 10.1371/journal.pbio.3000870.].

Obesity and related metabolic diseases show clear sex-related differences. The growing burden of these diseases calls for better understanding of the age- and sex-related metabolic consequences. High-throughput lipidomic analyses of population-based cohorts offer an opportunity to identify disease-risk-associated biomarkers and to improve our understanding of lipid metabolism and biology at a population level. Here, we comprehensively examined the relationship between lipid classes/subclasses and molecular species with age, sex, and body mass index (BMI). Furthermore, we evaluated sex specificity in the association of the plasma lipidome with age and BMI. Some 747 targeted lipid measures, representing 706 molecular lipid species across 36 classes/subclasses, were measured using a high-performance liquid chromatography coupled mass spectrometer on a total of 10,339 participants from the Australian Diabetes, Obesity and Lifestyle Study (AusDiab), with 563 lipid species being validated externally on 4,207 participants of the Busselton Health Study (BHS). Heat maps were constructed to visualise the relative differences in lipidomic profile between men and women. Multivariable linear regression analyses, including sex-interaction terms, were performed to assess the associations of lipid species with cardiometabolic phenotypes. Associations with age and sex were found for 472 (66.9%) and 583 (82.6%) lipid species, respectively. We further demonstrated that age-associated lipidomic fingerprints differed by sex. Specific classes of ether-phospholipids and lysophospholipids (calculated as the sum composition of the species within the class) were inversely associated with age in men only. In analyses with women alone, higher triacylglycerol and lower lysoalkylphosphatidylcholine species were observed among postmenopausal women compared with premenopausal women. We also identified sex-specific associations of lipid species with obesity. Lysophospholipids were negatively associated with BMI in both sexes (with a larger effect size in men), whilst acylcarnitine species showed opposing associations based on sex (positive association in women and negative association in men). Finally, by utilising specific lipid ratios as a proxy for enzymatic activity, we identified stearoyl CoA desaturase (SCD-1), fatty acid desaturase 3 (FADS3), and plasmanylethanolamine Δ1-desaturase activities, as well as the sphingolipid metabolic pathway, as constituent perturbations of cardiometabolic phenotypes. Our analyses elucidate the effect of age and sex on lipid metabolism by offering a comprehensive view of the lipidomic profiles associated with common cardiometabolic risk factors. These findings have implications for age- and sex-dependent lipid metabolism in health and disease and suggest the need for sex stratification during lipid biomarker discovery, establishing biological reference intervals for assessment of disease risk.

Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.

Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD.

Summary: Exercise-induced heart growth provides protection against cardiovascular disease, whereas disease-induced heart growth leads to heart failure. These distinct forms of growth are associated with different molecular profiles (e.g., mRNAs, non-coding RNAs, and proteins), and targeting differentially regulated genes has therapeutic potential for heart failure. The effects of exercise on the cardiac and circulating lipidomes in comparison to disease are unclear. Lipidomic profiling was performed on hearts and plasma of mice subjected to swim endurance training or a cardiac disease model (moderate or severe pressure overload). Several sphingolipid species and phospholipids containing omega-3/6 fatty acids were distinctly altered in heart and/or plasma with exercise versus pressure overload. A subset of lipids was validated in an independent mouse model with heart failure and atrial fibrillation. This study highlights the adaptations that occur to lipid profiles in response to endurance training versus pathology and provides a resource to investigate potential therapeutic targets and biomarkers. : Tham et al. utilized a HPLC-ESI/MS/MS targeted lipidomics approach to show distinct remodeling of the cardiac and plasma lipidomes in response to exercise in comparison to heart disease stimuli. Differentially altered lipids were validated in a model with heart failure and atrial fibrillation and represent potential biomarkers and drug targets. Keywords: lipids, heart, exercise, physiological hypertrophy, pathological hypertrophy, atrial fibrillation, phospholipids, sphingolipids, biomarkers, treatment

Abstract Background As prednisone and ciclosporin can have immunosuppressive effects and have been considered potential predisposing factors for skin infections, we investigated the impact of these drugs on the diversity of the cutaneous microbiota, the abundance of Malassezia and infection with Papillomaviruses. Results Six atopic, asymptomatic Maltese-beagle dogs were treated with ciclosporin for one month and then with prednisone for another month, with a one-month wash-out between treatments. The dogs were sampled on the abdomen and pinna before and after each treatment using a swab. Samples for Papillomavirus detection were obtained with cytobrush sticks. The bacterial microbiota was characterized using 16S amplicon high-throughput sequencing. Malassezia populations were quantified with nested real-time PCR targeting the ribosomal internal transcribed spacer 1. The diversity and composition of cutaneous microbiota was not impacted in a detectable manner by any of the treatments. As observed for the bacterial microbiota, Malassezia populations were not affected by treatment. Three dogs were positive for Papillomavirus at more than one timepoint, but an association with treatment was not apparent. Conclusions Ciclosporin and prednisone at doses used for the treatment of atopic dermatitis do not impact the canine cutaneous microbiota in a detectable manner.

Lipid metabolism is tightly linked to adiposity. Comprehensive lipidomic profiling offers new insights into the dysregulation of lipid metabolism in relation to weight gain. Here, we investigated the relationship of the human plasma lipidome and changes in waist circumference (WC) and body mass index (BMI). Adults (2653 men and 3196 women), 25–95 years old who attended the baseline survey of the Australian Diabetes, Obesity and Lifestyle Study (AusDiab) and the 5-year follow-up were enrolled. A targeted lipidomic approach was used to quantify 706 distinct molecular lipid species in the plasma samples. Multiple linear regression models were used to examine the relationship between the baseline lipidomic profile and changes in WC and BMI. Metabolic scores for change in WC were generated using a ridge regression model. Alkyl-diacylglycerol such as TG(O-50:2) [NL-18:1] displayed the strongest association with change in WC (β-coefficient = 0.125 cm increment per SD increment in baseline lipid level, p = 2.78 × 10−11. Many lipid species containing linoleate (18:2) fatty acids were negatively associated with both WC and BMI gain. Compared to traditional models, multivariate models containing lipid species identify individuals at a greater risk of gaining WC: top quintile relative to bottom quintile (odds ratio, 95% CI = 5.4, 3.8–6.6 for women and 2.3, 1.7–3.0 for men). Our findings define metabolic profiles that characterize individuals at risk of weight gain or WC increase and provide important insight into the biological role of lipids in obesity.

Plasmalogens or alkenylphospholipids are a sub-class of glycerophospholipids with numerous biological functions and are thought to have protective effects against metabolic disease. Dietary supplementation with alkylglycerols (AKGs) has been shown to increase endogenous plasmalogen levels, however effective modulation of different molecular plasmalogen species has not yet been demonstrated. In this study, the effects of an orally-administered AKG mix (a mixture of chimyl, batyl and selachyl alcohol at a 1:1:1 ratio) on plasma and tissue lipids, including plasmalogens, was evaluated. Mice on a Western-type diet were treated with either an AKG mix or vehicle (lecithin) for 1, 2, 4, 8 and 12 weeks. Treatment with the AKG mix significantly increased the total plasmalogen content of plasma, liver and adipose tissue as a result of elevations in multiple plasmalogen species with different alkenyl chains. Alkylphospholipids, the endogenous precursors of plasmalogens, showed a rapid and significant increase in plasma, adipose tissue, liver and skeletal muscle. A significant accumulation of alkyl-diacylglycerol and lyso-ether phospholipids was also observed in plasma and tissues. Additionally, the dynamics of plasmalogen-level changes following AKG mix supplementation differed between tissues. These findings indicate that oral supplementation with an AKG mix is capable of upregulating and maintaining stable expression of multiple molecular plasmalogen species in circulation and tissues.

In biological systems lipids generate membranes and have a key role in cell signaling and energy storage. Therefore, there is a wide diversity of molecular lipid expressed at the compositional level in cell membranes and organelles, as well as in tissues, whose lipid distribution remains unclear. Here, we report a mass spectrometry study of lipid abundance across 7 rat tissues, detecting and quantifying 652 lipid molecular species from the glycerolipid, glycerophospholipid, fatty acyl, sphingolipid, sterol lipid and prenol lipid categories. Our results demonstrate that every tissue analyzed presents a specific lipid distribution and concentration. Thus, glycerophospholipids are the most abundant tissue lipid, they share a similar tissue distribution but differ in particular lipid species between tissues. Sphingolipids are more concentrated in the renal cortex and sterol lipids can be found mainly in both liver and kidney. Both types of white adipose tissue, visceral and subcutaneous, are rich in glycerolipids but differing the amount. Acylcarnitines are mainly in the skeletal muscle, gluteus and soleus, while heart presents higher levels of ubiquinone than other tissues. The present study demonstrates the existence of a rat tissue-specific fingerprint.

Macrophages are abundant within adipose tissue depots where they are exposed to fatty acids, leading to lipid accumulation. Herein, we have determined the effects of various fatty acids on the macrophage lipidome. Using targeted mass-spectrometry, we were able to detect 641 individual lipid species in primary murine macrophages treated with a variety of saturated fatty acids and an un-saturated fatty acid, either alone or in combination. The most pronounced effects were observed for the long-chain saturated fatty acid palmitate, which increased the total abundance of numerous classes of lipids. While other medium- and long-chain saturated fatty acids, as well as the long-chain unsaturated fatty acid, had less pronounced effects on the total abundance of specific lipid classes, all fatty acids induced marked alterations in the abundance of numerous lipid species within given lipid classes. Fatty acid treatment markedly altered overall phospholipid saturation status; these effects were most pronounced for phosphatidylcholine and ether-phosphatidylcholine lipid species. Finally, treatment of macrophages with either palmitate or stearate in combination with oleate prevented many of the changes that were observed in macrophages treated with palmitate or stearate alone. Collectively, our results reveal substantial and specific remodelling of the macrophage lipidome following treatment with fatty acids.

Multiple rotating annular reactors were seeded with biofilms flushed from water distribution systems to assess (1) whether biofilms grown in bioreactors are representative of biofilms flushed from the water distribution system in terms of bacterial composition and diversity, and (2) whether the biofilm sampling method affects the population profile of the attached bacterial community. Biofilms were grown in bioreactors until thickness stabilized (9 to 11 weeks) and harvested from reactor coupons by sonication, stomaching, bead-beating, and manual scraping. High-throughput sequencing of 16S rRNA amplicons was used to profile bacterial populations from flushed biofilms seeded into bioreactors as well as biofilms recovered from bioreactor coupons by different methods. β diversity between flushed and reactor biofilms was compared to β diversity between (i) biofilms harvested from different reactors and (ii) biofilms harvested by different methods from the same reactor. These analyses showed that average diversity between flushed and bioreactor biofilms was double the diversity between biofilms from different reactors operated in parallel. The diversity between bioreactors was larger than the diversity associated with different biofilm recovery methods. Compared to other experimental variables, the method used to recover biofilms had a negligible impact on the outcome of water biofilm analyses based on 16S amplicon sequencing. Results from this study show that biofilms grown in reactors over 9 to 11 weeks are not representative models of the microbial populations flushed from a distribution system. Furthermore, the bacterial population profile of biofilms grown in replicate reactors from the same flushed water are likely to diverge. However, four common sampling protocols, which differ with respect to disruption of bacterial cells, provide similar information with respect to the 16S rRNA population profile of the biofilm community.

Liver disease is the greatest cause of death related to alcohol and a major public health problem. While excessive alcohol intake results in hepatosteatosis in most individuals, this can progress in some to more severe forms of liver disease including fibrosis and cirrhosis. An ongoing challenge in the management of alcoholic liver disease is the identification of liver injury early in the disease process such that intervention strategies can prevent serious long term outcomes. Given that excessive alcohol consumption results in dysregulation of lipid metabolism we applied lipid profiling technology to characterise and compare serum lipid profiles from excessive chronic drinkers with no liver disease to those with advanced alcoholic cirrhosis. In a cohort of 59 excessive drinkers (31 with liver cirrhosis and 28 with no evidence of liver disease) we used electrospray ionisation tandem mass spectrometry to measure over 300 individual lipid species in serum, including species of the major phospholipid, sphingolipid, glycerolipid and sterol classes. Six of the 25 lipid classes and subclasses were significantly associated with alcoholic liver cirrhosis; these included dihexosylceramide, trihexosylceramide, alkylphosphatidylcholine, lysoalkylphosphatidylcholine, phosphatidylinositol and free cholesterol. Multivariate classification models created with only clinical characteristics gave an optimal model with an AUC of 0.847 and an accuracy of 79.7%. The addition of lipid measurements to the clinical characteristics resulted in models of improved performance with an AUC of 0.892 and accuracy of 81.8%. The gain in AUC and accuracy of the combined models highlight the potential of serum lipids as markers of liver injury in alcoholic liver disease.