Journal of Lipid Research

Cholesteryl esters (CEs) are central intermediates in cholesterol storage and transport, yet the enzymes responsible for their hydrolysis in mammals remain poorly defined. While lysosomal acid lipase is the only well-established acidic CE hydrolase, the molecular identity of physiologically relevant neutral CE hydrolases has remained unresolved. Here, we systematically profiled CE hydrolase activity across mouse tissues and blood using substrate-based LC-MS assays, tissue fractionation, and inhibitor screening. We observed robust CE hydrolase activity in multiple tissues and circulation, with activity predominantly enriched in membrane fractions and strongly sensitive to broad-spectrum metabolic serine hydrolase inhibitors. Pharmacological screening excluded previously proposed neutral CE hydrolases, including NCEH1 and LIPE, and identified tetrahydrolipstatin-sensitive lipoprotein lipase (LPL) as a candidate CE hydrolase. Competitive activity-based protein profiling analyses in RAW264.7 macrophages further supported selective enrichment and inhibition of LPL. Biochemical characterization demonstrated that recombinant wild-type LPL, but not the catalytic S159A variant, efficiently hydrolyzed CEs in vitro. Importantly, this activity required co-expression of the lipase maturation factor 1, indicating that LPL-mediated CE hydrolysis is dependent on proper enzymatic maturation. Together, these findings identify LPL as a previously unrecognized mammalian CE hydrolase and expand its functional role beyond triglyceride metabolism.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids to meet cellular energetic and signaling needs. As a unique monolayer structure, LDs arise from the endoplasmic reticulum (ER) and acquire proteins and lipids through their membrane contact sites (MCSs) with the ER. In this study, we exogenously induce ER-LD MCSs using a dimerization-dependent fluorescent protein (ddFP) system. Strikingly, inducing these MCSs increases LD size without influencing LD total amount per cell, in a manner that is distinct from LD biogenesis induced by the dietary fatty acid oleic acid. By examining the trafficking of the triacylglycerol synthesis enzyme DGAT2 under ddFP induction, we found that artificial tethering recruits LD proteins to the ER-LD interface but not to the LD surface, unlike oleic acid supplementation. However, by supplementing ddFP-transfected cells with fluorescent fatty acids, we found that ddFP-positive LDs preferentially incorporate exogenous lipid, suggesting that inducing MCSs can facilitate ER-to-LD lipid transfer. These results demonstrate ddFPs as a tool for manipulating LD MCSs and elucidate the role of ER-LD MCSs following LD biogenesis to continue to promote LD growth.

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

BACKGROUND: Perfluorooctane sulfonate (PFOS) is a widely distributed persistent organic pollutant in the environment and has been associated with an increased risk of atherosclerosis. However, the underlying pathogenic mechanisms remain largely unclear. This study aimed to investigate the effects of PFOS on atherosclerosis and its associated gut-vascular axis. METHODS: Pseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to determine the role of the gut microbiota in PFOS-induced atherosclerosis. Metagenomic sequencing was performed to characterize alterations in gut microbial composition following PFOS exposure, and targeted metabolomics was used to assess bile acid profiles in the ileum and plasma. Transcriptomic analysis of Bacteroides caecimuris (B.caecimuris) was conducted to explore the reasons for the increased abundance of B.caecimuris after PFOS exposure. In addition, intestinal transcriptomics and ChIP-qPCR were performed to validate transcriptional regulation within the FXR-TLR3 signaling axis. RESULTS: Among 127 participants with paired serum and fecal samples, including 82 patients undergoing coronary angiography with Gensini scores (GS score), fecal PFOS levels were significantly associated with lipid profiles and GS score, whereas serum PFOS showed no such association. Mechanistically, PFOS exposure promotes intestinal enrichment of B. caecimuris by upregulating its tolC gene, thereby enhancing efflux capacity. This microbial shift was accompanied by reduced levels of tauro-ursodeoxycholic acid (TUDCA) and aberrant activation of intestinal FXR signaling. Further analyses demonstrated that FXR activation upregulated TLR3 expression and promoted inflammatory responses and atherosclerosis progression via the TLR3-NF-{kappa}B signaling axis. Both intestinal epithelial-specific FXR deficiency (Fxr{Delta}IE) and TUDCA supplementation significantly suppressed pathway activation and alleviated disease phenotypes.Functional experiments identified TLR3 as a key downstream effector of FXR. Overexpression of TLR3 abolished the protective effects observed in Fxr{Delta}IE mice. Moreover, pharmacological inhibition of TLR3 using CU CPT-4a significantly improved established atherosclerotic lesions in vivo. CONCLUSIONS: This study identifies a gut microbiota-driven FXR-TLR3 signaling axis that mediates PFOS-induced atherosclerosis. These findings provide new mechanistic insights into environmentally induced cardiovascular disease and suggest potential targets for risk assessment and therapeutic intervention.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied this method to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

Vascular ischemia is characterized not only by hypoxia but also by acidosis, which affects endothelial cells (ECs) due to increased H+ production from glycolysis and a deficit in H+ washout. We recently documented that an acidic environment facilitates the flip-flop transport of the non-ionized form of fatty acids (FAs) across the plasma membrane of cancer cells. In this study, we investigated how acidosis influences the capacity of highly glycolytic ECs to manage FAs and participates to endothelial dysfunction. We first tracked lipid droplet (LD) formation using Oil Red O staining and holotomographic microscopy. Purified monounsaturated oleate but also a mixture of FAs that reflect in vivo serum composition, resulted in dose- and time-dependent LD accumulation through FA transporter-independent mechanisms. Acid-exposed ECs exhibited enhanced mitochondrial respiration fueled by FAs, and endoplasmic reticulum (ER) stress, as indicated by the expression of ATF4 and CHOP. This phenotype was further associated with elevated reactive oxygen species production, which correlated with reduced nitric oxide (NO) availability. FA removal from EC culture media promoted lipolysis from LDs, supported by ATGL lipase induction which however slowed under acidic conditions. While ER stress persisted upon FA washout, NO availability was restored to levels comparable to those in FA-unexposed ECs. This observation coincided with dynamic mobilization of antioxidant defenses in acid-exposed ECs, as evidenced by low levels of reduced glutathione and enhanced cystine uptake, alongside a decrease in carnitine and FA-fueled mitochondrial respiration. Collectively, these data underscore the vulnerability of ECs to passive FA capture promoted by local acidosis, thereby contributing to a silent source of endothelial dysfunction in the postprandial state or during chronic exposure to elevated lipid levels.

BACKGROUND: Erythrophagocytosis constitutes a major pathogenic mechanism of metabolic dysfunction associated fatty liver disease (MAFLD). Our previous research established a quantitative thin-layer chromatography (TLC) technique for sphingomyelin, revealing reduced levels in the red blood cells (erythrocytes) of patients with metabolic dysfunction associated fatty liver disease (MAFLD). This reduction was accompanied by erythrocyte sphingosine accumulation, a driver of pro-inflammatory erythrophagocytosis, though sphingosine 1-phosphate release remained stable. To better understand erythrocyte sphingosine metabolism, we adapted our quantitative TLC method to analyze sphingosine within the erythrocyte-conditioned media (ECM) of MAFLD patients. Methodology Separation was performed on 10X10cm Silica gel 60 F254 plates using a mobile phase of chloroform, methanol, acetic acid, and water (60:50:1:4 v/v/v/v). The dynamic range, linearity, and range of linearity were assessed by analysing sphingosine levels from 0.1 to 10microg/spot. We validated the system precision and sensitivity by performing triplicate analyses of sphingosine standards (1.25, 2.5, and microg). The limits of detection and quantification were derived from the calibration curve slope and standard deviation (3.3 XSD/slope for LOD; 10 XSD/slope for LOQ). Accuracy was assessed via recovery tests at 100%, 200%, and 300% of a 2.5microg load. We confirmed specificity by evaluating the retention factors against other lipid species. This protocol was applied to Folch-extracted lipids from the ECM (5 X 107 cells/ml) of four MAFLD patients and four healthy controls, spiked with 5microg of sphingosine. Findings The calibration model, based on combined Green and Blue color intensities, followed the linear equation y = -11.171x + 353.25(R2 = 0.94). Interday precision values were 0.21%, 1.65%, and 0.44%, while recovery rates (accuracy) ranged from 94.5% to 98.7%. The measured LOD and LOQ were 0.75microg and 1.21microg, respectively. The sensitivity was calculated at 90ng. Statistical analysis showed no significant variance in sphingosine concentrations in erythrocyte-conditioned media between the MAFLD group and the control group. Summary The described thin layer chromatography is accurate, precise, sensitive, with good limits of detection and quantification, and most importantly is low-cost and time-efficient. Using this method, we show that while erythrocytes of MAFLD patients exhibit sphingosine accumulation, the utilisation of exogenous sphingosine from their erythrocytes is not affected. This suggests that the metabolic shift may be driven by increased sphingosine supply from the plasma.

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

Background and aimsAlcohol-associated hepatitis (AH) is characterized by excessive inflammation and blunted antiviral interferon (IFN) responses. We hypothesized that specific gut microbiome-derived metabolites could selectively enhance interferon signaling while limiting NF-{kappa}B mediated inflammation, thereby restoring immune balance in AH. Our goal is to identify microbiome-derived metabolites that differentially regulate the NF-{kappa}B and IFN signaling pathways. Methods and resultsWe used human monocytic THP1-Dual cells, which secrete reporters for NF-{kappa}B and IFN signaling, to model innate immune responses and screened a library of 152 gut microbiome-derived metabolites. From the metabolite screen, 4-hydroxyphenylacetic acid (4-HPAA) emerged as a unique immunomodulator: in LPS-challenged cells, 4-HPAA selectively increased IFN signaling with minimal NF-{kappa}B activation. 4-HPAA was evaluated in vivo using a NIAAA-model, with 4-HPAA supplementation (0.4mg/ml) added to the diet. In the NIAAA-model, dietary 4-HPAA did not induce liver injury and was associated with enhanced interferon-stimulated gene expression. Simultaneously, 4-HPAA reduced pro-inflammatory markers such as Il1{beta}, Ly6g and F4/80 compared to the group exposed to ethanol alone. Metabolomic profiling of mouse cecal contents revealed 4-HPAA supplementation counteracted ethanols metabolic effects, selectively reducing triglyceride-associated lipids that had accumulated with ethanol feeding. Conclusions4-HPAA enhances interferon signaling and antiviral gene induction while dampening NF-{kappa}B-driven inflammation in the presence of LPS, both in vitro and in vivo. In an acute-on-chronic alcohol injury model, 4-HPAA attenuated hepatic inflammation, reduced immune cell recruitment, and activated antioxidant defenses, reflecting a shift toward a more hepatoprotective effect. 4-HPAA treatment was associated with reduced pro-inflammatory markers and modest attenuation of ethanol-induced liver injury. Additionally, 4-HPAA reversed ethanol-induced lipid-dysregulation, particularly triglyceride accumulation, highlighting its metabolic benefit in alcohol-fed mice. In conclusion, 4-HPAA rebalances immune and metabolic pathways by enhancing IFN signaling, suppressing NF-{kappa}B inflammation, and reversing alcohol-induced hepatic injury and lipid accumulation.

BackgroundDietary sodium restriction is a common nutritional and physiological challenge that activates electrolyte-conserving endocrine pathways, but its impact on systemic lipid metabolism remains incompletely defined. We examined whether short-term dietary sodium deprivation alters the circulating lipidome and identifies lipid signatures of metabolic adaptation. MethodsMale Sprague-Dawley rats were maintained on sodium-sufficient (NaS) or sodium-deprived (NaD) diets for 7 days (n=3 per group). Serum lipids were profiled by untargeted LC-MS/MS in positive and negative ion modes. Lipidomic differences were evaluated using class-level and species-level analyses, principal component analysis, volcano plots, heatmaps, and pathway-oriented interpretation. ResultsNaD rats exhibited a distinct serum lipidomic profile compared with NaS controls, indicating global remodeling of circulating lipid composition. Sodium deprivation produced class-specific and species-resolved changes, including selective depletion of subsets of neutral lipid species, prominent wax ester remodeling, increased phosphatidylcholine and lysophosphatidylcholine abundance, and altered acylcarnitine profiles. These signatures are consistent with coordinated changes in lipid storage, membrane phospholipid turnover, and mitochondrial fatty-acid handling. ConclusionsDietary sodium deprivation induces coordinated serum lipidome remodeling in rats, supporting the concept that nutritional electrolyte status can influence systemic lipid metabolism. These exploratory findings identify sodium deprivation as a metabolic stressor linked to neutral lipid mobilization, phospholipid remodeling, and altered mitochondrial substrate handling, and provide a foundation for future mechanistic studies.

Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy posing significant diagnostic challenges, particularly in distinguishing it from other adrenal tumors, such as adenoma and pheochromocytoma, due to overlapping imaging and biochemical features. Improved non-invasive tools are critically needed for earlier, more accurate classification of this rare cancer. This pilot study analyzed serum lipidomic profiles in ACC, pheochromocytoma, and adenoma patients versus healthy volunteers. The most significant alterations occurred in sphingomyelins (SM) and diacylglycerols (DG). All tumor samples showed reduced very-long odd-chain SM (e.g., SM 39:1, SM 41:1, SM 41:2) and elevated DG (e.g., DG 34:1, DG 34:2, DG 36:2). These abnormalities were most pronounced in malignant tumors: ACC and metastases (AUC = 0.933), followed by pheochromocytoma (AUC = 0.800) and adenoma (AUC = 0.711). ACC patients also exhibited specific lipid signatures with decreased alkyl/alkenyl phospholipids (e.g., PE O-38:5) and lysophosphatidylcholines (e.g., LPC 20:5, LPC 18:2) versus healthy volunteers, not observed in pheochromocytoma or adenomas. Ceramide species (e.g., Cer 42:2;O2, Cer 34:1;O2) were increased in ACC compared to the other tumor types. Incorporating lipid-to-lipid ratios (Cer/SM, Cer/DG) further improved statistical model accuracy. Compared to clinical biochemistry/oxidative stress (OS) parameters, lipidomic profiling showed superior discriminatory power in adrenal tumor diagnosis. The presented study shows the serum lipidomic profiling as a promising non-invasive method for distinguishing adrenal tumor subtypes (ACC, pheochromocytoma, and adenoma) from healthy individuals, with strong diagnostic potential for ACC.

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD) and associated dyslipidemia is a growing health issue that gives rise to cardiovascular risk. Men are more prone to development of MASLD than women. Understanding mechanisms underlying sex differences in MASLD may lead to improved prevention and treatment approaches. Cholesteryl ester transfer protein (CETP) is a lipid transfer protein that shuttles triglycerides and cholesteryl esters between blood lipoproteins and tissues. In this study investigate the impact of hepatic CETP expression on MASLD. Hepatic CETP expression (L-HuCETP) was achieved by injecting liver-targeted CETP-expressing adeno-associated virus into C57BL/6J mice. In females, L-HuCETP improved glucose tolerance, consistent with our prior clamp results in global human CETP transgenic mice. Whereas in males, L-HuCETP worsened glucose metabolism and impaired insulin signaling. Correspondingly, L-HuCETP expression reduced the expression of gluconeogenic pathway genes in females but upregulated these genes in males. In males, L-HuCETP mice exhibited increased hepatic lipid droplet accumulation, lipogenesis proteins and these changes were not observed in females. L-HuCETP expression resulted in sex-specific hepatic responses, with increased expression of inflammation and fibrosis related genes in male, but decreased expression of these genes in females. Mechanistic studies indicate that L-HuCETP had sex specific effects on transcription factors ChREBP and HNF4, which are important for glucose and lipid metabolism. Our studies suggest that sex-specific roles of L-HuCETP with regard to liver metabolic adaptation and MASLD risk in obesity, highlighting CETP-mediated pathways as potential targets for sex-specific precision medicine approaches to improve MASLD.

Systemic inflammatory diseases can be influenced by dietary intake, with gastrointestinal dysfunction driving both metabolic and behavioural changes mirroring the altered inflammatory profile. Additionally, the use of live biotherapeutic products (LBPs) shows promise for treating metabolic and inflammatory diseases, but their efficacy is limited by poor persistence in inflamed gut environments. Designed to utilize inflammatory byproducts, the LBP EcN::ttr has proven efficacy in the treatment of acute and chronic colitis, however its effects on the metabolic and behavioural patterns remain uncharacterized. We evaluated the effects of EcN::ttr on mice fed a proinflammatory omega-6 PUFA-rich diet. EcN::ttr-treated mice exhibited notable changes in the gut, including an improved expression of tight junction protein occludin, accompanied by reduced serum lipopolysaccharide (LPS) - binding protein, indicating protection against endotoxemia. EcN::ttr improved insulin sensitivity compared to the parental strain, associated with increased hepatic insulin receptor expression and reduced GSK3{beta} activation and endoplasmic reticulum stress. Secondary bile acids in mice treated with EcN::ttr were more abundant, with increases in those associated with resolving diarrhea and bile acid detoxification. Behavioural assessment highlighted a normalization of long-term memory along with a reduction of stress management behaviours. Altogether, EcN::ttr restores gut-liver-brain axis function through coordinated modulation of inflammation, barrier integrity, and bile acid metabolism. HighlightsO_LILive Biotherapeutic Product EcN::ttr, designed with a fitness advantage to survive inflammation, and provides protection against a proinflammatory omega 6-rich diet C_LIO_LIAdministration of EcN::ttr improved metabolic outcomes including increasing insulin sensitivity C_LIO_LIEcN::ttr increased the abundance of secondary bile acids including those that modulate bile acid detoxification C_LIO_LIBehavioural parameters were normalized in mice given EcN::ttr C_LIO_LIEcN::ttr partially normalizes gut-liver-brain axis through restoring barrier integrity, modulating inflammation and improving secondary bile acid metabolism C_LI

Sex differences strongly influence susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD), yet the regulatory mechanisms underlying these differences remain incompletely understood. To examine sex-specific hepatic adaptation to a high-fat (HF) diet mouse model of MASLD, we integrated proteomics, transcriptomics, and Oxford Nanopore direct RNA sequencing for transcriptome-wide m6A profiling in male and female mouse livers. Female mice were relatively protected from HF diet-induced hepatic steatosis and exhibited distinct proteome remodeling enriched for peroxisomal pathways. In contrast, transcriptomic responses in females were dominated by inflammatory signatures and did not recapitulate the metabolic adaptations observed at the protein level, revealing extensive RNA-protein discordance and post-transcriptional remodeling. Integrated RNA-protein analyses identified female-specific amplification of peroxisomal proteins despite modest transcript-level changes. HF diet also induced sex-specific remodeling of m6A RNA methylation and altered regulation of the m6A methylation system. Notably, reduced 3' UTR m6A methylation of peroxisomal transcripts inversely correlated with increased protein abundance relative to RNA expression in female mice. Together, these findings implicate m6A-associated post-transcriptional regulation in sex-specific hepatic adaptation to HF diet exposure and the basis for discordance between many of the mRNAs and proteins in the liver.

Background The systemic biochemical diversity of circulating metabolites and lipids reflects the integrated effects of genetic variation and environmental exposure. Metabolite quantitative trait locus (mQTL) studies in humans have established gene-metabolite associations, but genetic contributions can be obscured by sex, diet, age, medication use, and environmental exposures. Genetically diverse model systems offer a powerful complementary strategy to isolate genetic contributions to the biochemical diversity of the circulating metabolome. Methodology/Principal Findings We applied mass spectrometry profiling to serum samples in 541 mice from the Diversity Outbred (DO) population and identified 1,933 mQTL across 240 metabolites, 561 lipids, 43 oxylipins, and 4,465 MS/MS features. Co-mapping QTL, i.e., QTL hotspots, on chromosomes 8 and 17 implicated carboxyl esterase gene clusters (Ces1 and Ces2) as major regulators of circulating lipid remodeling and demonstrated genetic control of circulating protein/peptide-like features at the major histocompatibility complex and complement C3 loci. QTL hotspots on chromosomes 9 and 10 revealed previously unknown genetic drivers of lipid and amino acid metabolism. Comparisons with matched red blood cell mQTL revealed widespread compartment-specific genetic control. Conclusions/Significance Collectively, these findings provide a high-resolution map of the genetic regulation of the circulating metabolome, offering mechanistic insights that complement and extend human metabolic genetics.

During household storage, expressed human milk can develop odor and flavor changes that trigger infant refusal and lead caregivers to discard their saved milk supply. We show that typical refrigeration and freezing conditions disrupt the milk fat globule membrane (MFGM), exposing milk lipids to lipases that catalyze hydrolysis and oxidation. A pectin-based formulation (PBF) maintains MFGM integrity during storage and following lipase challenge, suppressing production of glycerol, free fatty acid, and oxylipin byproducts without broadly affecting milk macronutrients, the proteome, and culturable microbial burden. Across an independent cohort of lactating individuals, lipase activity varied but tracked with maternal milk lipase gene expression, implicating endogenous lipolysis in stored-milk deterioration. In a blinded olfactory panel, PBF-treated, lipase-challenged milk smelled more like fresh milk than untreated controls. Together, these findings show that stabilizing the MFGM can protect stored human milk from lipase-driven deterioration, preserve sensory quality, and support use for infant feeding.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Background Besides hyperglycemia, type 2 diabetes (T2D) is characterized by dyslipidemia, which is typically assessed using traditional clinical lipid measurements. However, molecular plasma lipids beyond these traditional markers can provide additional information about an individuals health status. For molecular lipids to be used effectively, certain characteristics, such as their temporal variability, need to be determined. Methods We analyzed the plasma lipidome for three consecutive time points, each three months apart, of 51 individuals with T2D using targeted liquid chromatography coupled to mass spectrometry (LC-MS). 513 lipid species across 25 (sub)classes were quantified by this approach and the temporal variability were calculated. Moreover, to identify sex differences in the plasma lipidome, we analyzed 914 samples of a cross-sectional T2D cohort with the same approach. Results Neutral lipids and phosphatidylserine had the highest temporal variability which was independent of their platform-specific variability. In contrast, glycosphingolipids were found to be relatively stable over time in individuals with T2D. Acyl-chain analysis revealed generally similar variability in the acyl-chain groups but indicated a higher temporal variability in medium-length acyl-chains. Lipid-sex association analysis showed markedly higher sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines in women and higher acylcarnitines in men. Overall, approximately one-third of measured lipids showed significant sex differences independent of age, BMI, diabetes duration, glycemic control, and medication use. Conclusions Our findings provide insights into temporal variability of molecular lipids. This variability should be considered when assessing novel lipid biomarkers. Likewise, sex differences in these lipids need to be considered in precision medicine for diabetes management.

Abstract Background: Sarcopenia is associated with anabolic resistance, a blunted muscle protein synthesis response to protein ingestion. Here, we hypothesized that anabolic resistance may be associated with a delayed postprandial decline in circulating plasma amino acids following protein ingestion. We therefore wanted to investigate whether an oral protein tolerance test (OPTT) combined with untargeted plasma metabolomics can detect age-related or sarcopenia-related differences in amino acid time courses consistent with altered postprandial amino acid handling, which could potentially reflect reduced anabolic sensitivity. Moreover, we investigated whether metabolites other than amino acids reacted to the OPTT. Methods: Twelve young healthy adults (controls: 22-28 years) and 12 older adults with clinically diagnosed probable or confirmed sarcopenia (70-91 years) ingested 20 g of whey protein after an overnight fast. We collected venous blood at baseline, 1 h, and 2 h post-ingestion and analyzed the samples by untargeted LC-HRMS plasma metabolomics. Linear mixed-effects models were fitted for 2,968 metabolic features with Benjamini-Hochberg FDR correction. For each category (branched-chain amino acid, essential amino acid [EAA], total amino acid) we summed the within-subject log2 fold changes (FC); fold changes (FC) of the constituent amino acids. This composite is reported as the summed log2FC. Results: 201 metabolites were structurally annotated including 58 amino acid-related metabolites and 97 lipids. Fourteen of 17 proteinogenic amino acids increased significantly after protein ingestion (FDR<0.05). In young controls, essential amino acids rose more steeply at 1 h than in sarcopenic individuals (+10.06 +/- 1.05 vs. +7.84 +/- 1.58 summed log2FC) and declined more between 1 and 2 h (-4.93 +/- 1.29 vs. -0.20 +/- 2.27 summed log2FC). Leucine exemplified this pattern best, rising 1.74 log2FC in controls and declining to 0.96 at 2 h, while remaining elevated at 1.61 log2FC in the sarcopenic group at 2 h (p=0.009). Beyond amino acids, whey protein lowered circulating free fatty acids in both groups (FA 18:2, FA 18:1, FA 16:0; all FDR<0.05). Medium- and long-chain acylcarnitines (Car 8:0, Car14:2) declined postprandially in controls but remained elevated in sarcopenic individuals (p<0.05), suggesting altered postprandial lipid metabolism. Conclusion: In this proof-of-concept study, an OPTT showed that plasma EAAs declined more slowly from their postprandial peak in older adults with sarcopenia than in young adults, consistent with altered postprandial amino acid handling that may reflect anabolic resistance. Whey protein ingestion additionally modulates lipid and acylcarnitine metabolism in an age-dependent manner, suggesting broader alterations in postprandial metabolic regulation in older adults with sarcopenia.

Severe infection with Streptococcus (S.) pneumoniae is a leading cause of acute lung injury, multiorgan failure, and death despite appropriate antibiotic therapy. A major contributor to tissue damage is pneumolysin (PLY), a cholesterol-dependent pore-forming toxin that is released in large amounts during bacterial lysis. Because antibiotic treatment enhances PLY release, understanding the mechanisms that control PLY-induced host injury is critical for the development of strategies that limit tissue damage without impairing antibacterial defense. Using isolated perfused lungs and in situ two-photon imaging, we show that S. pneumoniae induces platelet and leukocyte recruitment, pulmonary edema, perfusion failure, and intravascular bacterial trapping within pulmonary arterioles. These responses required PLY and were associated with endothelial Ca{superscript 2} influx and plasma membrane permeabilization. The oxysterol 25-hydroxycholesterol (25-HC) prevented PLY-dependent Ca{superscript 2} entry, vascular leakage, and perfusion failure by reducing accessible membrane cholesterol through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Unexpectedly, pneumococcal infection suppressed expression of cholesterol-25-hydroxylase (CH25H), the enzyme responsible for 25-HC synthesis, although induction of this pathway would be predicted to protect against toxin-mediated injury. We show that this downregulation results from inhibition of platelet-activating factor (PAF) receptor (PAFR) signaling by pneumococcal phosphocholine-containing cell wall components. Remarkably, both PAF itself and the anti-phosphocholine antibody TEPC directly inhibited PLY pore formation independently of PAFR, while preserving intravascular coagulation and bacterial trapping. Thus, although 25-HC protects against toxin-mediated vascular injury, its suppression may allow coagulation-dependent bacterial containment, with PAF acting as a compensatory antagonist of PLY. These findings identify a lipid-based host defense system in which 25-HC and PAF differentially regulate PLY activity to balance tissue protection with bacterial clearance and suggest new therapeutic approaches to limit the harmful effects of antibiotic-induced PLY release during pneumococcal infection.