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.

Previous research has shown that mammalian fatty acids (FAs) can influence antibiotic tolerance of Staphylococcus aureus, yet many of these studies overlook the sources of these FAs, which are primarily esterified into glycero- and phospholipids, and the impact S. aureus lipase activity has on host lipids. Here we attempt to gain insight into the complex interplay between the S. aureus lipidome and its environment using culture media supplemented tissue-specific phospholipid mixtures. Phospholipid profiles of heart, liver, and brain-derived lipids revealed distinct distributions of headgroup and fatty acyl tail structures within the phospholipids. Following the growth of S. aureus in lipid-enriched broth, PG species containing mono- and poly-unsaturated acyl tails were detected with abundances that correlated strongly with the FA profile of the tissue extract. We found that S. aureus cultured with liver-derived lipid extract, which yielded the most unsaturated PGs, promoted growth in high concentrations of the membrane-targeting antimicrobial daptomycin. To explore the influence of lipase activity on the extracellular lipids, comparative analysis of fresh versus spent media revealed that the lipase-mediated degradation of complex phospholipid mixtures was influenced by both head group structure and acyl tail linkage. Concurrently, the spent media contained elevated levels of mono- and polyunsaturated lysophospholipids that were predominantly of the 2-acyl form rather than the 1-acyl form observed in the fresh media. Together, these results demonstrate the extent to which the lipase activity of S. aureus remodels both its own lipidome as well as the structures of the phospholipids in the surrounding environment. IMPORTANCES. aureus releases a secreted glycerol ester hydrolase, Geh, into the extracellular environment, which enables the bacterium to generate free FA from glycerolipids, phospholipids, and cholesterol esters that are present in surrounding tissue of an infection. The liberated FAs can be incorporated into the phospholipids of S. aureus, thereby altering its membrane physiology with mono- and poly-unsaturated FAs it cannot otherwise synthesize. Simultaneously, the action of Geh on lipids in the host environment leads to higher levels of bioactive lysophospholipids that participate in mammalian signaling pathways. This work reveals the preferences of S. aureus Geh across phospholipids with different head group and acyl tail structures found within tissue-derived lipid extracts, as well as the fate of the liberated FAs within the staphylococcal membrane lipids. The impacts of these processes on both the host and bacterium have implications for the immune response to and antibiotic treatment of S. aureus infections.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

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.

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.

Enteroviruses are arguably the most numerous group of viruses infecting humans. While most enterovirus infections are benign and self-resolving, their sheer number inevitably increases the chances of multiple complications. The diversity of enteroviruses means that the development of vaccines is only economically feasible against a select few, and no direct-acting or host-targeted anti-virals are approved to treat enteroviral infections, largely due to the rapid development of resistance against all experimental drugs. Here, we explored a universal property of enterovirus infection - a massive upregulation of phospholipid synthesis as a target for anti-viral interventions. The increased phospholipid synthesis consumes endogenously- and exogenously-derived long-chain fatty acids (LCFA). We demonstrate that polyunsaturated LCFAs can have a broad anti-enteroviral effect, affecting multiple steps of the virus life cycle. The anti-viral activity of LCFAs did not strictly depend on the degree of unsaturation or their capacity to induce lipid peroxidation but significantly correlated with their conformation. This suggests that their incorporation into the phospholipid molecules makes the replication organelle membranes incapable of properly accommodating viral replication machinery. Accordingly, the inhibition of neutral lipid synthesis promoted LCFAs retargeting to the membranes in infected cells and increased their anti-viral potency. We show that this approach is effective against diverse enteroviruses in different cell types, including differentiated primary cells, and that attempts to establish viruses resistant to such treatment were unsuccessful.

Circadian rhythm disruption is associated with metabolic and inflammatory disorders; however, the mechanisms linking circadian dysfunction to endocannabinoidome (eCBome) signaling and mitochondrial metabolism remain unclear. In our previous in vivo study, constant light exposure altered hepatic eCBome profiles, reduced N-acylethanolamines (NAEs), increased monoacylglycerols (MAGs), and elevated inflammatory cytokines. Here, we investigated the underlying mechanisms using CRISPR/Cas9-generated BMAL1 knockout (KO) HepG2 cells as an in vitro model of circadian alteration. The BMAL1 KO model showed broad lipid remodeling characterized by increased fatty acids, prostaglandins, and MAGs together with reduced NAEs and enhanced lipid accumulation. These changes were accompanied by increased inflammatory signaling and cytokine production. Among the assessed genes, GPR110 was significantly altered in mice exposed to constant light (in vivo study) and BMAL1 KO model and emerged as a potential mediator linking circadian signaling to mitochondrial function. BMAL1 KO cells also exhibited significantly increased calcium (Ca{superscript 2}+) levels in mitochondria and the endoplasmic reticulum (ER), along with attenuation of mitochondrial and glycolytic ATP production. BMAL1KO did not abolish the rhythmicity of NAEs level over 24 hours from medium deprivation and read ministration except for N-docosahexaenoyl-ethanolamide (DHEA). Further, experiments showed that DHEA acts through GPR110 and suppress inflammatory lipid-associated pathways, enhances ATP production, and increases mitochondrial and ER Ca{superscript 2}+ accumulation and inflammatory signaling. Together, these mitochondrial Ca{superscript 2}+ signaling, and inflammation in hepatocytes, highlighting DHEA-GPR110 signaling as a potential regulator of hepatic metabolic homeostasis. HighlightsCircadian disruption increases hepatic monoacylglycerols and decreases N-acylethanolamines. Circadian disruption decreases ATP production and enhances mitochondrial and endoplasmic reticulum Ca{superscript 2}+ levels in hepatocytes DHEA-GPR110 signaling regulates hepatocytes mitochondrial Ca{superscript 2}+ dynamics and ATP production GPR110-mediated Ca{superscript 2}+ signaling significantly alters hepatocytes glycolysis and glycolytic ATP production

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.

Introduction: Lung cancer in never-smokers is a growing, biologically distinct entity lacking non-invasive markers. Established urinary markers - creatine riboside (CR) and N-acetylneuraminic acid (NANA) - report tumor-intrinsic metabolism, not carcinogen processing. We investigated 27-nor-5{beta}-cholestane-3,7,12,24R,25S-pentol glucuronide (CPG), a bile-acid glucuronide linked to aryl-hydrocarbon-receptor (AhR)/CYP xenobiotic metabolism. Methods: Urinary CPG was quantified by UPLC-tandem mass spectrometry in an exploratory (NCI-Maryland; n=846) and validation (Colorado; n=505) cohort of non-small-cell lung cancer cases and frequency-matched controls. Associations with case status, smoking stratum, survival, and discrimination were assessed, using tumor RNA sequencing (n=83) and gene-set enrichment analysis (GSEA). Results: Urinary CPG was higher in cases than controls in both cohorts (P<0.0001). In never-smokers, cases exceeded smoking-matched controls (P<0.001 and P<0.0001), indicating elevation independent of tobacco exposure. After mutual adjustment for CR and NANA, CPG remained independently associated with case status (exploratory OR 1.58, 95% CI 1.15-2.16; validation OR 3.92, 95% CI 2.47-6.29), with a modest gain in discrimination. High CPG identified never-smokers with worse survival in both cohorts (P<0.001 and P=0.04), remaining significant after multivariable adjustment only in the exploratory cohort. GSEA showed AhR/CYP xenobiotic and Nrf2 oxidative-stress enrichment in high-CPG tumors; the CPG aglycone carried disease-specific 24R,25S stereochemistry. Conclusions: Urinary CPG was associated with NSCLC in two retrospective case-control cohorts, including in a smoking-matched never-smoker comparison. High CPG also identified never-smokers with worse survival, remaining independently prognostic after adjustment in the exploratory cohort. Tumor expression does not establish tissue of origin. Prospective validation against CR and NANA is required.

Heterosexual genital HIV transmission is a major driver of new infections, particularly in women, making them disproportionately vulnerable to HIV acquisition. Previous studies have associated injectable hormonal contraceptives (HC) with increasing susceptibility to HIV. Yet, the underlying molecular mechanism remains incompletely understood. Given the structural and signaling role of lipids in the female genital tract, cervicovaginal lipidomic profiling has the potential to reveal the mechanistic interplay among HC, lipidome, and HIV susceptibility in the female genital tract. We conducted untargeted cervicovaginal lipidomics study in a cohort of high-risk, HIV-negative, Kenyan sex workers who were using injectable depot medroxyprogesterone acetate (DMPA), oral contraceptive pill (OCP), or no hormonal contraception (NH). Genital lipids were quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS) and bioinformatics platforms. A total of 1045 lipid species were identified in the cervicovaginal lavage samples. Injectable DMPA significantly downregulated major structural and signaling membrane lipids, including phospholipids, ceramides, sphingomyelins, and glycosphingolipids (p<0.001, FDR<0.05), and markedly upregulated storage lipids (triglycerides) and sterols when compared with OCP or NH groups. Interestingly, microbial communities contributed significantly to the DMPA-driven genital lipidomic shifting, suggesting the intricate interplay among sex hormones, microbiome, and lipidome. In contrast, OCP, which contains estrogen, consistently lowered genital triglycerides and increased the fatty acid and diacylglycerol substrates compared with the NH group, implying that estrogen affects genital lipid metabolism by contrasting the lipogenesis effect of DMPA. Differential lipidomic pathway analysis further confirmed that DMPA suppressed the de novo phospholipid and sphingolipid biosynthesis, but enhanced salvaging of various lipids from reaction intermediates and from cell membrane lipid pool, shifting them towards TG accumulation. Our findings of dysregulated depletion of several membrane and signaling phospholipids, ceramides, sphingomyelins, glycosphingolipids, along with upregulation of triglycerides in the DMPA users provides insights into the underlying molecular mechanism by which DMPA may increase in susceptibility to HIV infection by weakening the epithelial barrier and increasing chronic inflammation. These findings underscore the need to address cervicovaginal lipidomic dysregulation associated with HCs while designing interventions to improve the reproductive health of women. Key Words: Lipidomics, hormonal contraceptives, LC-MS, phospholipids, sphingolipids, HIV susceptibility, microbial lipids, pathway analysis

1.Cholesterol in the central nervous system (CNS) is largely unesterified (>99%) and is predominantly present in the myelin sheath ([~]70% of total CNS cholesterol). Damage to the myelin sheath can result in the conversion of cholesterol to cholesterol esters, which occurs in many neurological diseases, including multiple sclerosis. In this study, we measured longitudinal CNS free cholesterol and cholesterol ester levels in a genetic mouse model during postnatal myelination, demyelination, and remyelination using gas chromatography-mass spectrometry with single ion monitoring technique (GC-MS-SIM) and liquid chromatography mass spectrometry (LC-MS). Cholesterol levels in healthy mouse brains increased up to 38 weeks. In contrast, cholesterol in the healthy spinal cord increased during postnatal timepoints, but then remained steady out to 38 weeks. Interestingly, cholesterol esters in the spinal cord were highest at P1 and drastically reduced by P42, while the brain had similar levels during all postnatal time points. During demyelination, both brain and spinal cord cholesterol levels were significantly reduced as compared to healthy mice and failed to return to normal cholesterol levels even during remyelination. Absolute quantification of cholesterol esters during peak demyelination revealed that cholesterol esters comprise 19% of the total cholesterol pool in the brain and 65% in the spinal cord. The lack of recovery in CNS cholesterol levels after demyelination suggests that healthy de novo cholesterol synthesis pathways are disrupted in this model. Absolute quantification of CNS cholesterol is critical for revealing mechanisms of cholesterol regulation during disease and identifying targets for restoring cholesterol to promote myelin repair.

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.

Excessive alcohol intake is frequently associated with hypertriglyceridemia, a condition that increases the risk of severe complications including acute pancreatitis and cardiovascular disease. The very low-density lipoprotein (VLDL) receptor (VLDLR) promotes uptake of apoE-containing VLDL particles by peripheral tissues and plays an important role in maintaining plasma triglyceride (TG) homeostasis. Brown adipose tissue (BAT) is a major metabolic organ that contributes to circulating lipid clearance during thermogenic activation. It was reported that cold-induced thermogenesis upregulates VLDLR expression in BAT and reduces plasma TG via VLDL uptake. However, whether BAT VLDLR-mediated VLDL uptake regulates alcohol-induced hypertriglyceridemia remains unknown. Here, we generated BAT-specific fatty acid synthase (FASN) knockout mice (FASNBKO) and subjected them to binge and acute-on-chronic alcohol feeding paradigms. We found that BAT FASN deficiency enhanced thermogenic function and promoted VLDL uptake, resulting in attenuation of alcohol-induced elevations in plasma TG. Consistent with these findings, pharmacological inhibition of FASN by TVB3664 treatment in differentiated brown adipocytes (bADs) increased thermogenic gene expression and VLDL uptake under both control and alcohol-exposed conditions. In addition, FASNBKO mice were protected from alcohol-induced hepatic steatosis, which was accompanied by increased hepatic AMP-activated-protein kinase (AMPK) activation and enhanced {beta}-oxidation. Furthermore, FASNBKO mice exhibited upregulated FGF21 mRNA expression in the BAT and elevated circulating FGF21 levels. Similarly, TVB3664-treated differentiated bADs showed higher FGF21 expression and increased FGF21 content in culture medium. Taken together, these findings identify the important role of brown adipocyte FASN in regulating thermogenic function and TG homeostasis during alcohol exposure and suggest that enhancing thermogenic lipid utilization in BAT may represent a potential therapeutic strategy for mitigating alcohol-associated increases in plasma TG and hepatic fat accumulation.

Ferroptosis is driven by the accumulation of oxidatively damaged membrane phospholipids, making membrane lipid composition a central determinant of cell death sensitivity. While fatty acid chain length and degree of unsaturation are well-established regulators of ferroptosis, whether fatty acid stereochemistry contributes to ferroptosis susceptibility is mostly unexplored. Here, we systematically screened structurally diverse fatty acids for their ability to modulate ferroptosis and unexpectedly identified trans-unsaturated fatty acids as potent sensitizers. Compared with its cis counterpart linoleic acid, the trans polyunsaturated fatty acid (PUFA) linoelaidic acid more strongly enhanced lipid peroxidation and promoted the accumulation of ferroptosis-susceptible phospholipid species. Unexpectedly, the trans monounsaturated fatty acid petroselaidic acid also sensitized cells to ferroptosis, whereas its cis stereoisomer petroselinic acid suppressed ferroptosis. Mechanistically, petroselaidic acid required stearoyl-CoA desaturase-dependent conversion to a PUFA, directly demonstrating that double-bond geometry can redirect fatty acid metabolic fate through altered recognition by lipid metabolic enzymes. Although linoelaidic acid and petroselaidic acid followed distinct metabolic pathways, both converged on phospholipid remodeling that expanded pools of ferroptosis-susceptible membrane lipids. Together, our findings demonstrate that fatty acid double-bond geometry determines their metabolic fate and the membrane phospholipid composition, establishing lipid stereochemistry as a previously unrecognized structural determinant of ferroptosis sensitivity.

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.

The molecular mechanisms of lysosomal glycerophospholipid (GPL) catabolism are incompletely understood. Here, we report that acid phospholipase A1 (APLA1), formerly known as palmitoyl-protein thioesterase 2 (PPT2), is required for efficient GPL degradation. Deletion of APLA1 in human cells results in excess accumulation of phospholipids within lysosomes # a pathological condition termed phospholipidosis. APLA1 activity depends on interactions with negatively charged GPLs and is inhibited by phospholipidosis-inducing cationic amphiphilic drugs. Hydrolysis of zwitterionic, but not anionic, GPLs requires co-activation of APLA1 by the lysosome-specific lipid bis(monoacylglycero)phosphate. Upon pharmacological mTORC inhibition, which increases lysosomal GPL turnover, APLA1-deficient cells exhibit massive accumulation of multilamellar membranes in lysosomes and reduced cytosolic triacylglycerol stores. APLA1 acts in concert with lysosomal phospholipase A2 (PLA2G15). Combined APLA1/PLA2G15-deficiency leads to a severe reduction in acid phospholipase A1/A2 activity, thereby exacerbating phospholipidosis. Our observations provide detailed mechanistic insights into lysosomal GPL catabolism, a crucial pathway for maintaining lipid homeostasis.

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.

Altered cholesterol metabolism is a recognized hallmark of cancer, but systemic modulation has not yet delivered significant clinical results. Accumulating evidence shows that cholesterol plays distinct roles across diverse cellular membranes, suggesting that site-specific modulation may produce superior therapeutic outcomes. Cholesterol is associated with gastric cancer (GC), but the mechanistic link is complex and no effective cholesterol-targeted therapy has been developed. Here, we report that cholesterol levels in GC cells are site-specifically elevated in the inner leaflet of the plasma membrane (IPM). This elevated IPM cholesterol constitutively activates Wnt-{beta}-catenin signaling to drive cell survival and proliferation. Mechanistically, Niemann-Pick C1-like 1 (NPC1L1), which is highly expressed in GC patient tissues and cell lines, acts as a cholesterol flippase to raise IPM cholesterol levels, facilitating ligand-independent {beta}-catenin signalosome formation. Ezetimibe, a clinically approved NPC1L1 inhibitor, blocks this flippase activity, lowers IPM cholesterol levels, and suppresses {beta}-catenin signaling. Ezetimibe treatment induces apoptosis in GC cells while sparing normal primary gastric epithelial cells, which exhibit low levels of NPC1L1 and IPM cholesterol. Collectively, these results suggest that site-specific modulation of cellular cholesterol is a viable approach to developing safe and effective therapies for cancers linked to local cholesterol elevation.

Microglial inflammatory activation is accompanied by extensive molecular remodeling, yet proteomic, lipidomic, and metabolomic responses are often analyzed independently. Here, we applied an integrated mass spectrometry-based multiomic workflow to characterize proteomic, lipidomic, and polar metabolomic remodeling from matched BV-2 biological samples following stimulation with interferon-{gamma} and lipopolysaccharide (IFN-{gamma} and LPS). Inflammatory activation was confirmed by increased nitrite accumulation, elevated TNF- and IL-6 secretion, and treatment-associated morphological changes. Discovery proteomics quantified 8,676 proteins and identified 562 significantly altered proteins, including 344 increased and 218 decreased proteins. Increased proteins were enriched for interferon-responsive, innate immune, inflammatory effector, and antigen-associated pathways, whereas decreased proteins were associated with cellular organization, protein biogenesis, vesicular trafficking, and metabolic regulation. Targeted lipidomics identified 237 significantly altered lipid features out of 356 measured lipids, including increased triacylglycerols and diacylglycerols and broad remodeling of glycerophospholipids, lysophospholipids, and sphingolipid-related species. Targeted polar metabolomics identified 75 significantly altered metabolites out of 98 measured metabolites, including changes in nucleotide/NAD-related metabolism, amino acid metabolism, methylation-associated metabolites, acylcarnitine abundance, phospholipid precursors, polyamine metabolism, arginine/nitric oxide-associated metabolism, and redox-associated metabolites. Process-level integration of significant features revealed coordinated remodeling of inflammatory protein programs with lipid storage, membrane remodeling, nucleotide metabolism, amino acid availability, phospholipid precursor abundance, nitric oxide-associated metabolism, and redox/osmolyte pathways. These findings demonstrate that IFN-{gamma} and LPS-induced activation of BV-2 cells involves integrated immune, lipid, and metabolic adaptation rather than isolated induction of canonical inflammatory mediators. This integrated multiomic framework provides a resource for investigating how lipid and metabolic remodeling regulate microglial inflammatory states. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/741024v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@11dd431org.highwire.dtl.DTLVardef@155f107org.highwire.dtl.DTLVardef@1430e89org.highwire.dtl.DTLVardef@16f4a25_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alpha-1 antitrypsin deficiency (AATD) caused by the Z variant leads to hepatic accumulation of misfolded AAT polymers and liver disease. Although proteotoxic stress is well established, its impact on lipid metabolism, mitochondrial function, and organelle homeostasis remains incompletely understood. The effects of Z-AAT accumulation were investigated in Z-HepG2 cells and 3D patient-derived ZZ hepatic organoids through protein aggregation, lipid storage, mitochondrial structure and function, peroxisomal dynamics, and comprehensive transcriptomic and proteomic analyses. Z-AAT expression led to intracellular polymer accumulation and reduced secretion, together with lipid accumulation, mitochondrial structural abnormalities, increased mitochondrial number but impaired respiratory capacity. Metabolic profiling revealed reduced oxidative phosphorylation and partial reliance on glucose metabolism. Peroxisomes displayed increased mass, consistent with altered lipid handling. Multi-omics analysis demonstrated widespread transcriptional and proteomic reprogramming related to protein synthesis, lipid metabolism, and mitochondrial function. Proteomic analysis confirmed proteotoxic stress-induced mitochondrial dysfunction, impaired lipid handling, and activation of stress response, inflammatory and vesicular trafficking pathways. Importantly, lipid supplementation elicited adaptive mitochondrial transcriptional responses in control cells, whereas Z-HepG2 cells showed a blunted response to lipid challenge. In conclusion, Z-AAT accumulation disrupts hepatic lipid processing and impaired mitochondrial and peroxisomal homeostasis, producing diminished metabolic flexibility likely contributing to AATD-associated liver disease.