Lipotoxicity refers to the harmful effects of excess fatty acids on metabolic health, and it can vary depending on the type of fatty acids involved. Saturated and unsaturated fatty acids exhibit distinct effects, though the precise mechanisms behind these differences remain unclear. Here, we investigated the lipotoxicity of palmitic acid (PA), a saturated fatty acid, compared with oleic acid (OA), a monounsaturated fatty acid, in the hepatic cell line HuH7. Our results demonstrated that PA, unlike OA, induces lipotoxicity, endoplasmic reticulum (ER) stress, and autophagy inhibition. Compared with OA, PA treatment leads to less lipid droplet (LD) accumulation and a significant reduction in the mRNA and protein level of diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of triacylglycerol synthesis. Using modulators of ER stress and autophagy, we established that DGAT1 downregulation by PA is closely linked to these cellular pathways. Notably, the ER stress inhibitor 4-phenylbutyrate can suppress PA-induced DGAT1 downregulation. Furthermore, knockdown of DGAT1 by siRNA or with A922500, a specific DGAT1 inhibitor, resulted in cell death, even with OA. Both PA and OA increased the oxygen consumption rate; however, the increase associated with PA was only partially coupled to ATP synthesis. Importantly, treatment with GW7647 a specific PPARα agonist mitigated the lipotoxic effects of PA, restoring PA-induced ER stress, autophagy block, and DGAT1 suppression. In conclusion, our study highlights the crucial role of DGAT1 in PA-induced lipotoxicity, broadening the knowledge of the mechanisms underlying hepatic lipotoxicity and providing the basis for potential therapeutic interventions.

Phages are ubiquitous in bacteria, including clinical Staphylococcus aureus, where Sfi 21/Sa3 phages often integrate into the hlb gene, which encodes Hlb sphingomyelinase. This integration acts as a rapid regulatory switch for Hlb production. Our findings suggest that Sfi 21/Sa3 prophages and Hlb activity influence S. aureus fitness by modulating the incorporation of the toxic linoleic acid (C18:2) from serum into the bacterial membrane. This process relies on C18:2 derived from 1,3-diglyceride, facilitated by the FakB1 kinase subunit. Palmitic acid (C16), primarily released from serum through Hlb activity, competes with C18:2 for FakB1. This mechanism contributes to adaptation to AFN-1252, an antibiotic inhibiting the fatty acid synthesis pathway (anti-FASII). Since S. aureus relies on exogenous fatty acids for growth, AFN-1252 treatment leads to increased proportion of C18:2 in the membrane. Furthermore, Hlb inhibition, whether by prophage insertion, gene inactivation, or enzyme inhibition, delays S. aureus adaptation, resulting in a higher proportion of C18:2 in the membrane. This study sheds light on the role of lipid environments in infections and may contribute to the accurate prediction of infection risks and therapeutic efficacy. Moreover, since both anti-FASII agent and Hlb inhibitor enhance C18:2 incorporation, they represent potential candidates for combined strategies against S. aureus.

Mice lacking monoacylglycerol acyltransferase 2 (mMGAT2) are resistant to diet-induced fatty liver, suggesting hMOGAT2 inhibition is a viable option for treating metabolic dysfunction-associated steatotic liver disease (MASLD)/metabolic dysfunction-associated steatohepatitis (MASH). We generated humanized hMOGAT2 mice (HuMgat2) for use in pre-clinical studies testing the efficacy of hMOGAT2 inhibitors for treating MASLD/MASH. HuMgat2 mice developed MASH when fed a steatotic diet. Computer-aided histology revealed the presence of hepatocyte cell ballooning, immune cell infiltration, and fibrosis. Hepatocytes accumulated Mallory-Denk bodies containing phosphorylated p62/sequestosome-1-ubiquintinated protein aggregates likely caused by defects in autophagy. Metainflammation and apoptotic cell death were seen in the livers of HuMgat2 mice. Treating HuMgat2 mice with elafibranor reduced several MASH phenotypes. RNASeq analysis predicted changes in bile acid transporter expression that correlated with altered bile acid metabolism indicative of cholestasis. Our results suggest that HuMgat2 mice will serve as a pre-clinical model for testing hMOGAT2 inhibitor efficacy and toxicity and allow for the study of hMOGAT2 in the context of MASH.

Metabolic dysfunction-associated steatohepatitis (MASH) is a severe form of metabolic dysfunction-associated fatty liver disease (MAFLD), characterized by hepatic steatosis, inflammation, and fibrosis. This study investigates the role and potential mechanisms of metallothionein 1B (MT1B) in MASH through bioinformatics analysis and experimental validation. qRT-PCR and western blot analyses confirm that MT1B expression is significantly downregulated in liver tissues of MASH patients, in high-fat diet (HFD)-induced mouse models, and in hepatocytes induced by free fatty acids (FFA). Further functional experiments show that upregulation of MT1B reduces intracellular triglycerides and total cholesterol levels, lipid droplet formation, and pro-inflammatory factors. In vivo experiments demonstrate that specific downregulation of hepatic MT1B expression via AAV8-shMT1B injection significantly increases triglyceride and total cholesterol levels, exacerbates lipid accumulation, and markedly elevates liver fibrosis and inflammatory factor expression. RNA-seq and bioinformatics analyses show that the AKT/PI3K pathway is significantly suppressed in MT1B-overexpressing cells. Further experiments indicate that AKT inhibition can reverse the lipid metabolism disorders and inflammatory responses caused by MT1B downregulation. Additionally, Zinc can promote the nuclear translocation of MTF1, leading to its binding to the MT1B promoter, thereby upregulating MT1B expression and ultimately mitigating MASH progression. These findings suggest that zinc-regulated MT1B plays a critical role in lipid metabolism and inflammatory responses by regulating the AKT/PI3K signaling pathway, influencing MASH progression.

Triglyceride (TG) /High density lipoprotein cholesterol (HDL-C) ratio (THR) is a surrogate predictor of hyperinsulinemia. To identify novel genetic loci for THR change over time (ΔTHR), we conducted genome-wide association study (GWAS) and genome-wide linkage scan (GWLS) among non-diabetic Europeans from the Long Life Family Study (LLFS, n=1384).

Statins are the most effective drugs used worldwide to lower the serum LDL-c by inhibiting the rate-limiting step, HMG-CoA reductase, in cholesterol biosynthesis. Despite its prevalent use, statins are known to increase PCSK9 expression, hindering its efficiency. However, the underlying mechanisms remain elusive. In this study, we have unraveled the pleiotropic effects of statins on hypercholesterolemia via epigenetic regulation of PCSK9. We observed that atorvastatin increases the fold enrichment of H3K4me3 at the promoter of PCSK9 by elevating the expression of the SET1/COMPASS family of proteins like SET1b and MLL1 in HepG2. Additionally, atorvastatin also acetylates H3K9 by increasing the expression of acetyltransferases like CBP and PCAF. Similarly, in mice fed a high-fat diet, atorvastatin showed increased levels of H3K4me3 and H3K9ac in the liver. Furthermore, a pharmacological intervention that inhibits the H3K4me3 and H3K9ac enrichment resulted in the reversal of statin-induced upregulation of PCSK9. Combining statin and OICR-9429 or resveratrol improved the overall uptake of LDL by hepatocytes. Together, these findings suggest that statin induces the colocalization of H3K4me3 and H3K9ac to transcribe PCSK9 actively and that inhibiting these marks reduces PCSK9 expression and ultimately increases hepatocyte LDL uptake. Our study unveils a previously unknown epigenetic mechanism of PCSK9 regulation that may lead to statin resistance or futility in patients and provide a potential therapeutic solution.

Oxysterols and bile acids are interconnected bioactive lipids playing pivotal roles in diverse physiological and pathological processes. For this reason, they are increasingly studied together for their implications in various diseases. However, due to analytical challenges inherent to the nature of these analytes, very few methods have been developed for the simultaneous analysis of these lipids. We here report the development of a sensitive LC-MS/MS method for the combined quantification of 18 oxysterols, 11 unconjugated, 15 conjugated bile acids, and 1 bile acid precursor, using 8 isotope-labeled internal standards, addressing the need for a more comprehensive analysis of these interesting lipid families. During the method development, we investigated different extraction protocols, set up a purification step and achieved chromatographic separation for these lipids, overcoming challenges such as the large number of analytes, isomers, and wide range of polarity across the analytes. Finally, the method was successfully applied to the analysis of preclinical and clinical samples, quantifying 12 oxysterols and 14 bile acids in human plasma, 10 oxysterols and 18 bile acids in mouse plasma from the vena cava, and 10 oxysterols and 20 bile acids in mouse plasma from the portal vein within a single chromatographic run.

Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoring of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements is limited in many parts of the world.

Both age and diet can contribute to alterations in triglyceride metabolism and subsequent metabolic disease. In humans, plasma triglyceride levels increase with age. Diets high in saturated fats can increase triglyceride levels while diets high in omega-3 fatty acids decrease triglyceride levels. Here we asked how age and long-term diet altered triglyceride metabolism in mice. We fed male and female C57Bl/6 mice a low-fat diet, a western diet (WD), or a diet high in polyunsaturated and omega-3 fatty acids (n3D) for up to 2 years. We measured survival, body composition, plasma triglyceride levels, chylomicron clearance, and oral fat, glucose, and insulin tolerance. Triglyceride levels in mice did not increase with age, regardless of diet. Oral fat tolerance increased with age, while chylomicron clearance remained unchanged. Decreased survival was observed in WD-fed mice. Interestingly, n3D-fed mice diet gained more lean mass and had lower insulin levels than WD-fed or LFD-fed mice. Moreover, triglyceride uptake into the hearts of n3D-fed mice was strikingly higher than in other groups. Our data indicate that in C57Bl/6 mice, age-induced changes in triglyceride metabolism differ from those observed in humans. Mice, like humans, appeared to have decreased fat absorption with age, but in mice plasma triglyceride clearance did not decrease with age, resulting in lower plasma triglyceride levels and improved fat tolerance with age. Although a chronic diet high in omega-3 fatty acids increased insulin sensitivity and triglyceride uptake specifically into the heart, how these observations are connected is unclear.

High-density lipoprotein (HDL)-free cholesterol (FC) transfers to other lipoproteins and cells, the former by a spontaneous mechanism and the latter by both spontaneous and receptor-mediated mechanisms. Macrophages are an important cell type in all stages of atherosclerotic cardiovascular disease (ASCVD), and the magnitude of FC efflux from macrophages to HDL, a metric of HDL function, inversely associates with several metrics of ASCVD. Very high plasma HDL concentrations are associated with increased all cause and ASCVD mortality, suggesting that the reverse process, FC influx from HDL into macrophages, is atherogenic. We hypothesize that HDL-FC is a metric of dysfunctional HDL, and when combined with HDL particle number (HDL-P), is an ASCVD risk factor. The magnitude of FC influx from HDL to macrophages is expected to be a function of HDL-P and HDL-FC content. Here we show that plasma HDL-FC content varies 2-fold among normolipidemic human subjects and linearly correlates with low-density lipoprotein (LDL)-FC content. The influx of HDL-FC into macrophages and transfer to LDL increase linearly with HDL-FC. As expected, influx of HDL-FC into macrophages and transfer to LDL are positively correlated. These data support the hypothesis that high HDL FC content is a marker for dysfunctional HDL, resulting in greater influx into macrophages and greater HDL-FC transfer to LDL. HDL-FC transfer to LDL is a valid surrogate for influx into macrophages. This study of HDL composition and function of normolipidemic subjects provides the basis for further investigation and establishment of HDL-FC content as an ASCVD risk factor.

Lipid droplets (LDs) are transient lipid storage organelles that can be readily tapped to resupply cells with energy or lipid building blocks, and therefore play a central role in cellular metabolism. Double FYVE Domain Containing Protein 1 (DFCP1/ZFYV1) has emerged as a key regulator of LD metabolism, where the nucleotide-dependent accumulation of DFCP1 on LDs influences their size, number, and dynamics. Here we show that DFCP1 regulates lipid metabolism by directly modulating the activity of Adipose Triglyceride Lipase (ATGL/PNPLA2), the rate-limiting lipase driving the catabolism of LDs. We show through pharmacological inhibition of key enzymes associated with LD metabolism that DFCP1 specifically regulates lipolysis and, to a lesser extent, lipophagy. Consistent with this observation, DFCP1 interacts with and recruits ATGL to LDs in starved cells, irrespective of other known regulatory factors of ATGL. We further establish that this interaction prevents dynamic disassociation of ATGL from LDs and thereby impedes the rate of LD lipolysis. Collectively, our findings indicate that DFCP1 is a nutrient-sensitive regulator of LD catabolism.

The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily lipoxin A (LXA), there are expanding concerns about the reported biological formation, detection and signaling mechanisms ascribed to LXA and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. The generation and signaling actions of LXA and its primary 15-oxo metabolite were assessed in control, lipopolysaccharide-activated and arachidonic acid supplemented RAW264.7 and bone marrow-derived macrophages. Despite the expression of catalytically active enzymes required for LXA synthesis, both LXA and its 15-oxo-LXA metabolite were undetectable in all conditions. Moreover, synthetic LXA and the membrane permeable 15-oxo-LXA methyl ester, which rapidly de-esterified to 15-oxo-LXA, displayed no ligand activity for the putative LXA receptor FPR2. Alternatively, 15-oxo-LXA, an electrophilic α,β-unsaturated ketone, alkylates nucleophilic amino acids and can modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA activated nuclear factor (erythroid related factor 2)-like 2-regulated expression of anti-inflammatory and repair genes and inhibited NF-κB-regulated pro-inflammatory mediator expression. Synthetic LXA showed no impact on these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA formation and receptor-mediated signaling actions of synthetic LXA. Rather, if present in sufficient concentrations, LXA and other mono- and poly-hydroxylated unsaturated fatty acids synthesized by macrophages would be readily oxidized to electrophilic α,β-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.

Several oxylipins are regulators of inflammation. They are formed by enzymes such as lipoxygenases or cyclooxygenases, but also stereorandomly by autoxidation. Reversed-phase liquid chromatography-tandem-mass-spectrometry (LC-MS/MS) methods for oxylipin quantification do not separate enantiomers. Here, we combine sensitive and selective oxylipin analysis with chiral separation using two-dimensional (2D)-LC-MS/MS. By multiple heart-cutting, the oxylipin peaks are transferred onto a chiral column. 45 enantiomeric pairs of (di-)hydroxy-fatty acids are separated with full gradient elution within 1.80min, yielding lower limits of quantification <1pg on column. Concentrations as well as enantiomeric fractions of oxylipins can be determined, even at low concentrations or at high enantiomeric excess of one isomer. The developed achiral-chiral multiple heart-cutting 2D-LC-MS/MS method offers unprecedented selectivity, enabling a better understanding of the formation route of these lipid mediators. This is demonstrated by distinguishing the formation of hydroxy-fatty acids by (acetylated) cyclooxygenase-2 and radical-mediated autoxidation. Applying the method to human M2-like-macrophages, we show that the so-called specialized pro-resolving mediators (SPM) 5,15-DiHEPE and 7,17-DiHDHA as well as 5,15-DiHETE were present as (S,S)-enantiomers, supporting their enzymatic formation. In contrast, at least eight isomers (including protectin DX but not neutroprotectin D1) of 10,17-DiHDHA are present in immune cells, indicating formation by autoxidation. In human plasma of healthy subjects, none of these dihydroxy-fatty acids are not present. However, we demonstrate that all four isomers quickly form via autoxidation if the samples are stored improperly. Thus, dihydroxy-FA should only be reported as SPM, such as resolvin D5 or resolvin E4, if an enantioselective analysis has been carried out.

Patients with familial hypercholesterolemia (FH) exhibit a significant residual cardiovascular risk. A new cardiovascular risk factor is the susceptibility of individual LDL particles to aggregation. This study examined LDL aggregation and its relationship with LDL lipid composition and biophysical properties in patients with FH compared to controls. LDL aggregation was measured as the change in particle size, assessed by dynamic light scattering (DLS), after exposure to sphingomyelinase (SMase), which breaks down sphingomyelin in the LDL phospholipid layer. DLS and transmission electron microscopy (TEM) showed that LDL in FH patients exhibited smaller size and greater susceptibility to aggregation. Biochemical analyses revealed a higher cholesteryl ester (CE)/ApoB100 ratio in LDL from FH patients. Differential Scanning Calorimetry (DSC) showed that LDL from FH patients had higher transition temperatures, indicating a more ordered CE core. Fourier-transform infrared (FTIR) spectroscopy revealed fewer flexible α-helices (1658 cm⁻) and more stable α-helices (1651 cm⁻) in ApoB100 of LDL from FH patients. These structural changes correlated with higher CE content and increased LDL aggregation. In conclusion, a more ordered CE core in smaller LDL particles, combined with a higher proportion of stable α-helices in ApoB100, promotes LDL aggregation in FH patients. These findings suggest new potential therapeutic targets within LDL to reduce cardiovascular risk in FH patients.

Oxidized phospholipids (OxPLs) are increasingly recognized as toxic and proinflammatory mediators, which raises interest to the mechanisms of their detoxification. Circulating OxPLs are bound and neutralized by plasma proteins, including both antibodies and non-immunoglobulin proteins. The latter group of proteins is essentially not investigated because only three OxPC-binding plasma proteins are currently known. The goal of this work was to characterize a broad spectrum of plasma proteins selectively binding OxPLs. Using pull-down-proteomic analysis, we found about 150 non-immunoglobulin proteins preferentially binding oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphatidylcholine (OxPAPC) as compared to non-oxidized PAPC. To test if candidate proteins indeed can form a barrier isolating OxPLs from recognition by other proteins, we applied an immune masking assay. Oxidized LDL (OxLDL) immobilized in multiwell plates was used as a carrier of OxPLs, while mAbs recognizing OxPC or OxPE were used as "detectors" showing if OxPLs on the surface of OxLDL are physically accessible to external binding partners. Using an orthogonal combination of pull-down and masking assays we confirmed that previously described OxPL-binding proteins (non-fractionated IgM, CFH, and Apo-M) indeed can bind to and mask OxPC and OxPE on liposomes and OxLDL. Furthermore, we identified additional plasma proteins selectively binding and masking OxPC including Apo-D, Apo-H, pulmonary surfactant-associated protein B, and antithrombin-III. We hypothesize that in addition to circulating antibodies, also multiple non-immunoglobulin plasma proteins can bind OxPLs and modulate their recognition by innate and adaptive immunity and that an orthogonal combination of pull-down and masking assays is a useful approach for the identification of such proteins.

De novo lipogenesis (DNL) has been implicated in the development and progression of liver steatosis. Hepatic DNL is strongly influenced by dietary macronutrient composition with diets high in carbohydrate increasing DNL and while diets high in fat decrease DNL. The enzymes in the core DNL pathway have been well characterised, however less is known about other liver proteins that play accessory or regulatory roles. In the current study, we associate measured rates of hepatic DNL and fat content with liver proteomic analysis in mice to identify known and unknown proteins that may have a role in DNL. Male mice were fed either a standard chow diet, a semi-purified high starch or high fat diet. Both semi-purified diets resulted in increased body weight, fat mass and liver triglyceride content compared to chow controls and hepatic DNL was increased in the high starch and decreased in high fat fed mice. Proteomic analysis identified novel proteins associated with DNL that are involved in taurine metabolism, suggesting a link between these pathways. There was no relationship between proteins that associated with DNL and those associated with liver triglyceride content. Further analysis identified proteins that are differentially regulated when comparing a non-purified chow diet to either of the semi-purified diets which provide a set of proteins that are influenced by dietary complexity. Finally, we compared the liver proteome between 4- and 30-week diet-fed mice and found remarkable similarity suggesting metabolic remodelling of the liver occurs rapidly in response to differing dietary components.

Sphingolipids (SPLs) are major components of cell membranes with significant functions. Their production is a highly-regulated multi-step process with the formation of two major intermediates, long chain bases (LCBs) and ceramides. Homologous Orm proteins in both yeast and mammals negatively regulate LCB production by inhibiting serine palmitoyltransferase (SPT), the first enzyme in SPL de novo synthesis. Orm proteins are therefore regarded as major regulator of SPL production. Combining targeted lipidomic profiling with phenotypic analysis of yeast mutants with both ORM1 and ORM2 deleted (orm1/2Δ), we report here that Ypk1, an AGC family protein kinase, signaling is compromised in an LCB-dependent manner. In orm1/2Δ, phosphorylation of Ypk1 at its activation sites is reduced, so does its in vivo activity shown by reduced phosphorylation of Ypk1 substrate, Lac1, the catalytic component of ceramide synthase (CerS). A corresponding defect in ceramide synthesis was detected, preventing the extra LCBs generated in orm1/2Δ from fully converting into downstream SPL products. The results suggest that Orm proteins play a complex role in regulating SPL production in yeast S. cerevisiae by exerting an extra and opposite effect on CerS. Functionally, we define an endocytosis and an actin polarization defect of orm1/2Δ and demonstrate the roles of Ypk1 in mediating the effects of Orm proteins on endocytosis. Collectively, the results reveal a previously unrecognized complexity of SPL de novo synthesis pathway and point to a potential role of Orm proteins as upstream regulators to control Ypk1-mediated biological functions via regulating LCB production.

The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux is a more robust predictor of cardiovascular disease protection than HDL-cholesterol levels in plasma. Previously, we found that lipidated HDL containing both apolipoprotein A-I (APOA1) and A-II (APOA2) promotes cholesterol efflux via the ATP-binding cassette transporter (ABCA1). In the current study, we directly added purified, lipid-free APOA2 to human plasma and found a dose-dependent increase in whole plasma cholesterol efflux capacity (CEC). APOA2 likewise increased the CEC of isolated HDL with the maximum effect occurring when equal masses of APOA1 and APOA2 coexisted on the particles. Follow-up experiments with reconstituted HDL corroborated that the presence of both APOA1 and APOA2 were necessary for the increased efflux. Using limited proteolysis and chemical cross-linking mass spectrometry, we found that APOA2 induced a conformational change in the N- and C-terminal helices of APOA1. Using reconstituted HDL with APOA1 deletion mutants, we further showed that APOA2 lost its ability to stimulate ABCA1 efflux to HDL if the C-terminal domain of APOA1 was absent, but retained this ability when the N-terminal domain was absent. Based on these findings, we propose a model in which APOA2 displaces the C-terminal helix of APOA1 from the HDL surface which can then interact with ABCA1 - much like it does in lipid-poor APOA1. These findings suggest APOA2 may be a novel therapeutic target given this ability to open a large, high-capacity pool of HDL particles to enhance ABCA1-mediated cholesterol efflux.

Plasma high-density lipoprotein (HDL), originally studied for its role in lipid transport, is now appreciated to have wide-ranging biological functions that become defective during disease. While >200 lipids have collectively been detected in HDL, published HDL lipidomic analyses in different diseases have commonly been targeted to prespecified subsets of lipids. Here, we report the results of untargeted lipidomic analysis of HDL isolated from 101 subjects referred for computed tomographic coronary imaging for whom multiple additional clinical and lipoprotein metadata were measured. Unsupervised clustering of the total HDL lipidome revealed that the subjects fell into one of two discrete groups, herein referred to as HDL 'metabotypes'. Subjects in metabotype 1 were likelier to be female and tended to have a less atherogenic lipoprotein profile, higher HDL cholesterol efflux capacity (CEC), and lower-grade non-calcified burden on coronary imaging than metabotype 2 counterparts. Specific lipids were relatively enriched in metabotype 1 HDL. Linear modeling revealed that several of these lipids were positively associated with CEC, statin use, HDL size, and HDL particle number, and positively correlated with HDL apolipoprotein A-1, suggesting that they may be informative HDL biomarkers. Taken together, we posit a novel, clinically relevant categorization for HDL revealed by systems biology.