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Excess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation. Cholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits. Cholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH. SOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester. • Cholesterol overload induces cholesterol crystals in Hep3B hepatocytes. • Induced crystals are lipid droplet localized and can form lattice structures. • Cholesterol esterification via SOAT/ACAT activity mediates crystal formation. • SOAT-dependent crystals form in cultured RAW264.7 macrophage. • SOAT-dependent crystals form in mouse liver during MASH. [ABSTRACT FROM AUTHOR]

Abrupt aggregation of amyloid β 1 – 42 (Aβ 1 – 42) peptide in the frontal lobe is the expected underlying cause of Alzheimer's disease (AD). β-Sheet-rich oligomers and fibrils formed by Aβ 1 – 42 exert high cell toxicity. A growing body of evidence indicates that lipids can uniquely alter the secondary structure and toxicity of Aβ 1 – 42 aggregates. At the same time, underlying molecular mechanisms that determine this difference in toxicity of amyloid aggregates remain unclear. Using a set of molecular and biophysical assays to determine the molecular mechanism by which Aβ 1 – 42 aggregates formed in the presence of cholesterol, cardiolipin, and phosphatidylcholine exert cell toxicity. Our findings demonstrate that rat neuronal cells exposed to Aβ 1 – 42 fibrils formed in the presence of lipids with different chemical structure exert drastically different magnitude and dynamic of unfolded protein response (UPR) in the endoplasmic reticulum (ER) and mitochondria (MT). We found that the opposite dynamics of UPR in MT and ER in the cells exposed to Aβ 1 – 42 : cardiolipin fibrils and Aβ 1 – 42 aggregates formed in a lipid-free environment. We also found that Aβ 1 – 42 : phosphatidylcholine fibrils upregulated ER UPR simultaneously downregulating the UPR response of MT, whereas Aβ 1 – 42 : cholesterol fibrils suppressed the UPR response of ER and upregulated UPR response of MT. We also observed progressively increasing ROS production that damages mitochondrial membranes and other cell organelles, ultimately leading to cell death. • Amyloid β1-42 (Aβ1-42) oligomers and fibrils are the expected underlying cause of Alzheimer's disease (AD) • Lipids can uniquely alter the secondary structure and toxicity of Aβ1-42 aggregates. • Such aggregates have different unfolded protein response (UPR) in the endoplasmic reticulum and mitochondria damage. • Aβ1-42:phosphatidylcholine fibrils upregulated ER UPR simultaneously downregulating the UPR response of MT., (make this a new bullet) Aβ1-42:cholesterol fibrils suppressed the UPR response of ER and upregulated UPR response of MT. [ABSTRACT FROM AUTHOR]

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol. • The accumulation of Erg6p and Pet10p in pet10Δ and erg6Δ is reciprocal. • The disruption of PET10 affects several phenotypes of the erg6Δ cells. • The mobilization of steryl esters in erg6Δpet10Δ is completely lost. • The disruption of PET10 in erg6Δ alters the composition of ergosterol intermediates. • Decreased levels of SE in erg6Δpet10Δ is linked with the overexpression of steryl ester hydrolase Yeh1p. [ABSTRACT FROM AUTHOR]

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Raman-based molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation. • T cell activation involves lipid accumulation in perinuclear area and morphological changes in cellular and nuclear areas. • The ratio of 1041/1003 cm-1 in Raman spectra decreased after T cell activation indicating conformational changes in proteins. • Carotenoids, found exclusively in non-activated T cells, were no longer observed after the activation process. • Raman-based analysis can identify activated T cells in a specific, sensitive, and label-free manner. [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism (IEM) represent a heterogeneous group of more than 1800 rare disorders, many of which are causing significant childhood morbidity and mortality. More than 100 IEM are linked to dyslipidaemia, but yet our knowledge in connecting genetic information with lipidomic data is limited. Stable isotope tracing studies of the lipid metabolism (STL) provide insights on the dynamic of cellular lipid processes and could thereby facilitate the delineation of underlying metabolic (patho)mechanisms. This mini-review focuses on principles as well as technical limitations of STL and describes potential clinical applications by discussing recently published STL focusing on IEM. • Over 100 inborn errors of metabolism (IEM) cause dyslipidaemia, but clinical routine tests only cover few lipid parameters. • Stable isotope tracing studies of the lipid metabolism (STL) are performed in clinical research to study alterations in IEM. • Mass spectrometry allows the sensitive analysis by the use of stable isotope labelled lipid precursors to perform STL. • In addition to non-labelled lipidomics approaches, STL enable kinetic flux studies of the lipid metabolism. • STL aim to provide turnover rates of metabolic pathways as well as to identify metabolic aberrations in IEM. [ABSTRACT FROM AUTHOR]

This study explores the intricate relationship between the yeast vacuolar H+-ATPase (V-ATPase) and neutral lipid metabolism. We show that LD generation observed upon loss of V-ATPase activity is crucial for survival in lipotoxic conditions. Moreover, the study uncovers a link between V-ATPase function, inositol metabolism and the activation of the oxidative pentose phosphate pathway, highlighting its pivotal role in counteracting oxidative stress. This work provides foundational insights into metabolic adaptations triggered by V-ATPase dysfunction, shedding light on cellular adaptability under lipotoxic and oxidative stress conditions. [ABSTRACT FROM AUTHOR]

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion. [Display omitted] • Blood meal does not control Brummer lipase gene expression in female Rhodnius prolixus. • RhoprBmm knockdown resulted in higher triacylglycerol content in the female fat body. • Silenced females flew for shorter times and had decreased locomotor activity. • Knockdown females laid fewer eggs which showed lower hatching rates. • The chorion of the eggs laid by the knockdown females had ultrastructure defects. [ABSTRACT FROM AUTHOR]

In the oleaginous fungus Mucor circinelloides , lipid accumulation is regulated by nitrogen metabolism, which is regulated by the areA gene, a member of the GATA zinc finger transporter family and a major regulator for nitrogen metabolism. However, the role of areA in lipid accumulation in this fungus has not been reported. In order to explore the regulatory effect of areA gene on nitrogen metabolism and lipid accumulation in M. circinelloides , we constructed areA gene knockout and overexpression strains. Then, the recombinant strains were cultured and their biochemical indexes were measured. Simultaneously, transcriptomic studies on the recombinant strains were conducted to infer the regulatory mechanism of areA. The results showed that the areA knockout strain accumulated more lipid, which is 42 % higher than the control. While the areA overexpressing strain obtained the higher biomass accumulation (23 g/L) and used up the nitrogen source in the medium earlier than the control strain and knockout strain. Transcriptome data analysis showed that nr and nit-6 genes related to nitrogen metabolism were up-regulated. And the expression levels of key genes acc and aclY were higher in the areA knockout strain than others, which was positively correlated with the increased lipid accumulation. In addition, in knockout strains, protein catabolism tended to provide substrates for the lipid production, and the expression levels of the related genes were also higher than others. These results indicated that the areA gene not only controls the transcription level of genes related to nitrogen metabolism but also affects lipid accumulation. • The role of areA in lipid accumulation in this fungus has not been reported. • AreA gene can control the transcription level of nitrogen metabolism related genes. • The genetic modification of areA altered the capacity of lipid biosynthesis and affect protein synthesis, and degradation. [ABSTRACT FROM AUTHOR]

Hepatic inflammation is commonly identified in Wilson disease (WD), a genetic disease of hepatic and brain copper accumulation. Copper accumulation is associated with increased oxidative stress and reactive oxygen species generation which may result in non-enzymatic oxidation of membrane-bound polyunsaturated fatty acids (PUFA). PUFA can be oxidized enzymatically via lipoxygenases (LOX), cyclooxygenases (COX), and cytochrome P450 monooxygenases (CYP). Products of PUFA oxidation are collectively known as oxylipins (OXL) and are bioactive lipids that modulate hepatic inflammation. We examined hepatic OXL profiles at early stages of WD in two mouse models, the toxic milk mouse from The Jackson Laboratory (tx-j) and the Atp7b knockout on a C57Bl/6 background (Atp7b −/−B6). Targeted lipidomic analysis performed by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry showed that in both tx-j and Atp7b −/−B6 mice, hepatic OXL profiles were altered with higher thromboxane and prostaglandins levels. The levels of oxidative stress marker, 9-HETE were increased more markedly in tx-j mice. However, both genotypes showed upregulated transcript levels of many genes related to oxidative stress and inflammation. Both genotypes showed higher prostaglandins, thromboxin along with higher PUFA-derived alcohols, diols, and ketones with altered epoxides; the expression of Alox5 was upregulated and many CYP-related genes were dysregulated. Pathway analyses show dysregulation in arachidonic acid and linoleic acid metabolism characterizes mice with WD. Our findings indicate alterations in hepatic PUFA metabolism in early-stage WD and suggest the upregulation of both, non-enzymatic ROS-dependent and enzymatic PUFA oxidation, which could have implications for hepatic manifestations in WD and represent potential targets for future therapies. • Early-stage Wilson disease presents alterations in arachidonic acid and linoleic acid metabolism. • Oxidative stress marker 9-HETE is increased with possible genotype-related variations. • Cyclooxygenase pathways are upregulated in mice with Wilson disease. • Higher polyunsaturated fatty acids- alcohols, diols, and ketones suggest upregulated lipoxygenases activities. • Cytochrome P450 monooxygenase and soluble epoxide hydrolase activities are dysregulated in Wilson disease. [ABSTRACT FROM AUTHOR]

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoK ATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum. [Display omitted] • ROMK2 channel localizes in multiple cell compartments. • ROMK2 forms complexes with mitochondrial AGK and ER-localized DGKE. • ROMK2-AGK or ROMK2-DGKE complexes can be immunoprecipitated. • AGK and DGKE products, LPA and PA, activate ROMK2. • ROMK2's interaction with lipid kinases may regulate activity via localized anionic lipid synthesis. [ABSTRACT FROM AUTHOR]

Patatin-like phospholipase domain containing proteins (PNPLAs) play diverse roles in lipid metabolism. In this review, we focus on the enzymatic properties and predicted 3D structures of PNPLA1-5. PNPLA2-4 exert both catabolic and anabolic functions. Whereas PNPLA1 is predominantly expressed in the epidermis and involved in sphingolipid biosynthesis, PNPLA2 and 4 are ubiquitously expressed and exhibit several enzymatic activities, including hydrolysis and transacylation of various (glycero-)lipid species. This review summarizes known biological roles for PNPLA-mediated hydrolysis and transacylation reactions and highlights open questions concerning their physiological function. • PNPLA2-4 exert both catabolic and anabolic functions as hydrolases and transacylases. • PNPLA1 is only described as a transacylase and is crucial for acylceramide biosynthesis. • PNPLA5 is only described as a possible TAG hydrolase. • Open questions include the physiological relevance of trancylation processes of PNPLA2-4. • The regulation of the balance between hydrolysis and transacylation also needs to be investigated in more detail. • The role of transacylation in the biosynthesis of other bioactive lipids besides FAHFAs still requires further studies. [ABSTRACT FROM AUTHOR]