Lipids associated with atherosclerotic plaque instability revealed by mass spectrometry imaging of human carotid arteries.

BACKGROUND AND AIMS: Lipids constitute one of the main components of atherosclerosis lesions and are the mediators of many mechanisms involved in plaque progression and stability. Here we tested the hypothesis that lipids known to be involved in plaque development exhibited associations with plaque vulnerability. We used spatial lipidomics to overcome plaque heterogeneity and to compare lipids from specific regions of symptomatic and asymptomatic human carotid atherosclerotic plaques. METHODS: Carotid atherosclerotic plaques were collected from symptomatic and asymptomatic patients. Plaque lipids were analyzed with the spatial lipidomics technique matrix-assisted laser desorption/ionization mass spectrometry imaging, and histology and immunofluorescence were used to segment the plaques into histomolecularly distinct regions. RESULTS: Macrophage-rich regions from symptomatic lesions were found to be enriched in phosphatidylcholines (synthesized to counteract excess free cholesterol), while the same region from asymptomatic plaques were enriched in polyunsaturated cholesteryl esters and triglycerides, characteristic of functional lipid droplets. Vascular smooth muscle cells (VSMCs) of the fibrous cap of asymptomatic plaques were enriched in lysophosphatidylcholines and cholesteryl esters, know to promote VSMC proliferation and migration, crucial for the buildup of the fibrous cap stabilizing the plaque. CONCLUSIONS: The investigation of the region-specific lipid composition of symptomatic and asymptomatic human atherosclerotic plaques revealed specific lipid markers of plaque outcome, which could be linked to known biological characteristics of stable plaques.

Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.

HYPOTHESIS: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration. METHODS AND RESULTS: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS. CONCLUSION: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.

Aberrant lipid accumulation and retinal pigment epithelium dysfunction in PRCD-deficient mice.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd-/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and lipid deposits in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Furthermore, the RPE of Prcd-/- mice exhibit a 1.7-fold increase in the expression of lipid transporter gene ATP-binding cassette transporter A1 (Abca1). Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd-/- mice exhibited age-related pathological features such as lipofuscin accumulation, Bruch's membrane (BrM) deposits and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd-/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd-/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.

Butyrate Increases Heparin Synthesis and Storage in Human Mast Cells.

Sulphated glycosaminoglycans (GAGs) such as heparin are a major component of mast cell granules and form the matrix within which biogenic mediators are stored. Since GAGs released from mast cells also play an important role in helminth expulsion, understanding GAG storage can offer new insights into mast cell function. Sodium butyrate (NaBu), a short-chain fatty acid, causes ultrastructural changes within the granules of human mast cells (HMC-1) and increases their histamine content. Therefore, we hypothesized that NaBu treatment would also modify the storage of polysaccharides such as GAGs. NaBu (1 mM) significantly increased GAG content and granularity in a time- and concentration-dependent manner without affecting cell viability and metabolic activity. NaBu increased the expression of enzymes associated with heparin biosynthesis (GLCE, NDST1, NDST2, HS6ST1, and GALT1) in a time-dependent manner. A cholesteryl butyrate emulsion (CholButE) increased heparin content after 24 and 48 h and modestly altered the expression of genes involved in heparin biosynthesis. Similar to NaBu, CholButE reduced cell proliferation without significantly altering viability or metabolic activity. These data show that butyrate increases the synthesis and storage of heparin in human mast cells, perhaps by altering their metabolic pathways.

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

Analysis of Lipoprotein Signaling in Iris Melanocytes.

Lipids are compounds involved in many biologic functions including cell structure, metabolism, energy storage and are involved in signaling. A prominent lipid in these functions is cholesterol. Cholesterol also plays a part in the signaling of melanocytes, which contain melanosomes. The maturation of these melanosomes happens during melanocyte growth. The deficit of melanogenesis or melanosome maturation is associated with ocular albinism in the eye. Aberrations of melanosome maturation are also associated with pigment dispersion syndrome. Albinism and pigment dispersion manifestations are systemic. Both melanogenesis and melanocyte maturation are affected by cholesterol metabolism. Cholesterol signaling is a part of many pathways in the body, and evaluating these signals can have implications in systemic disease processes of melanogenesis and melanosome maturation, like ocular albinism and pigment dispersion. Cholesterol is carried by lipoprotein particles. Low-density lipoprotein (LDL) is usually the transport vehicle for cholesterol to reach tissues and organelles. The LDL uptake on cells often sends out a cascade of internal signaling within the cells. We describe here LDL signaling related to lipase activity changes using enzymatic methods with a kit. We describe analyses of cholesterol esters and free cholesterol with liquid chromatography and gas chromatography with or in tandem with mass spectrometry (GC-MS and LC-MS/MS). These analyses will provide insight into melanosome maturation and melanogenesis. The methods described here are applicable to all melanocytes within the body of a model mammalian organism.

Sterol O-acyltransferase (SOAT/ACAT) activity is required to form cholesterol crystals in hepatocyte lipid droplets.

OBJECTIVE: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

BACKGROUND: LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. METHODS: We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. RESULTS: As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. CONCLUSIONS: Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation.

BACKGROUND: Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. METHODS: Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FINDINGS: Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. INTERPRETATION: Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FUNDING: This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Regional Differences in the Small Intestinal Proteome of Control Mice and of Mice Lacking Lysosomal Acid Lipase.

The metabolic contribution of the small intestine (SI) is still unclear despite recent studies investigating the involvement of single cells in regional differences. Using untargeted proteomics, we identified regional characteristics of the three intestinal tracts of C57BL/6J mice and found that proteins abundant in the mouse ileum correlated with the high ileal expression of the corresponding genes in humans. In the SI of C57BL/6J mice, we also detected an increasing abundance of lysosomal acid lipase (LAL), which is responsible for degrading triacylglycerols and cholesteryl esters within the lysosome. LAL deficiency in patients and mice leads to lipid accumulation, gastrointestinal disturbances, and malabsorption. We previously demonstrated that macrophages massively infiltrated the SI of Lal-deficient (KO) mice, especially in the duodenum. Using untargeted proteomics (ProteomeXchange repository, data identifier PXD048378), we revealed a general inflammatory response and a common lipid-associated macrophage phenotype in all three intestinal segments of Lal KO mice, accompanied by a higher expression of GPNMB and concentrations of circulating sTREM2. However, only duodenal macrophages activated a metabolic switch from lipids to other pathways, which were downregulated in the jejunum and ileum of Lal KO mice. Our results provide new insights into the process of absorption in control mice and possible novel markers of LAL-D and/or systemic inflammation in LAL-D.

Regulation of lipid droplets and cholesterol metabolism in adrenal cortical cells.

The adrenal gland is composed of two distinctly different endocrine moieties. The interior medulla consists of neuroendocrine chromaffin cells that secrete catecholamines like adrenaline and noradrenaline, while the exterior cortex consists of steroidogenic cortical cells that produce steroid hormones, such as mineralocorticoids (aldosterone), glucocorticoids (cortisone and cortisol) and androgens. Synthesis of steroid hormones in cortical cells requires substantial amounts of cholesterol, which is the common precursor for steroidogenesis. Cortical cells may acquire cholesterol from de novo synthesis and uptake from circulating low- and high-density lipoprotein particles (LDL and HDL). As cholesterol is part of the plasma membrane in all mammalian cells and an important regulator of membrane fluidity, cellular levels of free cholesterol are tightly regulated. To ensure a robust supply of cholesterol for steroidogenesis and to avoid cholesterol toxicity, cortical cells store large amounts of cholesterol as cholesteryl esters in intracellular lipid droplets. Cortical steroidogenesis relies on both mobilization of cholesterol from lipid droplets and constant uptake of circulating cholesterol to replenish lipid droplet stores. This chapter will describe mechanisms involved in cholesterol uptake, cholesteryl ester synthesis, lipid droplet formation, hydrolysis of stored cholesteryl esters, as well as their impact on steroidogenesis. Additionally, animal models and human diseases characterized by altered cortical cholesteryl ester storage, with or without abnormal steroidogenesis, will be discussed.

Shotgun lipidomics of human subretinal fluids under rod-dominant retina reveals cone-dominated lipids.

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases' mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina.

TREM2 regulates microglial lipid droplet formation and represses post-ischemic brain injury.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor protein predominantly expressed in microglia within the central nervous system (CNS). TREM2 regulates multiple microglial functions, including lipid metabolism, immune reaction, inflammation, and microglial phagocytosis. Recent studies have found that TREM2 is highly expressed in activated microglia after ischemic stroke. However, the role of TREM2 in the pathologic response after stroke remains unclear. Herein, TREM2-deficient microglia exhibit an impaired phagocytosis rate and cholesteryl ester (CE) accumulation, leading to lipid droplet formation and upregulation of Perilipin-2 (PLIN2) expression after hypoxia. Knockdown of TREM2 results in increased lipid synthesis (PLIN2, SOAT1) and decreased cholesterol clearance and lipid hydrolysis (LIPA, ApoE, ABCA1, NECH1, and NPC2), further impacting microglial phenotypes. In these lipid droplet-rich microglia, the TGF-ß1/Smad2/3 signaling pathway is downregulated, driving microglia towards a pro-inflammatory phenotype. Meanwhile, in a neuron-microglia co-culture system under hypoxic conditions, we found that microglia lost their protective effect against neuronal injury and apoptosis when TREM2 was knocked down. Under in vivo conditions, TREM2 knockdown mice express lower TGF-ß1 expression levels and a lower number of anti-inflammatory M2 phenotype microglia, resulting in increased cerebral infarct size, exacerbated neuronal apoptosis, and aggravated neuronal impairment. Our work suggests that TREM2 attenuates stroke-induced neuroinflammation by modulating the TGF-ß1/Smad2/3 signaling pathway. TREM2 may play a direct role in the regulation of inflammation and also exert an influence on the post-ischemic inflammation and the stroke pathology progression via regulation of lipid metabolism processes. Thus, underscoring the therapeutic potential of TREM2 agonists in ischemic stroke and making TREM2 an attractive new clinical target for the treatment of ischemic stroke and other inflammation-related diseases.

A study on cholesterol-cholesteryl ester metabolic homeostasis and drug intervention in hyperlipidemic hamsters using UHPLC-MS/MS.

Hyperlipidemia is a global metabolic disorder characterized by dysregulation of lipid metabolism. This dysregulation is closely associated with the altered homeostasis of cholesterol-cholesteryl ester (CE) metabolism in systemic circulation, and some organs. Additionally, the relationship between oxidized cholesteryl ester (oxCE) and the disease has also gained attention. Currently, there is a lack of comprehensive research on the alterations in cholesterol-CE metabolism in the context of hyperlipidemia, as well as the characteristics of lipid-lowering agents in regulating this metabolic state. Therefore, 40 oxCEs were identified in the hamster liver sample, and novel ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methods were established for simultaneous analysis of cholesterol, 57 CEs, and 40 oxCEs in the serum, liver, adipose tissue, and intestine of hyperlipidemic hamsters. This study investigated the metabolic alterations between cholesterol-CE/oxCE in hyperlipidemic hamsters and those treated with lipid-lowering agents, including the Niemann-Pick-C1 like-1 protein (NPC1L1) inhibitor ezetimibe and the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe. The study findings demonstrate metabolic disorders in cholesterol-CE/oxCE homeostasis in hyperlipidemic hamsters. Lipid-lowering agent therapy can improve the metabolic dysregulation caused by hyperlipidemia, with distinct characteristics: ezetimibe is more effective in reducing cholesterol, while avasimibe is more effective in reducing CEs/oxCEs. Eight potential biomarkers indicating the dysregulation of cholesterol-CE metabolism caused by hyperlipidemia and its improvement by lipid-lowering agents have been identified in the serum. This study offers new insights into the hyperlipidemia pathophysiology and the mechanisms of lipid-lowering agents from a novel perspective on cholesterol-CE/oxCE metabolic homeostasis.

Total Outflow of High-Density Lipoprotein-Cholesteryl Esters from Plasma Is Decreased in a Model of 3/4 Renal Mass Reduction.

(1) Background: Previous studies have enriched high-density lipoproteins (HDL) using cholesteryl esters in rabbits with a three-quarter reduction in functional renal mass, suggesting that the kidneys participate in the cholesterol homeostasis of these lipoproteins. However, the possible role of the kidneys in lipoprotein metabolism is still controversial. To understand the role of the kidneys in regulating the HDL lipid content, we determined the turnover of HDL-cholesteryl esters in rabbits with a three-quarter renal mass reduction. (2) Methods: HDL subclass characterization was conducted, and the kinetics of plasma HDL-cholesteryl esters, labeled with tritium, were studied in rabbits with a 75% reduction in functional renal mass (Ntx). (3) Results: The reduced renal mass triggered the enrichment of cholesterol, specifically cholesteryl esters, in HDL subclasses. The exchange of cholesteryl esters between HDL and apo B-containing lipoproteins (VLDL/LDL) was not significantly modified in Ntx rabbits. Moreover, the cholesteryl esters of HDL and VLDL/LDL fluxes from the plasmatic compartment tended to decrease, but they only reached statistical significance when both fluxes were added to the Nxt group. Accordingly, the fractional catabolic rate (FCR) of the HDL-cholesteryl esters was lower in Ntx rabbits, concomitantly with its accumulation in HDL subclasses, probably because of the reduced mass of renal cells requiring this lipid from lipoproteins.

Variants in the CETP gene affect levels of HDL cholesterol by reducing the amount, and not the specific lipid transfer activity, of secreted CETP.

BACKGROUND: Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters in plasma from high density lipoprotein (HDL) to very low density lipoprotein and low density lipoprotein. Loss-of-function variants in the CETP gene cause elevated levels of HDL cholesterol. In this study, we have determined the functional consequences of 24 missense variants in the CETP gene. The 24 missense variants studied were the ones reported in the Human Gene Mutation Database and in the literature to affect HDL cholesterol levels, as well as two novel variants identified at the Unit for Cardiac and Cardiovascular Genetics, Oslo University Hospital in subjects with hyperalphalipoproteinemia. METHODS: HEK293 cells were transiently transfected with mutant CETP plasmids. The amounts of CETP protein in lysates and media were determined by Western blot analysis, and the lipid transfer activities of the CETP variants were determined by a fluorescence-based assay. RESULTS: Four of the CETP variants were not secreted. Five of the variants were secreted less than 15% compared to the WT-CETP, while the other 15 variants were secreted in varying amounts. There was a linear relationship between the levels of secreted protein and the lipid transfer activities (r = 0.96, p<0.001). Thus, the secreted variants had similar specific lipid transfer activities. CONCLUSION: The effect of the 24 missense variants in the CETP gene on the lipid transfer activity was mediated predominantly by their impact on the secretion of the CETP protein. The four variants that prevented CETP secretion cause autosomal dominant hyperalphalipoproteinemia. The five variants that markedly reduced secretion of the respective variants cause mild hyperalphalipoproteinemia. The majority of the remaining 15 variants had minor effects on the secretion of CETP, and are considered neutral genetic variants.

Developing a model to predict the early risk of hypertriglyceridemia based on inhibiting lipoprotein lipase (LPL): a translational study.

Hypertriglyceridemia (HTG) is an independent risk factor for atherosclerotic cardiovascular disease (ASCVD). One of the multiple origins of HTG alteration is impaired lipoprotein lipase (LPL) activity, which is an emerging target for HTG treatment. We hypothesised that early, even mild, alterations in LPL activity might result in an identifiable metabolomic signature. The aim of the present study was to assess whether a metabolic signature of altered LPL activity in a preclinical model can be identified in humans. A preclinical LPL-dependent model of HTG was developed using a single intraperitoneal injection of poloxamer 407 (P407) in male Wistar rats. A rat metabolomics signature was identified, which led to a predictive model developed using machine learning techniques. The predictive model was applied to 140 humans classified according to clinical guidelines as (1) normal, less than 1.7 mmol/L; (2) risk of HTG, above 1.7 mmol/L. Injection of P407 in rats induced HTG by effectively inhibiting plasma LPL activity. Significantly responsive metabolites (i.e. specific triacylglycerols, diacylglycerols, phosphatidylcholines, cholesterol esters and lysophospholipids) were used to generate a predictive model. Healthy human volunteers with the impaired predictive LPL signature had statistically higher levels of TG, TC, LDL and APOB than those without the impaired LPL signature. The application of predictive metabolomic models based on mechanistic preclinical research may be considered as a strategy to stratify subjects with HTG of different origins. This approach may be of interest for precision medicine and nutritional approaches.

Roles of peripheral lipoproteins and cholesteryl ester transfer protein in the vascular contributions to cognitive impairment and dementia.

This narrative review focuses on the role of cholesteryl ester transfer protein (CETP) and peripheral lipoproteins in the vascular contributions to cognitive impairment and dementia (VCID). Humans have a peripheral lipoprotein profile where low-density lipoproteins (LDL) represent the dominant lipoprotein fraction and high-density lipoproteins (HDL) represent a minor lipoprotein fraction. Elevated LDL-cholesterol (LDL-C) levels are well-established to cause cardiovascular disease and several LDL-C-lowering therapies are clinically available to manage this vascular risk factor. The efficacy of LDL-C-lowering therapies to reduce risk of all-cause dementia and AD is now important to address as recent studies demonstrate a role for LDL in Alzheimer's Disease (AD) as well as in all-cause dementia. The LDL:HDL ratio in humans is set mainly by CETP activity, which exchanges cholesteryl esters for triglycerides across lipoprotein fractions to raise LDL and lower HDL as CETP activity increases. Genetic and pharmacological studies support the hypothesis that CETP inhibition reduces cardiovascular risk by lowering LDL, which, by extension, may also lower VCID. Unlike humans, wild-type mice do not express catalytically active CETP and have HDL as their major lipoprotein fraction. As HDL has potent beneficial effects on endothelial cells, the naturally high HDL levels in mice protect them from vascular disorders, likely including VCID. Genetic restoration of CETP expression in mice to generate a more human-like lipid profile may increase the relevance of murine models for VCID studies. The therapeutic potential of existing and emerging LDL-lowering therapies for VCID will be discussed. Figure Legend. Cholesteryl Ester Transfer Protein in Alzheimer's Disease. CETP is mainly produced by the liver, and exchanges cholesteryl esters for triglycerides across lipoprotein fractions to raise circulating LDL and lower HDL as CETP activity increases. Low CETP activity is associated with better cardiovascular health, due to decreased LDL and increased HDL, which may also improve brain health. Although most peripheral lipoproteins cannot enter the brain parenchyma due to the BBB, it is increasingly appreciated that direct access to the vascular endothelium may enable peripheral lipoproteins to have indirect effects on brain health. Thus, lipoproteins may affect the cerebrovasculature from both sides of the BBB. Recent studies show an association between elevated plasma LDL, a well-known cardiovascular risk factor, and a higher risk of AD, and considerable evidence suggests that high HDL levels are associated with reduced CAA and lower neuroinflammation. Considering the potential detrimental role of LDL in AD and the importance of HDL's beneficial effects on endothelial cells, high CETP activity may lead to compromised BBB integrity, increased CAA deposits and greater neuroinflammation. Abbreviations: CETP - cholesteryl transfer ester protein; LDL - low-density lipoproteins; HDL - high-density lipoproteins; BBB - blood-brain barrier; CAA - cerebral amyloid angiopathy, SMC - smooth muscle cells, PVM - perivascular macrophages, RBC - red blood cells.