Hydrolysis Of Cholesterol Esters Research Articles

3AcFNP-59 for Positron Emission Tomography Imaging of Cholesterol Trafficking and Utilization.

Cholesteryl ester analogues of [18F]FNP-59 have the ability to provide information on cholesterol trafficking and utilization at earlier time points than those of [18F]FNP-59 or [131I]NP-59. It is well-known that free cholesterol and cholesteryl esters have differing distribution profiles and that they can be interconverted enzymatically. Substitution of the ester influences the rate of cholesterol ester hydrolysis and the subsequent mixing of cholesterol esters with the lipid pool in the body. This can be utilized by preparing esters that are more readily taken up by lipoprotein, are quickly hydrolyzed and mixed with the endogenous lipid pool and delivered to tissues of interest more quickly than free cholesterol analogues that require esterification for lipoprotein association. The acetyl ester of FNP-59 demonstrated the preferred uptake properties and response to adrenal cortical manipulation, indicating its ability to image hormone production. Finally, dosimetry studies were conducted in preparation for the clinical translation of [18F]3AcFNP-59.

Wolman’s Disease and GNE Myopathy Frequency to Religion

Complete absence or deficiency of Lysosomal acid lipase (LAL) leads to the rare infantile disorder Wolman's Disease [1]. Decreased function of GNE can lead to GNE myopathy [2]. Although both pathological phenotypes are rare, LAL deficiency only affects 1 in every 500,000 births and dysfunction of GNE affects 1 in every 1,000,000 births, there is a higher incidence in the religious groups of the Middle East; the incidence is projected to be as high as 1 in every 4,200 births [3,4]. The LIPA gene encodes for LAL while the GNE gene encodes for UDPN- acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase [4,5]. LAL catalyzes the hydrolysis of cholesterol esters and triglycerides [6]. GlcNAc and ManNAc are responsible for the biosynthesis of sialic acid [7]. Presence of variations in GNE genes are known to impair the enzymes and lead to pathological phenotypes. This study reports the prevalence of mutations c.260G>T; p.G87V and c.894G>A; E8SJM in the lipase gene (LIPA) and c.2228T>C p.M743T in GNE gene amongst different religions. We sequenced 1,354 samples for the presence of the variations in LIPA exon 4 and 8 along with GNE exon 13(formerly exon 12). The sample population comprised individuals of varying Middle Eastern religions such as Mizrahi Jewish, Muslims, Christians, Baha’i, Catholic, Buddhist and Zoroastrian. The other religions that were not Mizrahi Jewish were classified under “Non-Jewish” to have a more even distribution. LIPA gene exon 4 variation p.G87V had a heterozygous frequency of 22 in 1,354(1.62%) with 20 of 22(90.9%) heterozygotes being Mizrahi Jewish while 2 of 22(9.09%) heterozygotes were Non-Jewish. Exon 8 splice junction mutation E8SJM, G>A transition at position 1 had a frequency of 2 in 1,354 (0.14%) with 2 of 2(100%) heterozygotes being Mizrahi Jewish. GNE exon 13 variation p.M743T (formerly exon 12 p.M712T) had a mutation frequency of 58 in 1,354(4.28%) with 5 of these 58(8.62%) samples being homozygous while 53 of the 58(91.37%) were heterozygous. In 58 samples that yielded a mutation, 54 of them were Mizarhi jewish (93.1%).

In vitro Raman imaging of human macrophages: Impact of eicosapentaenoic acid on the hydrolysis of cholesterol esters in lipid droplets

Atherosclerosis – a cardiovascular disease and the primary cause of morbidity and mortality in industrialized countries – is linked to the existence of atherosclerotic plaques characterized by cholesterol-laden macrophages called foam cells. In these cells, cholesterol esters associated with triglycerides form lipid droplets (LD). The only way to remove this excess cholesterol is to promote free cholesterol efflux from macrophages to specific acceptors. It has been shown recently that eicosapentaenoic acid (EPA) reduces efflux on cholesterol-loaded THP-1 macrophages in vitro due to decreased cholesterol esters hydrolysis. These in vitro observations could reflect EPA's difficulty in facilitating in vivo the antiatherogenic process of cholesterol efflux within advanced atherosclerotic plaques.This work aims to study in vitro the impact of EPA on cholesterol esters hydrolysis in the LD of human THP-1 macrophages using vibrational Raman microspectroscopy. For this, we used deuterated EPA and recorded spectral images at the cell scale after different hydrolysis times. Resultsshowed that EPA is involved in forming triglycerides and phospholipids of LD. Hydrolysis kinetics slowed down after 24 h, triglycerides increased, and the intensity of the characteristic bands linked to deuteration decreased. The size of LD without hydrolysis (H0) is higher than that after 24 h (H1) or 48 h (H2) of hydrolysis. The size decrease is sharper when going from H0 to H1 than from H1 to H2. Principal component analysis illustrated data’ projection according to the cellular compartment, the hydrolysis time, and the supplementation of the medium.

The role of CYP1A1/2 in cholesterol ester accumulation provides a new perspective for the treatment of hypercholesterolemia.

Cholesterol is an important precursor of many endogenous molecules. Disruption of cholesterol homeostasis can cause many pathological changes, leading to liver and cardiovascular diseases. CYP1A is widely involved in cholesterol metabolic network, but its exact function has not been fully elucidated. Here, we aim to explore how CYP1A regulates cholesterol homeostasis. Our data showed that CYP1A1/2 knockout (KO) rats presented cholesterol deposition in blood and liver. The serum levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and total cholesterol were significantly increased in KO rats. Further studies found that the lipogenesis pathway (LXRα-SREBP1-SCD1) of KO rats was activated, and the key protein of cholesterol ester hydrolysis (CES1) was inhibited. Importantly, lansoprazole can significantly alleviate rat hepatic lipid deposition in hypercholesterolemia models by inducing CYP1A. Our findings reveal the role of CYP1A as a potential regulator of cholesterol homeostasis and provide a new perspective for the treatment of hypercholesterolemia.

Proteomic and lipidomic analyses of lipid droplets in Aurantiochytrium limacinum ATCC MYA-1381

Aurantiochytrium limacinum is a heterotrophic unicellular organism that develops cytosolic lipid droplets that store triacylglycerols (TGs) with a high docosahexaenoic acid (C22:6) content. Although the information on protein and lipid compositions in lipid droplets is useful for the understanding of storage lipid metabolism, it is not well known in this organism. In the present study, we purified lipid droplets by mild disruption of the cells using a hypo-osmotic buffer containing 0.05 % or 0.2 % NP-40 and analyzed the proteomics and lipidomics. In total, 29 putative lipid droplet-localizing proteins were identified. Approximately half of the lipid droplet protein content [54.0 % (w/w)] was accounted for by thraustochytrid-specific lipid droplet protein 1 (TLDP1), which might be a structural protein in the lipid droplet. The proteins included acyl-CoA synthetase (ACS), sterol 24-C methyltransferase (SMT1), diacylglycerol acyltransferase 2 (DGAT2), and heat-shock protein 70 kDa (HSP70), which are often found in lipid droplets isolated from other organisms. We also found putative orthologs of a lipid droplet-associated hydrolase which catalyzes the hydrolysis of cholesterol esters in macrophages and a monoglyceride lipase that has been identified in mammalian lipid droplets. The lipidomics showed that the major unsaturated TGs were TG60:12 [palmitic acid (C16:0)/C22:6/C22:6] and TG66:18 (C22:6/C22:6/C22:6). Phytosterol ester was more abundant in the lipid droplet fraction than the nonlipid droplet fraction. The accumulation of phytosterol ester in the lipid droplet might have a causal relationship to the localization of SMT1, which is considered to act in the phytosterol biosynthesis pathway. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present, but the lipid droplet fraction was deficient in lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Through the proteome and lipidome analyses, the characteristics of proteins and lipids in A. limacinum lipid droplets were shown.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic \u03b2-oxidation

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

Quantitative trait locus mapping identifies the Gpnmb gene as a modifier of mouse macrophage lysosome function

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.

Regulation of lipid droplets via the PLCβ2-PKCα-ADRP pathway in granulosa cells exposed to cadmium

In steroidogenic cells, steroids are synthesized de novo from cholesterol stored in lipid droplets (LDs). The size of LDs regulated by adipose differentiation-related protein (ADRP) is closely related to cholesterol ester hydrolysis. Many studies reported that cadmium (Cd) had dual effects on steroidogenesis in granulosa cells (GCs). However, the role of LD and its regulation in abnormal steroidogenesis caused by Cd exposure remain unknown. In current study, female rats were exposed to CdCl2 during gestation and lactation, and influence of such exposure was investigated in ovarian GCs of female offspring. The size of LDs was found much smaller than normal in GCs; ADRP was down-regulated and hormone-sensitive lipase (HSL) phosphorylation was increased, followed by up-regulation of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (CYP11A1); the expression of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-2 (PLCβ2) and protein kinase C alpha type (PKCα) were both decreased accompanying the ADRP down-regulation. This series of events resulted in a high level of progesterone in serum. Similar results were demonstrated in GCs treated with 20 μM CdCl2 for 24 h in vitro. The protein level of ADRP was decreased after gene silencing of PLCβ2/PKCα, and the knockdown of PLCβ2/PKCα/ADRP led to micro-sized LD formation. We found that Cd exposure down-regulated ADRP by inhibiting the PLCβ2-PKCα signaling pathway, reduced the size of LDs, and promoted HSL phosphorylation. StAR and CYP11A1 were both up-regulated following the hydrolysis of cholesterol ester, which led to a high production of progesterone. LD thereby is a target subcellular organelle for Cd to affect steroid hormone synthesis in ovarian GCs. These findings might help to uncover the mechanism of ovarian dysfunction and precocious puberty caused by Cd pollution.

Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

ObjectiveCholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (ER). In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport. MethodsCRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking. ResultsAblation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction. ConclusionsWe identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol esters, implicating the Aster-B protein as a novel regulator of steroidogenesis.

Retinoic acid receptor-related orphan receptor \u03b1 reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) catalyzes the hydrolysis of cholesterol ester (CE) in macrophages. Genetic ablation of NCEH1 promotes CE-laden macrophages and the development of atherosclerosis in mice. Dysregulation of NCEH1 levels is involved in the pathogenesis of multiple disorders including metabolic diseases and atherosclerosis; however, relatively little is known regarding the mechanisms regulating NCEH1. Retinoic acid receptor-related orphan receptor α (RORα)-deficient mice exhibit several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia and increased susceptibility to atherosclerosis.ResultsIn this study, inhibition of lipid droplet formation by RORα positively regulated NCEH1 expression in macrophages. In mammals, the NCEH1 promoter region was found to harbor putative RORα response elements (ROREs). Electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that RORα binds and responds to ROREs in human NCEH1. Moreover, NCEH1 was upregulated through RORα via a phorbol myristate acetate-dependent mechanism during macrophage differentiation from THP1 cells. siRNA-mediated knockdown of RORα significantly downregulated NCEH1 expression and accumulated lipid droplets in human hepatoma cells. In contrast, NCEH1 expression and removal of lipid droplets were induced by RORα agonist treatments and RORα overexpression in macrophages.ConclusionThese data strongly suggested that NCEH1 is a direct RORα target, defining potential new roles for RORα in the inhibition of lipid droplet formation through NCEH1.

Cholesterol: A Prelate in Cell Nucleus and its Serendipity.

Cholesterol is a chameleon bio-molecule in cellular multiplex. It acts as a prelate in almost every cellular compartment with its site specific characteristics viz. regulation of structural veracity and scaffold fluidity of bio-membranes, insulation of electrical transmission in nerves, controlling of genes by making steroid endocrines, acting as precursors of metabolic regulators and many more with its emerging prophecy in the cell nucleus to drive new cell formation. Besides the crucial legacy in cellular functionality, cholesterol is ostracized as a member of LDL particle, which has been proved responsible to clog blood vessels. LDL particles get deposited in the blood vessels because of their poor clearance owing to the non-functioning LDL receptor on the vessel wall and surrounding tissues. Blocking of blood vessel promotes heart attack and stroke. On the other hand, cholesterol has been targeted as pro-cancerous molecule. At this phase again cholesterol is biphasic. Although cholesterol is essential to construct nuclear membrane and its lipid-rafts; in cancer tumour cells, cholesterol is not under the control of intracellular feedback regulation and gets accumulated within cell nucleus by crossing nuclear membrane and promoting cell proliferation. In precancerous stage, the immune cells also die because of the lack of requisite concentration of intracellular and intranuclear cholesterol pool. The existence of cholesterol within the cell nucleus has been found in the nuclear membrane, epichromosomal location and nucleoplasm. The existence of cholesterol in the microdomain of nuclear raft has been reported to be linked with gene transcription, cell proliferation and apoptosis. Hydrolysis of cholesterol esters in chromosomal domain is linked with new cell generation. Apparently, Cholesterol is now a prelate in cell nucleus too ------ A serendipity in cellular haven.

Adrenal cortex hypoxia modulates aldosterone production in heart failure

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated β3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced β3-adrenergic receptor expression. Hypoxia-mediated β3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Targeting Foam Cell Formation in Atherosclerosis: Therapeutic Potential of Natural Products.

Foam cell formation and further accumulation in the subendothelial space of the vascular wall is a hallmark of atherosclerotic lesions. Targeting foam cell formation in the atherosclerotic lesions can be a promising approach to treat and prevent atherosclerosis. The formation of foam cells is determined by the balanced effects of three major interrelated biologic processes, including lipid uptake, cholesterol esterification, and cholesterol efflux. Natural products are a promising source for new lead structures. Multiple natural products and pharmaceutical agents can inhibit foam cell formation and thus exhibit antiatherosclerotic capacity by suppressing lipid uptake, cholesterol esterification, and/or promoting cholesterol ester hydrolysis and cholesterol efflux. This review summarizes recent findings on these three biologic processes and natural products with demonstrated potential to target such processes. Discussed also are potential future directions for studying the mechanisms of foam cell formation and the development of foam cell-targeted therapeutic strategies.

Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier.

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.

Morbid obesity-related changes in the expression of lipid receptors, transporters, and HSL in human sperm.

To study the location and expression of receptors (SR-BI/CLA-1, SR-BII, and LDLr) and transporter (ABCA1) involved in uptake and efflux of cholesterol in human spermatozoa and assess whether obesity alters its location/expression and whether this could be related to infertility. Observational study. None PATIENT(S): Ten controls and 20 obese patients. Anthropometric parameters. Serum and semen samples were collected. Spermatozoon concentration, immunolocalization, and protein expression in semen. Spermatozoon concentration and motility was decreased in morbidly obese patients. SR-BI/CLA-1, SR-BII, LDLr, and ABCA1 are located in the spermatozoon cell membrane and the localization does not change between obese patients and controls. Control spermatozoa showed high SR-BI expression, and less expression for the rest of the receptors analyzed, indicating that SR-BI/CLA-1 is relevant in human spermatozoon cholesterol uptake/efflux. On the contrary, spermatozoa of obese patients showed less SR-BI/CLA-1 expression than controls, and more intense positive staining for SR-BII, LDLr, and ABCA1. Finally, human sperm expresses the 130- and 82-kDa hormone-sensitive lipase (HSL) isoforms. The 130-kDa isoform is expressed in the control sperm, and the expression disappears in the obese patients. The presence of lipid receptors/transporters and HSL in human spermatozoa suggests their role in the process of maturation/capacitation. The changes in the expression of lipid receptors/transporters and the lack of the 130-kDa HSL isoform in obese patients prevent the hydrolysis of cholesterol esters internalized by these receptors, and favor their accumulation in the cytoplasm of the spermatozoa that could contribute to lipotoxicity and infertility.

Cholesteryl Ester Storage Disease – A Rare Presentation

Cholesteryl Ester Storage Disease (CESD) is a rare genetic disease characterized by accumulation of cholesteryl esters and triglycerides in many tissues due to deficiency of Lysosomal Acid Lipase (LAL/ LIPA) enzyme, which is essential for hydrolysis of triglycerides and cholesterol esters in lysosomes.1 The diagnosis is indicated by abnormal lipid profile, deposition of cholesterol crystals in internal organs and reduced acid lipase activity in leukocytes. Here we report a 16 year-old girl who presented with repeated episodes of hepatic encephalopathy with onset of first symptom at 9 years of age with history of consanguinity of marriage between parents. On examination, we found hepatosplenomegaly. Laboratory examination showed abnormal lipid profile and reduced activity of acid lipase enzyme in leukocytes. After exclusion of other possible pathological conditions and on the basis of lab criteria, we diagnose the case as Cholesteryl Ester Storage Disease (CESD).J MEDICINE JUL 2018; 19 (2) : 130-132

Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier Variant.

Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism. We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell-derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion. A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.

Identification of Lead Molecules in Garcinia mangostana L. Against Pancreatic Cholesterol Esterase Activity: An In Silico Approach.

Hypercholesterolemia is one of the major risk factors for the development and progression of atherosclerosis. Hence, inhibitors of cholesterol absorption have been investigated for decades as a strategy to prevent and treat cardiovascular diseases associated with hypercholesterolemia. Cholesterol esterase (CEase) in pancreatic juice plays a vital role in the hydrolysis of dietary cholesterol esters to cholesterol and fatty acids. Since inhibition of CEase might lead to a reduction of cholesterol absorption, an attempt is made in this study to identify lead molecules of Garcinia mangostana by the in silico approach. The study employed software applications viz., AutoDock 4.2 and GOLD Suite of Programs 5.2. The study revealed the efficacy of three compounds viz., epicatechin, euxanthone, and 1,3,5,6-tetrahydroxy-xanthone, which exhibited least binding energy in AutoDock and moderate scoring in GOLD. The molecular properties as well as biological activity of these three compounds were predicted by molinspiration prediction tool. The results show the crucial role of polyphenolic compounds to limit the activity of CEase. The drug-likeness prediction revealed the prospects of the identified lead molecules as potential drug candidates.

The Role of Macrophage Lipophagy in Reverse Cholesterol Transport.

Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.

Ursolic acid enhances macrophage autophagy and attenuates atherogenesis

Macrophage autophagy has been shown to be protective against atherosclerosis. We previously discovered that ursolic acid (UA) promoted cancer cell autophagy. In the present study, we aimed to examine whether UA enhances macrophage autophagy in the context of atherogenesis. Cell culture study showed that UA enhanced autophagy of macrophages by increasing the expression of Atg5 and Atg16l1, which led to altered macrophage function. UA reduced pro-interleukin (IL)-1β protein levels and mature IL-1β secretion in macrophages in response to lipopolysaccharide (LPS), without reducing IL-1β mRNA expression. Confocal microscopy showed that in LPS-treated macrophages, UA increased LC3 protein levels and LC3 appeared to colocalize with IL-1β. In cholesterol-loaded macrophages, UA increased cholesterol efflux to apoAI, although it did not alter mRNA or protein levels of ABCA1 and ABCG1. Electron microscopy showed that UA induced lipophagy in acetylated LDL-loaded macrophages, which may result in increased cholesterol ester hydrolysis in autophagolysosomes and presentation of free cholesterol to the cell membrane. In LDLR(-/-) mice fed a Western diet to induce atherogenesis, UA treatment significantly reduced atherosclerotic lesion size, accompanied by increased macrophage autophagy. In conclusion, the data suggest that UA promotes macrophage autophagy and, thereby, suppresses IL-1β secretion, promotes cholesterol efflux, and attenuates atherosclerosis in mice.