Cholesterol Ester Metabolism Research Articles

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored.MethodsThe expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured.ResultsResults showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1.ConclusionsNCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Untargeted lipidomics-based study reveals the treatment mechanism of Qingxue Bawei tablets on atherosclerotic in ApoE−/− mice

Qingxue Bawei (QXBW) tablets, a Mongolian medicine prescription, have proved to possess good lipid-lowering and antihypertensive effects in previous studies. However, the therapeutic effects and potential mechanisms of QXBW tablets on atherosclerosis (AS) have not been well studied yet. This study aimed to investigate the potential liver-protective mechanism of QXBW tablets on AS mice by hepatic lipidomics analysis. After 10 weeks of administration, serum and liver were collected for biochemical, histopathological, and lipid metabolomics analysis to evaluate the efficacy of the QXBW tablets on high-fat diet (HFD) induced mice. The experimental results indicated that QXBW tablets could ameliorate liver injury and inflammatory response in AS mice. Liver lipid data from different groups of mice were collected by UPLC-Q-Orbitrap-MS, and a total of 22 potential biomarkers with significant differences between the model and control groups were identified finally, of which 16 potential biomarkers were back-regulated after the QXBW tablets intervention. These 22 potential differential metabolic markers were mainly involved in glycerolipid metabolism, glycerophospholipid metabolism, and cholesterol ester metabolism pathways. The results of this study showed that serum inflammatory factors, liver function indices, and lipid metabolism disorders were positively alleviated in AS mice after QXBW tablets treatment.

Sea Cucumber Phospholipids Regulate Cholesterol Metabolism in High-Fat Diet-Induced ApoE−/− Mice

BackgroundSea cucumber phospholipids, marine-derived lipids with high nutritional functions, have been proven to exhibit various biological activities. However, it is unclear how sea cucumber phospholipids regulate cholesterol (Chol) metabolism in atherosclerosis. ObjectivesThis study aimed to investigate the effects and mechanism of sea cucumber phospholipids on the metabolism of Chol and cholesterol esters (CE) in ApoE−/− mice, including plasmenyl phosphatidylethanolamine (PE-P) and plasmanyl phosphatidylcholine (PC-O). MethodsMale ApoE−/− mice were fed with Chow diet, high-fat diet (HFD), and HFD supplemented with PC-O or PE-P, respectively. We integrated a targeted lipidomics strategy to classify and compare the cholesteryl esters according to their fatty acid types, and then analyzed the individual cholesteryl ester molecular species in the liver and serum of mice. Furthermore, the Chol metabolism-related genes and pathways were analyzed in high-fat-induced ApoE−/− mice. ResultsBiochemical analysis showed that sea cucumber phospholipids significantly inhibit the generation of arterial plaque in ApoE−/− mice. Compared with the HFD group, PE-P significantly reduced the contents of SFA-CE and MUFA-CE in mice liver (P < 0.05), whereas PC-O particularly upregulated CE20:5 and CE22:6 in the serum of mice (P < 0.001). Furthermore, PC-O and PE-P inhibited the Chol synthesis pathway (Cyp7A1 and Cyp27A1), as well as promoted the catabolism of Chol by upregulating gene expressions of bile acid synthesis (Abcb11) and lysosomal activity (Lamp1), respectively. ConclusionsSea cucumber phospholipids could ameliorate the atherosclerosis symptoms by regulating Chol metabolism. J Nutr 20xx;x:xx.

Deficiência de Lipase Ácida Lisossômica (LAL): Análise Enzimática em Papel-filtro como Ferramenta Diagnóstica em Paciente com Diagnóstico Prévio de Doença de Niemann-Pick tipo C

Lysosomal acid lipase deficiency (LAL-D) is a lysosomal storage disorder involved in cholesterol ester metabolism. It is a poorly understood genetic cause of cirrhosis, dyslipidemia and premature atherosclerotic disease in children and adults. As the manifestations of LAL-D may resemble those observed in other more common diseases, delayed diagnosis is not uncommon. Wolman disease is an early-onset LAL-D phenotype that is usually fatal in the first year of life, but the most common form of the disease may occur at all ages. Recently, enzyme replacement therapy (ERT) for LAL-D has become available. Case report: A 12- year-old male patient was referred for hepatomegaly. Initially diagnosed as having Niemann-Pick type C disease (NPC), it did not present confirmatory mutations of this disease. Later, with enzymatic dosage of lysosomal acid lipase on filter paper, the diagnosis was redirected to LAL-D (with confirmation in enzymatic dosage in leukocytes and fibroblasts). Discussion: Although LAL-D is considered a rare and uncommon cause of liver disease, recent studies point to a higher prevalence of this disease. Early diagnosis is essential, since specific treatment for this disease is already available. About half of patients with LAL-D die before age 21 in the absence of adequate treatment

Deficiência de Lipase Ácida Lisossômica (LAL): análise enzimática em papel-filtro como ferramenta diagnóstica em paciente com diagnóstico prévio de doença de Niemann-Pick tipo C

INTRODUCTION: Lysosomal acid lipase deficiency (LAL-D) is a lysosomal storage disorder involved in cholesterol ester metabolism. It is a poorly understood genetic cause of cirrhosis, dyslipidemia and premature atherosclerotic disease in children and adults. As the manifestations of LAL-D may resemble those observed in other more common diseases, delayed diagnosis is not uncommon. Wolman disease is an early-onset LAL-D phenotype that is usually fatal in the first year of life, but the most common form of the disease may occur at all ages. Recently, enzyme replacement therapy (ERT) for LAL-D has become available. CASE REPORT: A 12-year-old male patient was referred for hepatomegaly. Initially diagnosed as having Niemann-Pick type C disease (NPC), it did not present confirmatory mutations of this disease. Later, with enzymatic dosage of lysosomal acid lipase on filter paper, the diagnosis was redirected to LAL-D (with confirmation in enzymatic dosage in leukocytes and fibroblasts). DISCUSSION: Although LAL-D is considered a rare and uncommon cause of liver disease, recent studies point to a higher prevalence of this disease. Early diagnosis is essential, since specific treatment for this disease is already available. About half of patients with LAL-D die before age 21 in the absence of adequate treatment.

Transcriptome and lipidomics profiling of F2 generation female Chinese sturgeon (Acipenser sinensis) in response to different arachidonic acid diets

Our previous study indicated that dietary arachidonic acid (ARA) inclusion levels higher than 1% in the diet play key roles in ARA deposition, steroid and vitellogenin (Vtg) synthesis in F2 generation female Chinese sturgeon, but the regulatory mechanism remains to be elucidated. As follow-up to our previous study (Aquaculture Reports, 21, 100818), the study further aimed to investigate the possible mechanisms of the effects of dietary ARA promoted steroid hormone synthesis in the ovary by transcriptomics and in the serum by lipidomics. RNA-Seq and lipidomics technology were employed to profile the ovary transcriptome and serum lipidomics after feeding four ARA supplemented diets (named CT, LT, MT and HT), and the differentially expressed genes (DEGs) and key differential metabolites were identified. A total of 1018 DEGs, 853 DEGs, 2680 DEGs, 303 DEGs, 1185 DEGs and 409 DEGs were identified in the LT versus CT, MT versus CT, HT versus CT, MT versus LT, HT versus LT and HT versus MT comparisions, respectively. Gene ontology showed that the DEGS between the ARA-supplemented diet group and the unsupplemented diet group were enriched in regulation of cellular process, development process and reproduction. Pathways concerning steroid hormone biosynthesis and cholesterol biosynthesis in lipid metabolism and ovarian steroidogenesis as well as the nuclear-initiated estrogen signaling pathway in endocrine system were enriched. Furthermore, weighted correlation network analysis (WGCNA) identified six hub genes that regulate gonad development and cholesterol synthesis, namely, mitogen-activated protein kinase (MAPK14), cyclin-dependent kinase 2 (CDK2), heat shock protein 8 (HSPA8), two forkhead boxes (FOXP3 and FOXJ3) and cytochrome P450 sterol 14α-demethylase (CYP51A1). Collectively, the molecular mechanism by which ARA regulates the synthesis of steroids, may be related to enhanced cholesterol ester metabolism and facilitation of steroidogenesis gene transcription, which ultimately benefits ovary development in F2 generation Chinese sturgeon.

Lysosomal Acid Lipase, a Novel Biomarker for Hepatic Fibrosis

Background: Hepatic fibrosis (HF) can be easily developed irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in cholesterol ester metabolism and is considered a biomarker for liver cirrhosis. However, the association between LAL and HF, as well as the role of LAL in HF development are poorly understood. In this study, we aimed to determine whether LAL served a novel biomarker for HF development and reveal its underlying mechanism. Methods: The expression of LAL was surveyed in the liver tissues of CCl 4 and high-fat diet (HFD)-induced HF animals. Alpha smooth muscle actin level, collagen and Gata family expression was analyzed by Real-time PCR and Western blot. The identification of putative transcription factor binding sites in the DNA sequences of Lipa was located by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed using Desmond to explore the protein-ligand interaction. Findings: Our results showed that LAL expression was reduced in TGF-β1-induced HSC-T6 cells and liver tissues. Gata 3, a member in the Gata family that transcribes Lipa, was involved in the pathological process of HF. More importantly, morroniside, an active compound of Cornus officinalis Sieb. et Zucc., regulated the GATA3/Lipa axis, inhibited HSCs activation and enhanced extracellular matrix (ECM) degradation. These findings, for the first time, suggest a novel biomarker for HF, which is also a molecular target of morroniside for its anti-HF effects. Interpretation: This study provides strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for HF prevention and treatment. Funding: This work was supported by the Basic and Applied Basic Research Fund Project of Guangdong Province (NO.2019A1515110202), the China Postdoctoral Science Foundation (NO.2020M672604), The National Natural Science Foundation of China (82074019 and 81930114), Characteristic innovation projects of universities in Guangdong Province (2020KTSCX030), Guangdong Key Laboratory for Translational Cancer research of Chinese Medicine (2018B030322011), Key-Area Research and Development Program of Guangdong Province (No. 2020B1111100004). Research Grant Council of HKSAR (HKBU-22103017-ECS), Innovation & Technology Commission (PRP/015/19FX), National Natural Science Foundation of China (SCM-2016-NSFC-003) and Natural Science Foundation of Guangdong Province (2018A0303130122). Declaration of Interest: None to declare. Ethical Approval: All animals received humane care, and the experimental protocol was approved by the Committee of Laboratory Animals and was performed according to institutional guidelines.

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases −3 and −4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition

Background and aimsThe transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). MethodsWe used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). ResultsWe showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4−/− BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. ConclusionsOur results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.

Abstract 4378: The association of enzymes related to cholesterol esters metabolism with hepatocellular carcinoma

Abstract Objectives: The roles of cholesteryl esters (CE) in the pathogenesis of hepatocellular carcinoma (HCC) have not been completely elucidated. This study is aimed to investigate differences in the expression level of enzymes involving in synthesis and degradation of CEs between benign hapatic tissues (BHT) and HCC, and between male and female patients from African American (AA) and Caucasian American (CA). Methods: TMAs were constructed with 97 BHT and 91 HCC samples from patients enrolled in the University of Mississippi Medical Center from 2010 to 2017. Immunochemistry (IHC) was performed on the TMAs for enzymes involved in CE synthesis (acyl CoA:cholesterol acyltransferase 1, ACAT1 and acyl CoA:cholesterol acyltransferase 2, ACAT2), and in CE degradation (lysosomal acid lipase, LAL and neutral cholesterol ester hydrolase 1, NCEH1) following the manufacturer’s instructions. The expression level for each enzyme was expressed as IHC score calculated with a scoring system. Two-tailed Student’s t-test was used to compare the difference in the expression level of each enzyme between two groups. Significant p value was set at p&amp;lt;0.05. Results: The IHC score of ACAT2 was significantly higher in HCC than in BHT in all population (6.21±2.42 vs (4.89.4±2.40, P=0.0006) and in stratified male population (6.15±2.33 vs 5.08±2.32, p=0.046), but not in stratified women, AA and CA populations. There was no significant difference in IHC score for ACAT1 between HCC and BHT in all population and populations stratified by race and gender. The IHC score of LAL was significantly higher in HCC as compared to BHT in all population (3.57±2.31 vs 2.40±1.30, P =0.0001) and in male population (3.33±2.01 vs 2.39±1.16, p=0.015), but not in stratified women, AA or CA population. The IHC score of NCEH1 was not significantly different between HCC and BHT in all studied population and population stratified by gender. However, it was disparate between AA and CA populations: The IHC score of NCEH1 was significantly lower in HCC than in BHT in AA population (5.59±2.04 vs 6.66±1.74, p=0.031) but it was higher in HCC than in BHT in CA population (6.84±1.96 vs 7.12±1.70, p&amp;gt;0.05). Conclusions: These results suggest that alterations in the expression levels of enzymes involving CE metabolism are associated with pathogenesis, progression and disparities in race and gender of HCC. This study also suggest that ACAT2 predominates CE synthesis as compared to ACAT1, and that LAL plays major role in CE degradation as compared to NCEH1 in association with HCC. Citation Format: Zhirong Liu, Akram Shalaby, Charu Subramony, Jinghe Mao, Xinchun Zhou. The association of enzymes related to cholesterol esters metabolism with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4378.

Lysosomal acid lipase promotes cholesterol ester metabolism and drives clear cell renal cell carcinoma progression.

Clear cell renal cell carcinoma (ccRCC) is characterized histologically by accumulation of cholesterol esters, cholesterol and other neutral lipids. Lysosomal acid lipase (LAL) is a critical enzyme involved in the cholesterol ester metabolism. Here, we sought to determine whether LAL could orchestrate metabolism of cholesterol esters in order to promote ccRCC progression. Quantitative reverse-transcription PCR and western blots were conducted to assess the expression of LAL in human ccRCC tissues. We analysed the relationship between LAL levels and patient survival using tissue microarrays. We used cell proliferation assays, colony formation assays, cell death assays, metabolic assays and xenograft tumour models to evaluate the biological function and underlying mechanisms. LAL was up-regulated in ccRCC tissue. Tissue microarray analysis revealed higher levels of LAL in advanced grades of ccRCC, and high LAL expression indicated lower patient survival. Suppressing LAL expression not only blocked the utilization of cholesterol esters but also impaired proliferation and cellular survival. Furthermore, immunohistochemistry staining showed that LAL expression was correlated with Akt phosphorylation. Suppressing LAL expression decreased the phosphorylation level of Akt and Src and reduced the level of 14,15-epoxyeicosatrienoic acids in ccRCC cells. Supplement of 14,15-epoxyeicosatrienoic acids rescued proliferation in vitro and in vivo. LAL promoted cell proliferation and survival via metabolism of epoxyeicosatrienoic acids and activation of the Src/Akt pathway.

Common functional variant LIPA T16P affects HDL composition among patients with heterozygous familial hypercholesterolemia

Aim: Lysosomal acid lipase (LAL), coded by LIPA gene, has critical role in cholesterol-ester metabolism in LDL receptor pathway. LIPA is a susceptibility gene for CAD in GWAS studies, as LIPA SNP with increased expression shows increased CAD risk. LAL activates SREBP and LXR, and LAL deficiency shows non-HDL overproduction and HDL reduction. LAL showed lipotoxicity in pancreas ß-cells. However, little is known about physiological LAL activity.

Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2–4X) and HDL (5–10X). Halftimes for [14C]CE hydrolysis were 3.0±0.2, 4.4±0.6 and 5.4±0.7h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~50% by 8h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.

Abstract 326: Streptococcal Serum Opacity Factor Product, Cholesteryl Ester Rich Particles, Induce Cholesterol Ester Metabolism and Bile Acid Secretion after Uptake by Human Huh7 Hepatocytes

Plasma concentrations of HDL-cholesterol (C) negatively correlate with atherosclerotic cardiovascular disease. Nevertheless, current evidence suggests that this correlation is not axiomatic and that some forms of HDL are dysfunctional and atherogenic. Interventions that raise HDL-C have not been uniformly successful suggesting that mechanisms by which HDL-C is increased determine its anti atherogenic potency. Additionally, mice overexpressing the HDL receptor, SR-BI, have lower plasma HDL-C levels and less atherosclerosis. Thus, new strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. Serum opacity factor (SOF) disrupts HDL and forms three products, including a cholesteryl ester-rich microemulsion (CERM) containing apo E and the CE of ~400,000 HDL particles. Hepatic CE uptake in Huh7 cells is faster when delivered by CERM than by HDL, and cleared both in vitro and in vivo, in part, via the LDL-receptor (LDLR). We investigated the therapeutic potential of SOF in promoting RCT by comparing the final RCT steps, hepatic uptake, CE metabolism to cholesterol and bile salts, and secretion, of [ 14 C]-CE-HDL, -CERM and [[Unable to Display Character: &amp;#8211;]]LDL. Cells were pulse-labeled for 2 h with 20-50 ug/mL HDL protein (6-17 ug/mL [ 14 C]-CE), or the CE-equivalent as CERM or LDL, and chased for 0, 2 or 6 h. Cells, pulse media and chase media were analyzed for sterols by beta-counting, TLC and solvent partitioning. [ 14 C]CE uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [ 14 C]CE hydrolysis were 3.0 ± 0.2, 4.4 ± 0.6 and 5.4 ± 0.7 h respectively for HDL, CERM and LDL-CE. Bile acids in cells after uptake from HDL or CERM were low, &lt;0.2% of total sterol but higher in cells after uptake from LDL, 4.2 ± 2.2 % of total sterol ( p = 0.03 ) at the 2 h chase time. The fraction of sterols secreted as bile acids was comparable for all three donor particles. After the 2 h chase, ~40% of the secreted sterols were bile acids, and after 6 h, ~ 50% were bile acids. These ratios were the same for cells treated with 14 C-CE-HDL, CERM or LDL. Thus, the rates of hepatic metabolism of CERM-CE to free cholesterol, to bile acids and secretion are intermediate between those for HDL- and LDL-CE, supporting the therapeutic potential of SOF as a promoter of RCT.

Selenium bioavailability from shrimps (Penaeus vannamei Boone) and its effect on the metabolism of phospholipid and cholesterol ester

We evaluated selenium bioavailability from shrimps, and its effect on the metabolism of phospholipid (PL) and cholesterol ester (CE) using a mouse model. Experimental mice were categorized into 4 groups received different doses of Se from shrimps for 4 consecutive weeks. It was calculated that the bioavailability of Se from shrimps was ∼86–88% based on the apparent absorption. Comparing with control group, Se content in tissues and Glutathione peroxidase (GPx) activities in liver and blood increased significantly from selenium supplemented groups. The lipidomics analysis showed that there was no difference in detectable total species of PLs and CE in intestine samples, but the total content and the relative percentage of PLs and CE increased proportionally to the dose of Se intake. The results indicate that selenium supplementation lead to an increase in tissue Se concentration and GPx activity as well as changes in intestine PLs and CE, species and abundance of individual lipid fractions.

Physiological Difference in Autophagic Flux in Macrophages From 2 Mouse Strains Regulates Cholesterol Ester Metabolism

DBA/2 apoE(-/-) mice have ≈10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells

In late-stage atherosclerosis, much of the cholesterol in macrophage foam cells resides within enlarged lysosomes. Similarly, human macrophages incubated in vitro with modified LDLs contain significant amounts of lysosomal free cholesterol and cholesteryl ester (CE), which disrupts lysosomal function similar to macrophages in atherosclerotic lesions. The lysosomal cholesterol cannot be removed, even in the presence of strong efflux promoters. Thus, efflux of sterol is prevented. In the artery wall, foam cells interact with triglyceride-rich particles (TRPs) in addition to modified LDLs. Little is known about how TRP metabolism affects macrophage cholesterol. Therefore, we explored the effect of TRP on intracellular CE metabolism. Triglyceride (TG), delivered to lysosomes in TRP, reduced CE accumulation by 50%. Increased TG levels within the cell, particularly within lysosomes, correlated with reductions in CE content. The volume of cholesterol-engorged lysosomes decreased after TRP treatment, indicating cholesterol was cleared. Lysosomal TG also reduced the cholesterol-induced inhibition of lysosomal acidification allowing lysosomes to remain active. Enhanced degradation and clearance of CE may be explained by movement of cholesterol out of the lysosome to sites where it is effluxed. Thus, our results show that introduction of TG into CE-laden foam cells influences CE metabolism and, potentially, atherogenesis.-Ullery-Ricewick, J. C., B. E. Cox, E. E. Griffin, and W. G. Jerome. Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells.

Hormone-sensitive Lipase Expression and IHC Localization in the Rat Ovary, Oviduct, and Uterus

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.