Neutral Cholesteryl Ester Hydrolase Research Articles

Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer.

In clinically operable pancreatic cancer, regions distant from malignant cells already display proteomic changes related to lipid transport and metabolism that affect prognosis and may be pharmacologically targeted.

Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases

ObjectivesLysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2. MethodsWe performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2. ResultsPharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes. ConclusionsOur findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 μM.

Bioactive components to inhibit foam cell formation in atherosclerosis.

The production of lipid-laden cells in macrophages after significant ingestion of oxidized low-density lipoprotein is considered the most critical phase in the creation of atherosclerotic lesions, which is known as foam cell formation. Targeting foam cell development to find a potential therapeutic strategy for the management of atherosclerosis has yielded numerous promising outcomes. Multiple variables influence foam cell growth, including scavenger receptor expression, cholesterol transporter expression acyl CoA: cholesterol acyltransferase activity, and neutral cholesteryl ester hydrolase activity. Plants used during herbal therapy have been shown to assist with a variety of ailments. In this study, we found medicinal plants and their bioactive components suppress foam cell formation in a variety of ways; some inhibit cholesterol transporter and lectin-like oxidized low-density lipoprotein receptor-1 upregulation, while others inhibit the function of acyl CoA: cholesterol acyltransferase activity, and neutral cholesteryl ester hydrolase activity. Recent study findings related to the synthesis of the new active component from plant sources by focusing on the typical process involved in the generation of foam cells. We're also looking at using a cellular target-based therapeutic approach to generate novel plant-based medications for the cure of atherosclerosis.

Abnormal histidine metabolism promotes macrophage lipid accumulation under Ox-LDL condition

Distinct macrophage populations exert highly heterogeneity and perform various functions, among which, a crucial function of lipid metabolism is highlighted. However, the role of histidine metabolism disorder in macrophage lipid metabolism remains elusive. Addressed this question, we sorted and cultured the bone marrow-derived macrophages (BMDMs) of histidine decarboxylase (Hdc) knockout (Hdc−/-) mice with an in vitro oxidized low-density lipoprotein (ox-LDL) model, and detected the intracellular lipids by Oil Red O staining as well as lipid probe staining. Astemizole, a canonical and long-acting histamine H1 receptor (H1R) antagonist, was applied to elucidate the impact of antagonizing the H1R-dependent signaling pathway on macrophage lipid metabolism. Subsequently, the differential expressed genes were screened and analyzed in the bone marrow-derived CD11b+ immature myeloid cells of Hdc−/- and Hdc+/+ mice with a high fat diet by the microarray study. The expression levels of cholesterol metabolism-related genes were examined by qRT-PCR to explore underlying mechanisms. Lastly, we used a high-sensitivity histidine probe to detect the intracellular histidine in the BMDMs after oxidative stress. The results revealed that histidine metabolism disorder and histamine deficiency aggravated lipid accumulation in the ox-LDL-treated BMDMs. The expression level of H1R gene in the BMDMs was down-regulated after ox-LDL stimulation. The disruption of the H1R-dependent signaling pathway by astemizole further exacerbated ox-LDL-induced lipid deposition in the BMDMs partly by up-regulating scavenger receptor class A (SR-A) for lipid intake, down-regulating neutral cholesteryl ester hydrolase (nCEH) for cholesterol esterification and down-regulating ATP-binding cassette transporters A1 (ABCA1) and ABCG1 for reverse cholesterol transport. The intracellular histidine increased under ox-LDL condition, which was further increased by Hdc knockout. Collectively, these results partially reveal the relationship between histidine metabolism and lipid metabolism in the BMDMs and offer a novel strategy for lipid metabolism disorder-associated diseases.

Macrophage-mediated cholesterol handling in atherosclerosis.

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

Pioglitazone reduces lipid droplets in cholesterolosis of the gallbladder by increasing ABCA1 and NCEH1 expression.

As a cholesterol-induced metabolic disease, cholesterolosis of the gallbladder is often resected clinically, which could lead to many complications. The histopathology of cholesterolosis is due to excessive lipid droplet accumulation in epithelial and subcutaneous tissues. The main components of lipid droplets are cholesterol esters (CEs). Removal of CEs from gallbladder epithelial cells (GBECs) is very important for maintaining intracellular cholesterol homeostasis and for treating cholesterol-related diseases. In this study, pioglitazone was used to reduce intracellular CEs. To further elucidate the mechanism, cholesterolosis GBECs were treated with pioglitazone, 22-(R)-hydroxycholesterol (a liver X receptor α (LXRα) agonist), or peroxisome proliferator-activated receptor gamma (PPARγ) siRNA. Western blotting for PPARγ, LXRα, ATP-binding cassette transporter A1 (ABCA1), and neutral cholesteryl ester hydrolase 1 (NCEH1) was performed. At length, cholesterol efflux to apoA-I was measured, and oil red O staining was used to visualize lipid droplet variations in cells. In conclusion, we observed that pioglitazone increased ABCA1 expression in an LXR-dependent manner and NCEH1 expression in an LXRα-independent manner, which mobilized CE hydrolysis and cholesterol efflux to reduce lipid droplet content in cholesterolosis GBECs. Our data provide a plausible alternative to human gallbladder cholesterolosis.

Critical role of neutral cholesteryl ester hydrolase 1 in cholesteryl ester hydrolysis in murine macrophages

Hydrolysis of intracellular cholesteryl ester (CE) is the rate-limiting step in the efflux of cholesterol from macrophage foam cells. In mouse peritoneal macrophages (MPMs), this process is thought to involve several enzymes: hormone-sensitive lipase (Lipe), carboxylesterase 3 (Ces3), neutral CE hydrolase 1 (Nceh1). However, there is some disagreement over the relative contributions of these enzymes. To solve this problem, we first compared the abilities of several compounds to inhibit the hydrolysis of CE in cells overexpressing Lipe, Ces3, or Nceh1. Cells overexpressing Ces3 had negligible neutral CE hydrolase activity. We next examined the effects of these inhibitors on the hydrolysis of CE and subsequent cholesterol trafficking in MPMs. CE accumulation was increased by a selective inhibitor of Nceh1, paraoxon, and two nonselective inhibitors of Nceh1, (+)-AS115 and (-)-AS115, but not by two Lipe-selective inhibitors, orlistat and 76-0079. Paraoxon inhibited cholesterol efflux to apoA-I or HDL, while 76-0079 did not. These results suggest that Nceh1 plays a dominant role over Lipe in the hydrolysis of CE and subsequent cholesterol efflux in MPMs.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Abstract 18385: Serum Amyloid A is an Evolutionary Conserved Apolipoprotein That Functions to “Fine Tune” Reverse Cholesterol Transport During the Acute-Phase Response

In response to severe injury, the acute-phase protein, serum amyloid A (SAA) can replace apoA-I as the major apo-protein of HDL. The inclusion of SAA in HDL changes a number of HDL's properties. Our data indicates that SAA's presence reduces HDL's capacity to accept free cholesterol. SAA also increases HDL's binding affinity for macrophages and is dependent on the cell surface glycosaminoglycan, heparin sulfate (HS). SAA contains two HS binding sites and HS binding to SAA can cause it to dissociate from HDL, leaving behind lipid poor apoA-I rich oligomers, with enhanced cholesterol accepting potential. Under "amyloidogenic" condition this dissociated SAA can be readily converted into amyloid fibrils by macrophages and our data suggests this occurs largely on the cell surface. SAA can also be taken up by macrophages and once in the cytoplasm we have shown that it can stimulate neutral cholesteryl ester hydrolase (nCEH) and inhibit acetyl-coenzyme A acetyltransferase (ACAT) activities, respectively, with the net effect of mobilizing stored cholesteryl ester and making it available for export via the ABCA1/ABCG1 transporters. Under experimental conditions SAA can promote the export of over 70% of the cholesterol loaded into macrophages within 24 h. SAA's activities complement those of apoA-I/HDL by increasing the pool of exportable (de-esterified) cholesterol for efflux to an extracellular cholesterol acceptor like apoA-I/HDL and entry into the reverse cholesterol transport (RCT) pathway. The nCEH and ACAT modulating domains of SAA have been identified and as synthetic peptides have been also demonstrated to be potent promoters and inhibitors of nCEH and ACAT activities, respectively, with purified enzyme preparations, intact cells in culture and in vivo in mice, consistent with an allosteric mechanisms of action. When delivered in liposomes to a mouse apoE knockout model of atherosclerosis, these SAA peptides were shown to be highly effective in the prevention and regression of atherosclerotic lesions. We believe SAA has been evolutionary conserved to aid in the recycling of cellular cholesterol.

Rat Carboxylesterase ES-4 Enzyme Functions as a Major Hepatic Neutral Cholesteryl Ester Hydrolase

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30–40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Estrogen-dependent activation of neutral cholesterol ester hydrolase underlying gender difference of atherogenesis in apoE−/− mice

ObjectiveMechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery. MethodsFirst, male and female apoE−/− mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE−/− mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0μg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17β-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured. ResultsFirst, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II. ConclusionThese results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.

Seeking Novel Targets for Improving In Vivo Macrophage-Specific Reverse Cholesterol Transport: Translating Basic Science into New Therapies for the Prevention and Treatment of Atherosclerosis

Epidemiologic studies have demonstrated that increased high-density lipoprotein cholesterol (HDL-C) is a protective factor against cardiovascular disease. However, the beneficial therapeutic effects of raising HDL-C are proving difficult to confirm in humans. Macrophage-specific reverse cholesterol transport (RCT) is thought to be one of the most important HDL-mediated cardioprotective mechanisms. A new approach was developed to measure in vivo RCT from labeled cholesterol macrophages to liver and feces in mice. Since its original publication, this method has been extensively used to assess the effects of genetic manipulation of pivotal genes involved in HDL metabolism on this major HDL antiatherogenic function in mice. These studies indicate that in vivo macrophage-specific RTC is a strong predictor of atherosclerosis susceptibility compared with steady-state plasma HDL-C levels or other global RCT measurements. This review aims to identify the best molecular targets for improving this HDL antiatherogenic function. Strong evidence supports a positive effect of interventions on macrophage adenosine triphosphate-binding cassette transporter (ABC) A1 and neutral cholesteryl ester hydrolase, apolipoprotein (apo) A-I, apoE, liver scavenger receptor class B type I and ABCG5/G8 on in vivo macrophage-specific RCT and atherosclerosis susceptibility. However, other genetic modifications have yielded conflicting results. Several preclinical studies tested the effects on macrophage-specific RCT in vivo of promising new HDL-based therapeutic agents, which include cholesteryl ester transfer protein inhibitors, apoA-I-directed therapies, liver X receptor and peroxisome proliferator-activated receptor agonists, intestinal cholesterol absorption inhibitors, fish oil and phenolic acid intake, inflammatory modulation and non-nucleoside reverse transcriptase inhibitors. This review also discusses recent findings on the potential effects of these therapeutic approaches on macrophage RCT in mice and cardiovascular risk in humans.

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Insulin Suppresses HDL-Mediated Cholesterol Efflux from Macrophages Through Inhibition of Neutral Cholesteryl Ester Hydrolase and ATP-Binding Cassette Transporter G1 Expressions

We studied the effect of insulin on HDL-mediated cholesterol efflux from macrophages. The potential involvement of cholesteryl ester hydrolysis and membrane cholesterol transport was also addressed. Human monocyte-derived THP-1 cells were developed into macrophages. Cholesterol efflux was measured by incubating macrophages, labeled with [³H]-cholesterol, with HDL for 24 h. The cells were treated with insulin (0-500 nM) for 30 min prior to the addition of HDL. To investigate the molecular mechanisms of the effect of insulin, the expressions of neutral cholesteryl ester hydrolase (nCEH) and ATP-binding cassette transporter (ABC) G1 were analyzed. Insulin inhibited, in a concentration-dependent manner, HDL-mediated cholesterol efflux from macrophages. Insulin also inhibited the enzyme activity of nCEH and its mRNA and protein expression in cells. Insulin also suppressed the expressions of mRNA and protein for ABCG1. Insulin inhibits HDL-mediated cholesterol efflux from macrophages, which may result from the suppression of nCEH and ABCG1 expressions. Our findings show part of the potential molecular mechanism of atherogenesis in type 2 diabetes with hyperinsulinemia.

Subcellular localization of carboxylesterase‐4 in rat liver cells and its role as neutral cholesteryl ester hydrolase

The rat carboxylesterases, ES‐2 (serum esterase), ES‐3 (pI 5.6 esterase), ES‐4 (pI 6.2/6.4 esterase), ES‐10 (pI 6.0/6.1 esterase), and ES‐15 (pI 5.0/5.2 esterase) comprise a multi‐gene family, and function in hydrolysis of a variety of ester‐ and amide‐ containing chemicals to their respective free acids. Using realtime PCR, in the rat hepatic cell line McArdle‐RH7777 (McA) we observed ES‐4 mRNA had the highest level of expression compared to ES‐2 and ES‐3, while ES‐10 was not detectable. The subcellular location of ES‐4 in McA cells was investigated using confocal microscopy. ES4 co‐localized with ER Tracker dye, a maker for the endoplasmic reticulum (ER). Immunohistochemical studies with rat liver demonstrated the presence of ES‐4 within hepatocytes. ES‐4 also functions as a rat hepatic cholesteryl ester hydrolase. McA cells overexpressing ES‐4 showed decreased cholesteryl ester (CE) levels compared to control cells indicating that ES‐4 can hydrolyze CE in intact cells. In cells where ES‐4 levels were suppressed by 75% using siRNA there were increased CE levels. Confocal microscopy, using Oil Red O and ES‐4 antibodies, revealed areas of association of ES‐4 with the CE droplets. The results show ES‐4 is expressed in hepatocytes and specifically in endoplasmic reticulum. ES‐4 is directly associated with CE droplets and functions as cholesteryl ester hydrolase. Supported by NIH‐DK044498.

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

Abstract 5476: Hepatic Over-Expression of Cholesteryl Ester Hydrolase Enhances Cholesterol Elimination and in vivo Reverse Cholesterol Transport

Neutral Cholesteryl ester hydrolase (CEH) mediated hydrolysis of cellular cholesteryl esters (CE) is required not only to generate free cholesterol for efflux from macrophages but also to release FC from lipoprotein delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH will regulate the hydrolysis of lipoprotein derived CE and enhance reverse cholesterol transport (RCT). Adenoviral mediated CEH over-expression led to a significant increase (>3 fold) in bile acid output (see Figure ). To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol loaded and [ 3 H]-cholesterol labeled J774 macrophages were injected intraperitoneally into mice and appearance of [ 3 H]-cholesterol in gall bladder bile and feces over 48 hours was quantified. Mice over-expressing CEH had significantly higher [ 3 H]-cholesterol in bile and feces and it was associated with bile acids. This CEH-mediated increased movement of [ 3 H]-cholesterol from macrophages to bile and feces was significantly attenuated in SR-BI−/−mice. These studies demonstrate that similar to macrophage CEH that rate limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output. This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [3H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [3H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [3H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [3H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.

Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) showed massive accumulation of CE in the liver compared with Lep(ob/ob)/HSL(+/+) mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL(-/-) mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL(-/-) mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL(-/-) mice showed reduced activity of CE hydrolase and contained more CE than those from HSL(+/+) mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.