Neutral Cholesterol Ester Hydrolase Activity Research Articles

Neutral Cholesterol Ester Hydrolases in Macrophages: Still a Matter of Debate

HomeCirculation ResearchVol. 108, No. 10Neutral Cholesterol Ester Hydrolases in Macrophages: Still a Matter of Debate Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBNeutral Cholesterol Ester Hydrolases in Macrophages: Still a Matter of Debate Dagmar Kratky Dagmar KratkyDagmar Kratky Institute of Molecular Biology and Biochemistry Medical University of Graz Graz, Austria E-mail (Kratky) Search for more papers by this author Originally published13 May 2011https://doi.org/10.1161/CIRCRESAHA.111.245829Circulation Research. 2011;108:e13To The Editor:In a recent study, Igarashi et al1 reported knockdown experiments using adenoviral short hairpin RNA constructs targeting human neutral cholesterol ester hydrolase-1 (NCEH1, originally named KIAA1363) and the potential cholesterol ester hydrolase CES1. The authors concluded that “NCEH1 is the only enzyme that requires attention when dealing with neutral cholesterol ester hydrolase activity in human macrophages,” reinforcing similar observations by the same group on murine macrophages.2 This created a controversy,3 because our group demonstrated identical neutral cholesterol ester hydrolase activity in wild-type and Nceh1 knockout mice,4 which argues against a critical role of NCEH1 as cholesterol ester hydrolase. The validity of our observations was questioned5 by claiming that we failed to present “data demonstrating the clean knockout of KIAA1363 of this model by Western and/or Northern blot analyses.” This is at variance with Figure 4F of our report,4 which demonstrated the lack of NCEH1 in macrophages of Nceh1 knockout mice by Western blotting with a specific anti-NCEH1 antibody. Moreover, Figure 1 of the supplemental material of our study confirmed homologous recombination by Southern blot analysis and PCR. Thus, we provided comprehensive information on the generation of the Nceh1 knockout mice and compelling evidence that we used macrophages from a “complete and clean” Nceh1 knockout mouse model for our cholesterol ester hydrolase activity assays.The major conclusion drawn by Igarashi et al1 that “NCEH1 is quantitatively the most important neutral cholesterol ester hydrolase in human macrophages and atherosclerosis” is insufficiently supported by the data presented. Despite an almost complete knockdown of NCEH1 in human monocyte-derived macrophages, neutral cholesterol ester hydrolase activity was reduced by only 50%, which leads us to question the conclusion that “NCEH1 is the only enzyme that requires attention when dealing with neutral cholesterol ester hydrolase activity in human macrophages.” In contrast to NCEH1, CES1 knockdown lacked any reduction in cholesterol ester hydrolase activity. Knockdown of CES1, however, was less efficient, and substantial amounts of CES1 protein were present in CES1-silenced macrophages.The claim that NCEH1 (KIAA1363) is the neutral cholesterol ester hydrolase in human1 and murine2 macrophages, therefore, continues to be at variance with observations made by us4 and others.3Dagmar Kratky Institute of Molecular Biology and Biochemistry Medical University of Graz Graz, Austria E-mail [email protected]Sources of FundingD.K. is supported by the Austrian Science Fund (SFB-LIPOTOX F30, DK-MCD, and P19186) and the Austrian Federal Ministry of Science and Research (GEN-AU project GOLD).DisclosuresNone.FootnotesLetters to the Editor will be published, if suitable, as space permits. They should not exceed 1000 words (typed double-spaced) in length and may be subject to editing or abridgment.

Important Considerations for Evaluating the Data Presented by Igarashi et al

To The Editor: The major conclusion drawn in the article by Igarashi et al,1 that cholesteryl ester hydrolase (gene symbol CES1) does not contribute to neutral cholesterol ester (CE) hydrolase activity, is based on the inability of the authors to detect CE hydrolase activity associated with the cDNA clone that they obtained by PCR amplification and by the inability of the authors to demonstrate change in intracellular CE hydrolytic activity by short hairpin (sh)RNA-mediated knockdown of CES1. The authors have not provided the sequence of the clone obtained and do not unequivocally show its identity to the reported CES1 with established role not only in intracellular CE hydrolysis,2 mobilization,3 and cholesterol efflux4 but also in attenuation of diet-induced atherosclerosis and lesion necrosis.5 It is noteworthy that the sense primer used in this study also shows a 95% homology to human CES4 (17/18 bp match; accession no. NR_003276). Single amino …

Abrogation of neutral cholesterol ester hydrolytic activity causes adrenal enlargement

We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.

The Critical Role of Neutral Cholesterol Ester Hydrolase 1 in Cholesterol Removal From Human Macrophages

Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis. We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. NCEH1 is expressed in human atheromatous lesions, where it plays a critical role in the hydrolysis of CE in human macrophage foam cells, thereby contributing to the initial part of reverse cholesterol transport in human atherosclerosis.

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.

Effect of sex, ovariectomy and female sex hormones on neutral cholesterol ester hydrolase in rat liver.

The effects of sex, ovariectomy and female sex hormones on neutral cholesterol ester hydrolase activity were investigated in rat liver cytoplasm. Hydrolase activity was determined at pH 7.45 with an acetone dispersed cholesterol oleate substrate preparation. Neutral cholesterol esterase activity was higher in male than in female rats and the ovariectomy decreased the enzyme activity. The administration of only estradiol or combined with progesterone to ovariectomized rats for three consecutive days resulted in an increase in esterase activity to the level observed in intact animals. By contrast, the only progesterone resulted in no significant changes of activity. In intact female rats, the short-term treatments of gonadal hormones did not produce any alteration on cholesterol esterase. Examination of the effects of a single dose of estradiol and progesterone for intervals between 1 and 24 h showed that both hormones promoted early and opposite effects on neutral ester hydrolase. Activity was decreased in 3-4 h by the estradiol and was markedly increased throughout 3-4-6 h by the progesterone injection. In vitro, the two female sex hormones behaved as potent inhibitors of the enzyme, competing with the substrate for the active centre of the hydrolase.

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.

Hormone-sensitive lipase deficiency in mouse islets abolishes neutral cholesterol ester hydrolase activity but leaves lipolysis, acylglycerides, fat oxidation, and insulin secretion intact.

Lipids are thought to serve as coupling factors in insulin secretion. Hormone-sensitive lipase (HSL) is expressed in pancreatic beta-cells and could potentially regulate insulin secretion via mobilization of stored triglycerides. Here, we examined the impact of HSL deficiency on fuel metabolism and insulin secretion in mouse islets. Lack of HSL resulted in abrogation of neutral cholesterol ester hydrolase activity, whereas diglyceride lipase activity remained intact. Although glucose stimulates lipolysis in rat islets, elevation of glucose with or without addition of cAMP failed to increase lipolysis in mouse islets regardless of genotype, as indicated by release of glycerol from islets. Storage of lipids, assayed as total acylglycerides, was unaltered in HSL null islets, and oxidation of fatty acids or glucose was not different. The intracellular rise in Ca(2+) triggered by glucose and its subsequent oscillations was unaffected in HSL null islets. Accordingly, insulin secretion in static incubations of islets, in response to fuel- and nonfuel secretagogues, was in no instance significantly different between wild-type and HSL null mice. The lacking impact of HSL deficiency on insulin secretion may be attributed to the failure of insulin secretagogues to stimulate lipolysis. Consequently, a regulatory function of lipid mobilization in insulin secretion in the mouse appears unlikely.

Oxidative tyrosylation of high density lipoproteins impairs cholesterol efflux from mouse J774 macrophages: role of scavenger receptors, classes A and B.

Studies were designed to test whether tyrosylation of high-density lipoprotein (HDL(T)) modifies its metabolic features. HDL(T) was less effective than native HDL in promoting cholesterol efflux from J774-AI macrophages. Cell association with fluorescent HDL(T)-apolipoprotein and the uptake of HDL(T)-[(3)H]cholesteryl hexadecyl ether were enhanced by 50% in comparison with native HDL. In addition, neutral cholesterol ester hydrolase (nCEH) activity in J774-AI, which controls the hydrolysis of cholesteryl ester stores to provide free cholesterol for cellular release, declined in the presence of HDL(T). In vitro displacement experiments revealed the ability of HDL(T) to compete with oxidized and acetylated LDL, known as ligands of scavenger receptor (SR) class B type I/II. Similarly, treatment with a blocking antibody to SR-BI/II reduced the cell association of HDL(T) and native HDL by 50%. The addition of polyinosinic acid, an inhibitor of SR class A, reduced the cell association of HDL(T) without affecting that of native HDL. These findings provide evidence that HDL(T) can compete with modified LDL, bind SR-BI/BII and internalize cholesterol ester. Furthermore, the impaired capacity of HDL(T) in promoting cholesterol efflux from J774-AI was accompanied by diminished nCEH and enhanced recognition by SR-AI/II, which appears to involve the transport of cholesterol into cells.

Glucose-dependent Regulation of Cholesterol Ester Metabolism in Macrophages by Insulin and Leptin

Insulin resistance, obesity, and diabetes are characterized by hyperglycemia, hyperinsulinemia, and hyperleptinemia and are associated with increased risk of atherosclerosis. In an effort to understand how this occurs, we have investigated whether these factors cause disregulation of cholesterol ester metabolism in J774.2 macrophages. Raising glucose levels alone was sufficient to increase uptake of acetylated low density lipoprotein but did not stimulate synthesis of cholesterol esters. In the presence of high glucose, both insulin and leptin increased the rate of cholesterol ester synthesis, although they did not further increase uptake of acetylated low density lipoprotein. However, in the presence of high glucose both insulin and leptin caused a significant increase in the activity of acyl-CoA: cholesterol O-acyltransferase (ACAT) combined with a significant reduction in the level of hormone-sensitive lipase (HSL). Because ACAT is the main enzyme responsible for cholesterol ester synthesis and HSL contributes significantly to neutral cholesterol ester hydrolase activity, this suggests that glucose primes the J774.2 cells so that in the presence of high insulin or leptin they will store cholesterol esters. This contrasts with 3T3-L1 adipocytes, where HSL activity and expression are increased by insulin in high glucose conditions. These findings may provide an explanation for the observation that in conditions characterized by hyperglycemia, hyperleptinemia, and hyperinsulinemia, triglyceride lipolysis in adipocytes is increased while hydrolysis of cholesterol esters in macrophages is decreased, contributing to foam cell formation.

Elimination of Cholesterol Ester from Macrophage Foam Cells by Adenovirus-mediated Gene Transfer of Hormone-sensitive Lipase

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol.

Suppression of Neutral Cholesterol Ester Hydrolase Activity by Antisense DNA of Hormone-Sensitive Lipase

In order to investigate the role of the hormone-sensitive lipase (HSL) gene in the activity of neutral cholesterol ester hydrolase (NCEH) from a molecular perspective, Chinese hamster ovary (CHO) cells were transfected with rat antisense hormone-sensitive lipase cDNA, and three different cell lines, designated as anti-HSL, were established. NCEH activity in anti-HSL cells was reduced to approximately 50% of that in control CHO cells. The concentration of cellular esterified cholesterol increased and the concentration of free cholesterol decreased in the anti-HSL cell lines. These results suggest that the HSL gene has a function of NCEH as well.

Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse

Acute inflammation results in a profound change in the apolipoprotein composition of high density lipoprotein (HDL). Several isoforms of the serum amyloid A (SAA) family, SAA 1 and SAA 2, become major components of HDL. This structural relationship has suggested that acute phase SAA plays some as yet unidentified role in HDL function, possibly related to cholesterol transport, during the course of acute inflammation. Using subcutaneous AgNO 3 to induce a sterile abscess changes in plasma cholesterol and SAA were monitored over the subsequent 144 h. Total plasma cholesterol began to increase within 12 h of the induction of inflammation and reached a peak in 24 h. Thereafter its plasma levels fell returning to normal values by 96–120 h. The bulk of the increase in plasma cholesterol was found in the free cholesterol fraction of HDL. This pattern of cholesterol increase corresponds to the established temporal changes for acute phase SAA (AP-SAA). AP-SAA levels increased within 8 h of the induction of inflammation and reached a peak at 24 h. They began to decrease by 48 h with small quantites still present 120 h later. In concert, but inversely, with the changes in AP-SAA the apoA-I, apoA-II, and apo-E, content of HDL decreased during the AP-SAA increases and increased as AP-SAA levels fell. The plasma appearance of cholesterol from the periphery, and central parts of the inflammatory site was assessed by the use of radiolabelled cholesterol. The peripherally placed cholesterol rapidly reached a peak plasma concentration within 24 h of injection. Cholesterol placed in the central part of the sterile abscess, a site relatively inaccessible to the vasculature required 48 h to reach its peak and was 5-times lower than that placed peripherally. The influence of AP-SAA on neutral cholesterol ester hydrolase (nCEH) activity in mouse liver homogenates, mouse peritoneal macrophage homogenates, and a purified porcine pancreatic enzyme with nCEH activity was also assessed. Following optimization with regard to pH, bile salt concentration, protein concentration and incubation time, mouse peritoneal macrophages had a significantly higher nCEH specific activity than that found in liver (7–8 fold). Purified AP-SAA, assessed over a concentration range of 0–10 μg/ml, enhanced nCEH activity at concentrations above 2 μg/ml. The nCEH activity, regardless of its source, increased by 3–7 fold in the presence of AP-SAA. Equivalent concentrations of apolipoprotein A-I (apo A-I) and bovine serum albumin (BSA) failed to alter the activity of nCEH. The effect of AP-SAA on a purified form of nCEH suggests that AP-SAA may have a direct effect on the activity of this enzyme. The temporal correlation of circulating AP-SAA and plasma cholesterol and the significant stimulation of nCEH by AP-SAA (but not apoA-I or BSA) provides further evidence that AP-SAA plays a role in cholesterol metabolism during the course of acute inflammation.

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.

Androgenic Modulation of Lipid Metabolism at Subcellular Sites in Cholestatic Rats

The present studies examine the modulation of lipid metabolism at subcellular sites in the liver of control and cholestatic rats by a male sex hormone. Subcutaneous injection of testosterone enanthate (5 mg/kg body weight), increased triacylglycerol lipase (TG-lipase) and neutral cholesterol ester hydrolase (CE-hydrolase) activity in endosomes of normal rats. During induced cholestasis, TG-lipase and CE-hydrolase activity in endosomal fractions were decreased compared to those in endosomes of age-matched sham-operated female controls. Following testosterone administration to cholestatic rats, endosomal lipolytic enzyme activity increased to approximate the levels of normal controls. These observations may provide insight into the alterations in lipid metabolism which accompany cholestatic liver disease.

The Hypolipidemic Activity of Metal Complexes of AmineCarboxyboranes in Rodents

The metal complexes of amine-carboxyborane including copper, chromium, zinc, calcium amd cobalt were effective hypolipidemic agents lowering both serum cholesterol and triglyceride levels significantly in mice at 8 mg/kg/day, I.P. after 16 days. The agents reduced acetyl CoA synthetase, ATP-dependent citrate lyase, acyl CoA cholesterol acyl transferase, sn-glycerol-3-phosphate acyl transferase activities of rat liver and small intestinal mucosa after 14 days treatment. The neutral cholesterol ester hydrolase activity was elevated by the agents in both tissues. The metal complexes altered lipid levels in the bile of rats after treatment as well as the bile acid composition after 14 days administration, orally. The agents blocked enterohepatic absorption of cholesterol from rat isolated intestinal loops.

Hypolipidemic Effects of 2‐Furoic Acid in Sprague‐Dawley Rats

2-Furoic acid was shown to be effective in lowering both serum cholesterol and serum triglyceride levels significantly in rats with an elevation of HDL cholesterol level at 20 mg/kg/day orally. LDL receptor activity was reduced in hepatocytes, aorta foam cells, small intestinal epithelium cells and fibroblasts. HDL receptor activity was elevated in the rat hepatocytes and small intestinal cells. These activities were correlated with inhibition of acyl CoA cholesterol acyl transferase activity. Neutral cholesterol ester hydrolase activity was elevated in rat hepatocytes and human fibroblasts. Thus, 2-furoic acid appears to interfere directly with activity of intracellular enzymes rather than affecting high affinity-mediated lipoprotein membrane receptors. In vivo treatment with 2-furoic acid led to reduction in the liver and small intestine ATP dependent citrate lyase, acetyl CoA synthetase, acyl CoA cholesterol acyl transferase, sn-glycerol 3-phosphate acyl transferase, phosphatidylate phosphohydrolase and heparin induced lipoprotein lipase activities. 2-Furoic acid reduced biliary cholesterol levels but the agent increased bile salts which are lithogenic. Acute toxicity studies in mice suggest that the agent has some hepatic toxicity effects. The LD50 was relatively low at 250 mg/kg IP in mice.

Phosphorylation and activation of hormone-sensitive lipase in isolated macrophages

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesterol ester hydrolase activity in macrophages. Incubation of intact WEHI macrophages or mouse peritoneal macrophages leads to phosphorylation of HSL, which is increased by incubation with either dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine or okadaic acid. Correspondingly, these agents also activate neutral cholesterol ester hydrolase activity in intact WEHI cells. Regulation of mobilisation of esterified cholesterol in macrophages may be of antiatherogenic value, which this model system now allows us to investigate further.

Stimulation of a neutral cholesteryl ester hydrolase by cAMP system in P388D 1 macrophages

Cholesteryl ester laden foam cells in atherosclerotic lesions derive, in part, from macrophages. Mobilization of stored cholesteryl esters involves hydrolysis by a neutral cholesteryl ester hydrolase. Incubation of intact P388D 1 macrophages with dibutyryl cAMP in the presence of 1-methyl-3-isobutylxanthine resulted in a dose-dependent increase in neutral cholesteryl ester hydrolase activity of up to 50% (ED 50 = 0.1 mM). Incubation with prostaglandin E 1 in the presence of 1-methyl-3-isobutylxanthine also increased neutral cholesterol ester hydrolase activity by about 50%. In cell-free preparation, cAMP-dependent protein kinase caused about a 2-fold activation of the neutral cholesteryl ester hydrolase. Activation was blocked by protein kinase inhibitor. These data suggest that the P388D 1 macrophage may be a useful model for studying the hormonal regulation of cholesteryl ester mobilization in macrophage-derived foam cells.

Hormone-sensitive lipase is responsible for the neutral cholesterol ester hydrolase activity in macrophages

Anti-hormone-sensitive lipase (HSL) immunoglobulin selectively immunoprecipitates a single 84 kDa 32P-phosphoprotein from macrophage homogenates previously phosphorylated by cyclic AMP-dependent protein kinase in the presence of [γ- 32P]ATP-Mg. This immunoglobulin also completely removes the neutral cholesterol ester hydrolase activity from macrophage homogenates. These data demonstrate that HSL is responsible for the neutral cholesterol ester hydrolase activity in macrophages and hence plays a key role in cholesterol metabolism in these cells.