Neutral Cholesteryl Ester Hydrolase Activity Research Articles

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.

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.

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.

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.

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.

Carboxylesterase 3 (EC 3.1.1.1) Is a Major Adipocyte Lipase

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like peptide 1.

We previously reported decreased glucose-stimulated insulin secretion (GSIS) in hormone-sensitive lipase-null mice (HSL(-/-)), both in vivo and in vitro. The focus of the current study was to gain further insight into the signaling role and regulation of lipolysis in islet tissue. The effect of glucagon-like peptide 1 (GLP-1) on GSIS was also studied, as GLP-1 could augment GSIS via protein kinase A activation of HSL and lipolysis. Freshly isolated islets from fasted and fed male HSL(-/-) and wild-type (HSL(+/+)) mice were studied at ages 4 and 7 months. Neutral cholesteryl ester hydrolase activity was markedly reduced in islets from both 4- and 7-month-old male HSL(-/-) mice, whereas a marked deficiency in triglyceride lipase activity became evident only in the older mice. The deficiencies in lipase activities were associated with higher islet triglyceride content and reduced lipolysis at basal glucose levels. Lipolysis was stimulated by high glucose in islets of both wild-type and HSL-null mice. Severe deficiencies in GSIS were found, but only in islets from 7-month-old, fasted, male HSL(-/-) mice. GSIS was less affected in 4-month-old fasted male HSL(-/-) mice and not reduced in female mice. Exogenous delivery of free fatty acids (FFAs) rescued GSIS, supporting the view that the lack of endogenous FFA supply for lipid-signaling processes in HSL(-/-) mice was responsible for the loss of GSIS. GLP-1 also rescued GSIS in HSL(-/-) mice, indicating that signaling via HSL is not a major pathway for its incretin effect. Thus, the secretory phenotype of HSL-null mice is gender dependent, increases with age, and is influenced by the nutritional state. Under most circumstances, the major determinant of lipolytic flux in the beta-cell involves an enzyme(s) other than HSL that is acutely activated by glucose. Our results support the view that the availability of endogenous FFA through HSL and an additional enzyme(s) is involved in providing lipid moieties for beta-cell signaling for secretion in response to glucose.

Hormone-sensitive lipase is required for high-density lipoprotein cholesteryl ester-supported adrenal steroidogenesis.

Steroid hormones are synthesized using cholesterol as precursor, with a substantial portion supplied by the selective uptake of lipoprotein-derived cholesteryl esters. Adrenals express a high level of neutral cholesteryl ester hydrolase activity, and recently hormone-sensitive lipase (HSL) was shown to be responsible for most adrenal neutral cholesteryl ester hydrolase activity. To determine the functional importance of HSL in adrenal steroidogenesis, adrenal cells were isolated from control and HSL-/- mice, and the in vitro production of corticosterone was quantified. Results show that, even though adrenal cholesteryl ester content was substantially elevated in both male and female HSL-/- mice, basal corticosterone production was reduced approximately 50%. The maximum corticosterone production induced by dibutyryl cAMP, and lipoproteins was approximately 75-85% lower in adrenal cells from HSL-/- mice compared with control. There is no intrinsic defect in the conversion of cholesterol into steroids in HSL-/- mice. Dibutyryl cAMP-stimulated conversion of high-density lipoprotein cholesteryl esters into corticosterone was reduced 97% in HSL-/- mice. An increase in low-density lipoprotein receptor expression appears to be one of the compensatory mechanisms for cholesterol delivery in HSL-/- mice. These findings suggest that HSL is functionally linked to the selective pathway and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis.

Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein: FACILITATION OF CHOLESTEROL TRANSFER IN ADRENAL

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.

Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles

The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n−6 PUFA) or fish oil (rich in n−3 PUFA) were prepared in vivo and oxidised by incubation with CuSO 4. The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants (<2-fold). Triacylglycerol synthesis, as measured by the incorporation of [ 3H]oleate and [ 3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [ 3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n−6 PUFA as compared with n−3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation.

Hormone-Sensitive Lipase Is Not Required for Cholesteryl Ester Hydrolysis in Macrophages

Storage of cholesteryl esters in the cytoplasm of macrophages is one of the earliest and most ubiquitous event observed in the development of arteriosclerosis. Macrophages have an enormous capacity to uptake and store cholesterol in the form of cytosolic cholesteryl ester droplets. These stores are mobilized by the action of a neutral cholesteryl ester hydrolase (NCEH), producing free cholesterol that is either secreted to extracellular acceptors or reesterified. It has been proposed that hormone-sensitive lipase (HSL) is responsible for the NCEH activity in macrophages. The present work shows, however, that peritoneal macrophages from HSL null mice hydrolyze cytosolic stores of cholesteryl esters at a comparable rate to that of peritoneal macrophages from wild-type mice, therefore demonstrating that HSL is not the main NCEH in macrophages.

Adrenal neutral cholesteryl ester hydrolase: identification, subcellular distribution, and sex differences.

Adrenals express a high level of neutral cholesteryl ester hydrolase (CEH) activity, and male rats have greater activity than females; however, the identity of the enzyme(s) responsible for this activity and the basis for the sex differences are unknown. Using mice in which hormone-sensitive lipase (HSL) was inactivated by homologous recombination (HSL -/-), neutral CEH activity was reduced more than 98% compared with controls. Female HSL -/- mice showed a reduction in stimulated corticosterone values. Mechanical separation of rat adrenals revealed less HSL in the outer than the inner cortex. Examination of subfractions of rat adrenals showed that immunoreactive HSL was prominently expressed in microsomes, with lesser amounts in the cytosol and little to no HSL in mitochondrial and nuclear fractions or the lipid droplet. Four- to 10-fold more neutral CEH activity was in the microsomal fraction than any other fraction. No sex differences in the expression or subcellular distribution of HSL protein were found; however, neutral CEH activity was lower in the microsomal fraction of females, and female adrenals contained more cholesteryl esters. Thus, HSL appears to be responsible for most, if not all, of adrenal neutral CEH activity, is prominently expressed in microsomes, and its activity is influenced by sex.

Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

Cholesteryl ester transfer protein (CETP) mediates triglyceride and cholesteryl ester (CE) transfer between lipoproteins, and its activity is strongly modulated by dietary cholesterol. To better understand the regulation of CETP synthesis and the relationship between CETP levels and cellular lipid metabolism, we selected the SW872 adipocytic cell line as a model. These cells secrete CETP in a time-dependent manner at levels exceeding those observed for Caco-2 or HepG2 cells. The addition of LDL, 25OH-cholesterol, oleic acid, or acetylated LDL to SW872 cells increased CETP secretion (activity and mass) up to 6-fold. In contrast, CETP production was decreased by almost 60% after treatment with lipoprotein-deficient serum or beta-cyclodextrin. These effects, which were paralleled by changes in CETP mRNA, show that CETP biosynthesis in SW872 cells directly correlates with cellular lipid status. To investigate a possible, reciprocal relationship between CETP expression and cellular lipid homeostasis, CETP biosynthesis in SW872 cells was suppressed with CETP antisense oligonucleotides. Antisense oligonucleotides reduced CETP secretion (activity and mass) by 60% compared with sense-treated cells. When CETP synthesis was suppressed for 24 h, triglyceride synthesis was unchanged, but cholesterol biosynthesis was reduced by 20%, and acetate incorporation into CE increased 31%. After 3 days of suppressed CETP synthesis, acetate incorporation into the CE pool increased 3-fold over control. This mirrored a similar increase in CE mass. The efflux of free cholesterol to HDL was the same in sense and antisense-treated cells; however, HDL-induced CE hydrolysis in antisense-treated cells was diminished 2-fold even though neutral CE hydrolase activity was unchanged. Thus, CETP-compromised SW872 cells display a phenotype characterized by inefficient mobilization of CE stores leading to CE accumulation. These results strongly suggest that CETP expression levels contribute to normal cholesterol homeostasis in adipocytic cells. Overall, these studies demonstrate that lipid homeostasis and CETP expression are tightly coupled.

Role of the cyclic AMP-dependent pathway in free radical-induced cholesterol accumulation in vascular smooth muscle cells

We have previously reported that free radical-treated vascular smooth muscle cells (SMC) lead to cholesterol accumulation in vitro. In the current study, we investigated the effects of oxidative stress on cyclic AMP concentration and cAMP-dependent enzymes involved in cholesterol homeostasis in A7r5 cells. Under our conditions of a mild oxidative stress, namely with no change in cell viability, we found that free radicals, initiated using azobis-amidinopropane dihydrochloride (AAPH), resulted in a dose-dependent decrease in cellular cAMP which was opposed by vitamin E preincubation. Although the addition of adenylate cyclase activators (carbacyclin and forskolin) increased cAMP levels it did not succeed in restoring the AAPH-induced decrease. The oxidative stress-induced increase in activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and of acyl coenzyme A: cholesterol acyltransferase and the decrease in neutral cholesteryl ester hydrolase activity were suppressed by addition of dibutyryl cAMP. Taken together, these results strongly suggest that free radicals reduce cAMP concentrations by altering cell membrane adenylate cyclase activity. The changes of cAMP-dependent enzymes induced by oxidative stress resulting in cholesterol accumulation might be one of the processes leading to SMC-derived foam cells depicted in atheroma plaque. Moreover, if extrapolated to in vivo, these data may explain in part the beneficial effects of antioxidants in the reduction of cardiovascular diseases.

Low level expression of hormone-sensitive lipase in arterial macrophage-derived foam cells: potential explanation for low rates of cholesteryl ester hydrolysis

Conversion of arterial macrophages into foam cells is a key process involved in both the initiation and progression of atherosclerotic lesions. Foam cell formation involves the progressive accumulation and storage of lipoprotein-derived cholesteryl esters. The resulting imbalance in cholesterol metabolism in arterial foam cells may be due in part to an inadequately low level of cytoplasmic neutral cholesteryl ester hydrolase (NCEH) activity. In this study, we have demonstrated that hormone-sensitive lipase (HSL) mRNA is expressed at very low levels in macrophage-derived foam cells, using the unique approach of extracting mRNA from macrophage-derived foam cells purified from human and rabbit atherosclerotic plaques coupled with reverse transcriptase polymerase chain reaction (RT–PCR). We also demonstrate that macrophage-derived foam cells isolated from rabbit atherosclerotic lesions exhibit a resistance to high density lipoprotein (HDL)-mediated cholesterol efflux along with reduced levels of NCEH activity compared to lipid-loaded mouse peritoneal macrophages. Thus, low level expression of HSL may partially account for the reduced NCEH activity observed in arterial foam cells isolated from atherosclerosis-susceptible species.

Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-γ

The Th1-derived cytokine gamma interferon, IFN-γ, is present within the microenvironment of an atheromatous lesion and likely contributes to lesion progression through macrophage activation. While the inflammatory effects of IFN-γ are well known, the role of this cytokine in cholesterol metabolism in macrophage derived foam cells is unclear. In the present study, the incubation of foam cells with IFN-γ resulted in the reduction of HDL3-mediated cholesterol efflux. The decrease in cholesterol efflux was not observed with other macrophage-activating factors as colony-stimulating factors failed to demonstrate a similar effect. The reduction in cholesterol efflux was independent of apoE synthesis or SR-BI expression and was associated with a redistribution of intracellular cholesterol with an increase in cholesteryl ester accumulation. The increase in the esterified pool, primarily in cholesterol eicosapentadenoate, docosapentaenoate, arachidonate, and linoleate was associated with a 2-fold increase in acyl-CoA:cholesterol-O-acyltransferase, ACAT, activity and message without any change in neutral cholesteryl ester hydrolase activity. While CD36 message was reduced in IFN-γ-treated foam cells, the ability to reverse the decrease in efflux by the ACAT inhibitor A58035 in a dose-dependent manner suggests that the IFN-γ effect on efflux is primarily through the modulation of ACAT expression. Therefore, in addition to its inflammatory effects, IFN-γ can contribute to the progression of an atherosclerotic lesion by altering the pathway of intracellular cholesterol trafficking in macrophage derived foam cells. —Panousis, C. G., and S. H. Zuckerman. Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-γ.

Glucocorticoids stimulate cholesteryl ester formation in human smooth muscle cells.

The aim of the present study was to investigate the effect of synthetic glucocorticoid dexamethasone (Dex) on cholesterol esterification in cultured human smooth muscle cells (SMC). In labeled SMC, Dex stimulated the esterification of [3H]cholesterol in a dose-dependent manner. This effect was specific for glucocorticoid hormones and could be inhibited by cycloheximide (3 ng/mL), actinomycin D (10(-5) mol/L), and the specific glucocorticoid antagonist RU 486 (10(-8) mol/L). When plasma membrane was selectively labeled with trace quantities of [3H]cholesterol (0.25 microCi/mL, 1 hour, 10 degrees C), Dex (10(-8) mol/L) caused a net flux of free [3H]cholesterol into the cells. Moreover, Dex (10(-8) mol/L, 24 hours) stimulated the esterification of sterols, newly synthesized from [14C]mevalonate (10 microCi/mL, 4 hours) and lowered the amount of [14C]sterols susceptible for cholesterol oxidase. The incorporation of [14C]oleic acid into cholesteryl esters was markedly higher in Dex-pretreated SMC than in the control cells (2.1 +/- 0.07 and 1.4 +/- 0.1 pmol/h/microgram protein, respectively, P < .01). At the time, cholesteryl ester hydrolysis in Dex-treated cells was reduced (72 +/- 8 pmol cholesteryl esters/h per milligram versus 130 +/- 10 in the control cells). HDL3-mediated [3H]cholesterol efflux was also inhibited in Dex-treated cells; moreover, HDL3 (40 micrograms/mL, 24 hours) had practically no effect on [3H]cholesteryl ester content in Dex-treated SMC but caused a 50% reduction of [3H]cholesteryl esters in the control cells. Thus, in human SMC glucocorticoids alter the redistribution of cholesterol between the pools of free and esterified cholesterol, paralleled by the change in acyl coenzyme A: cholesteryl acyltransferase and neutral cholesteryl ester hydrolase activities, leading to the impaired HDL3-mediated cholesterol efflux.

Anti-hyperlipidemic and anti-atherosclerotic actions of shosaikoto (kampo medicine).

We investigated the anti-atherosclerotic action shown by Shosaikoto, a Kampo medicine, using hypercholesterolemic mice. Oral administration of Shosaikoto significantly suppressed the elevation of serum cholesterol in C57BL/6 mice fed a 1.25% cholesterol-enriched diet for four weeks and improved the T cell ratio in peripheral blood, which decreased with the increase of the serum cholesterol level. In addition, Shosaikoto reduced the accumulation of cholesteryl oleate, which alters macrophages into foam cells, after the treatment of macrophages with oxidized or acetylated low density lipoprotein (LDL). Enzymatic study revealed that the treatment of macrophages with oxidized LDL enhanced acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity and markedly reduced neutral cholesteryl ester hydrolase (NCEase) activity. Shosaikoto treatment prevented a decrease in the NCEase activity, however due to the oxidized LDL treatment, although it slightly augmented ACAT activity. Thus, Shosaikoto, which is known to modulate the immune system, improves macrophage and lymphocyte functions diminished by hypercholesterolemia, resulting in an anti-atherosclerotic action.