Cholesterol O-acyltransferase Research Articles

Statin Therapy Alone and in Combination with an Acyl-CoA:Cholesterol O-Acyltransferase Inhibitor on Experimental Atherosclerosis

The ability to modify the enzymatic processes involved in promoting atherosclerotic plaque disruption and to serially monitor atherosclerotic evolution could provide novel information in the management of patients with atherosclerosis. We studied the effects of a statin (atorvastatin) and its combination with an acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor (avasimibe) on atherosclerotic regression and plaque stability as measured by matrix metalloproteinase 1 and 3 (MMP-1 and MMP-3) levels. Watanabe Heritable Hyperlipidemic (WHHL) rabbits treated with atorvastatin alone experienced an attenuated increase in atherosclerotic burden versus controls as determined by MR imaging. The mean vessel wall area (VWA) prior to drug therapy was 5.57 ± 0.01 mm². The VWA increased to 6.71 ± 0.03 and 7.16 ± 0.03 mm², respectively, in atorvastatin-treated and control groups (p < 0.0001 for both). The combination of atorvastatin and avasimibe induced a significant regression of the previously established atherosclerotic lesions, with the VWA decreasing to 4.54 ± 0.04 mm² (p = 0.009). Atorvastatin alone induced a nonsignificant reduction in the percent staining of MMP-1 in atherosclerotic lesions, but the combination treatment with avasimibe led to a significant reduction versus controls (p = 0.005). However, a reduction in MMP-3 staining was significant for rabbits treated with both atorvastatin alone (p = 0.007) and in combination with avasimibe (p = 0.04) versus controls. In this animal model, the addition of avasimibe to atorvastatin has beneficial effects on both atherosclerotic plaque regression and stabilization.

Dietary Fat–Induced Alterations in Atherosclerosis Are Abolished by ACAT2-Deficiency in ApoB100 Only, LDLr −/− Mice

The enzyme acyl-coenzymeA (CoA):cholesterol O-acyltransferase 2 (ACAT2) in the liver synthesizes cholesteryl esters (CE) from cholesterol and fatty acyl-CoA, which get incorporated into apoB-containing lipoproteins that are secreted into the bloodstream. Dietary fatty acid composition influences the amount and fatty acid composition of CE within apoB-containing lipoproteins. We hypothesized that when ACAT2 activity is removed by gene deletion, hepatic CE synthesis and secretion would be minimal and, as a result, dietary fat-related differences in atherosclerosis would be eliminated. Groups of female apoB100 only, LDLr-/- mice with and without ACAT2 were fed diets enriched in either omega-3 or omega-6 polyunsaturated fat, saturated fat, and cis or trans monounsaturated fat. After 20 weeks on diet, mice fed diets enriched in monounsaturated or saturated fat exhibited significantly higher amounts of plasma cholesterol, larger LDL particles enriched in monounsaturated CE, and more atherosclerosis than mice fed polyunsaturated fat. The dietary fat-induced shifts in plasma cholesterol, LDL size, LDL CE composition, and atherosclerosis were not observed in ACAT2-/- mice. Regardless of the diet fed, the ACAT2-/- mice were protected from atherosclerosis. The results indicate that in apoB100 only, LDLr-/- mice, ACAT2 plays an essential role in facilitating dietary fat type-specific atherosclerosis through its various effects on plasma lipoprotein concentration and composition.

Development of a method for simultaneous determination of eflucimibe and its three major metabolites in rat plasma by liquid chromatography/electrospray tandem mass spectrometry: a preliminary study

A high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the simultaneous determination of eflucimibe, a powerful acyl-coenzyme A cholesterol O-acyltransferase (ACAT) inhibitor, and its main metabolites, in plasma. The ESI and MS/MS parameters were investigated and optimised for each of the four compounds in the positive ion mode. Plasma samples were deproteinised by precipitation with acetonitrile and directly analysed by HPLC/ESI-MS/MS in less than 4 min. Quantitation was performed in the multiple reaction monitoring (MRM) mode for highest sensitivity, selecting the protonated molecules [M+H](+) as precursor ions. The method was demonstrated to be specific and sensitive, and a linear response was observed within a 1-25 ng/mL concentration range. Correlation coefficients (r(2)) greater than 0.9960 were obtained by least-squares regression, and limits of detection down to 0.2 ng/mL were calculated. Therefore, this HPLC/ESI-MS/MS method appears to be an efficient tool, able to provide valuable information for a pharmacological purpose.

Crystallization of Eflucimibe Drug in a Solvent Mixture: Effects of Process Conditions on Polymorphism

Nucleation and metastability of the eflucimibe (S)-2‘,3‘,5‘-trimethyl-4‘-hydroxy-dodecylthiophenyl-acetanilide, a new acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) inhibitor, have been investigated according to a polythermal method. Eflucimibe exhibits two polymorphs, A and B, which crystallize simultaneously (concomitant polymorphs). The focus of this study was on the effect of the cooling rate, the initial concentration, and the seeds on metastability and polymorphism of eflucimibe. For nonseeded crystallization, it was found that the metastable zone width is large (primary nucleation) and fulfills well the linear relation between log ΔTmax and log(dT/dt). In addition, the X-ray powder diffraction and differential scanning calorimetry analyses of the powder obtained show that the desired polymorph (pure A form) is difficult to obtain. On the contrary, for seeded experiments, the metastable zone width is significantly reduced (secondary nucleation) and the occurrence of polymorphism is controlled by seeding with the desired polymorph.

Synthesis and structure–activity relationship studies on a novel series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT)

The synthesis and structure–activity relationships of N-phenyl-N′-[3-(4-phenylnaphthylidinoyl)]urea derivatives 3 as a novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring, together with a 1-N-nbutyl substitution, SM-32504 (3m), gave a potent ACAT inhibitor, in vitro, respectively. The most potent compound, SM-32504 (3m), decreased the serum cholesterol level significantly in a high fat and high cholesterol-fed mouse model.

Compared with Acyl-CoA:Cholesterol O-Acyltransferase (ACAT) 1 and Lecithin:Cholesterol Acyltransferase, ACAT2 Displays the Greatest Capacity to Differentiate Cholesterol from Sitosterol

The capacity of acyl-CoA:cholesterol O-acyltransferase (ACAT) 2 to differentiate cholesterol from the plant sterol, sitosterol, was compared with that of the sterol esterifying enzymes, ACAT1 and lecithin:cholesterol acyltransferase (LCAT). Cholesterol-loaded microsomes from transfected cells containing either ACAT1 or ACAT2 exhibited significantly more ACAT activity than their sitosterol-loaded counterparts. In sitosterol-loaded microsomes, both ACAT1 and ACAT2 were able to esterify sitosterol albeit with lower efficiencies than cholesterol. The mass ratios of cholesterol ester to sitosterol ester formed by ACAT1 and ACAT2 were 1.6 and 7.2, respectively. Compared with ACAT1, ACAT2 selectively esterified cholesterol even when sitosterol was loaded into the microsomes. To further characterize the difference in sterol specificity, ACAT1 and ACAT2 were compared in intact cells loaded with either cholesterol or sitosterol. Despite a lower level of ACAT activity, the ACAT1-expressing cells esterified 4-fold more sitosterol than the ACAT2 cells. The data showed that compared with ACAT1, ACAT2 displayed significantly greater selectively for cholesterol compared with sitosterol. The plasma cholesterol esterification enzyme lecithin:cholesterol acyltransferase was also compared. With recombinant high density lipoprotein particles, the esterification rate of cholesterol by LCAT was only 15% greater than for sitosterol. Thus, LCAT was able to efficiently esterify both cholesterol and sitosterol. In contrast, ACAT2 demonstrated a strong preference for cholesterol rather than sitosterol. This sterol selectivity by ACAT2 may reflect a role in the sorting of dietary sterols during their absorption by the intestine in vivo.

A Novel Action of Terpendole E on the Motor Activity of Mitotic Kinesin Eg5

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.

Inhibitory effects of N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N'-(3,4-dimethylphenyl)piperazine (YIC-C8-434), an acyl-CoA:cholesterol O-acyltransferase inhibitor, on cholesterol esterification in the intestine and liver.

The effects of an acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor, N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N'-(3,4-dimethylphenyl)piperazine (YIC-C8-434), on cholesterol esterification in the intestine and liver were investigated in vitro and in vivo. YIC-C8-434 inhibited the formation of cholesteryl [(3)H]oleate from [(3)H]oleic acid and cholesterol both in human colon adenocarcinoma Caco2 cells and in human hepatoma HepG2 cells with IC(50) values of 0.38 and 0.49 microM, respectively. However, it did not influence the incorporation of [(3)H]oleic acid into triacylglycerols and phospholipids. Oral administration of YIC-C8-434 at a dose of 8.3 mg/kg/d inhibited [(14)C]cholesterol absorption by 17% (p<0.01) in rats. YIC-C8-434 also significantly reduced the secretion of very low-density lipoprotein (VLDL) cholesterol from the liver into the plasma at an oral dose of 100 mg/kg/d after an intravenous injection of Triton WR-1339. These results suggest that oral administration of YIC-C8-434 reduces intestinal cholesterol absorption and hepatic VLDL cholesterol secretion by direct inhibition of ACAT in the intestinal epithelium and hepatocytes, respectively. However, the inhibitory action of YIC-C8-434 on cholesterol absorption rather than hepatic cholesterol secretion may play a more important role in its hypocholesterolemic activity, because the effective dose for the former was 12-fold lower than that for the latter.

Predicting Acyl-Coenzyme A: Cholesterol O-Acyltransferase Inhibitory Activity: Computational Approach Using Topological Descriptors

Relationship between the topological indices and acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) inhibitory activity of (aminosulfonyl)ureas has been investigated. Three topological indices, Wiener's index--a distance-based topological descriptor, molecular connectivity index--an adjacency-based topological index, and eccentric connectivity index--an adjacency-cum-distance-based topological descriptor, were used for the present investigations. A data set comprising 41 analogues of substituted (aminosulfonyl)ureas was selected for the present studies. The values of wiener's index, eccentric connectivity index, and molecular connectivity index for each of the 41 compounds comprising the data set were computed using an in-house computer program. Resultant data were analyzed and suitable models were developed after identification of active ranges. Subsequently, a biological activity was assigned to each compound using these models, which was then compared with the reported in vitro ACAT inhibitory activity. Accuracy of prediction using these models was found to vary from a minimum of approximately 83% to a maximum of approximately 91%.

Predicting Acyl-Coenzyme A: Cholesterol O-Acyltransferase Inhibitory Activity: Computational Approach Using Topological Descriptors

Relationship between the topological indices and acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) inhibitory activity of (aminosulfonyl)ureas has been investigated. Three topological indices, Wiener's index—a distance-based topological descriptor, molecular connectivity index—an adjacency-based topological index, and eccentric connectivity index—an adjacency-cum-distance–based topological descriptor, were used for the present investigations. A data set comprising 41 analogues of substituted (aminosulfonyl) ureas was selected for the present studies. The values of wiener's index, eccentric connectivity index, and molecular connectivity index for each of the 41 compounds comprising the data set were computed using an in-house computer program. Resultant data were analyzed and suitable models were developed after identification of active ranges. Subsequently, a biological activity was assigned to each compound using these models, which was then compared with the reported in vitro ACAT inhibitory activity. Accuracy of prediction using these models was found to vary from a minimum of ∼83% to a maximum of ∼91%.

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.

Interferon-gamma-mediated downregulation of cholesterol efflux and ABC1 expression is by the Stat1 pathway.

The pathological role of interferon-gamma (IFN-gamma) in atherosclerosis is mediated through effects on macrophages, foam cells, and other vascular cells. Recently, we reported that ATP-binding cassette transporter 1(ABC1) message and protein levels were decreased 3- to 4-fold in foam cells by IFN-gamma. In the present study, the pathway by which IFN-gamma inhibited ABC1 expression was investigated with signal transducers and activators of transcription (Stat1) knockout mice. IFN-gamma-stimulated, wild-type, macrophage-derived foam cells, as previously reported, exhibited a decrease in cholesterol efflux and ABC1 expression as well as an increase in acyl coenzyme A:cholesterol-O-acyltransferase activity. However, IFN-gamma treatment of foam cells from Stat1 knockout mice failed to demonstrate reductions in efflux or ABC1 expression at the message or protein levels, nor were there any increases in acyl coenzyme A:cholesterol-O-acyltransferase activity. However, ABC1 mRNA expression in macrophages from Stat1 knockout mice could still be demonstrated to be increased by lipid loading with acetylated low density lipoprotein. Finally, Stat1-independent gene activation by IFN-gamma was intact in the Stat1 KO macrophages, inasmuch as IFN-gamma was shown to stimulate increases in interleukin-6 production in the Stat1 KO macrophages that were comparable to those observed in the wild-type macrophages. Therefore, Stat1 signaling is necessary and sufficient for the inhibitory effects of IFN-gamma on cholesterol efflux and ABC1 expression.

Cholesterol absorption inhibitors for the treatment of hypercholesterolaemia.

The benefits of lipid lowering therapy on coronary heart disease have been clearly established in many clinical trials on primary and secondary prevention. Despite the availability of potent lipid lowering drugs, many patients do not reach the current treatment goals. This paper reviews new therapeutic approaches in lipid lowering drugs focusing on compounds which lower cholesterol absorption. The role of plant sterols and stanols, new acyl-CoA:cholesterol O-acyl transferase (ACAT) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, and ezetimibe are summarised. Although the lipid lowering effect of plant sterols and plant stanols is only moderate, their use as functional foods is beneficial for patients with mild hypercholesterolaemia and is able to enhance the lipid lowering effect of HMG-CoA reductase inhibitors (statins). The role of ACAT inhibitors that might also inhibit cholesterol absorption remains unclear. Avasimibe, the first oral bioavailable ACAT inhibitor, has entered phase III trials. However, the presently available data in humans do not indicate a clear clinical benefit. The role of MTP inhibitors, which exhibit remarkable effects on all plasma lipids, also remains unclear, as safety concerns must first be addressed. Ezetimibe, the first available 2-azetidinone, succeeded in phase III trials showing remarkable effects in inhibition of cholesterol absorption as well as cholesterol lowering. The synergistic effect of co-administration of ezetimibe with statins seemingly offers a new approach in reaching the therapeutic goals.

3 ACAT Inhibitors: The Search for a Novel and Effective Treatment of Hypercholesterolemia and Atherosclerosis

This chapter explores that many different clofibrate-type compounds soon appeared with some desirable features, and a unifying molecular mechanism now exists for these drugs, none of them are particularly efficacious with regard to cholesterol reduction, especially low-density lipoprotein-cholesterol (LDL-cholesterol). They have been most useful for triglyceride reduction and to some extent, elevation of high-density lipoprotein-cholesterol (HDL-cholesterol). Thus, other drugs were sought, resulting in the emergence of compounds that inhibit specific steps in the cholesterol biosynthetic pathway. This emphasis led to the discovery of the HMG-CoA reductase inhibitors, now the dominant class of drugs widely used for the treatment of hypercholesterolemia. The chapter describes the biology and pharmacology of another class of potential cholesterol-lowering agents—namely, inhibitors of the intracellular enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT). It discusses that ACAT catalyzes the conversion of unesterified (free) cholesterol to cholesteryl esters in virtually every tissue, especially liver, intestine, and the endocrine organs, and, thereby plays a major role in intracellular cholesterol homeostasis. The chapter focuses on lipoprotein-producing tissues, with an additional emphasis on specific cells within developing atherosclerotic lesions. The biology of ACAT is briefly discussed for these tissues, and comprehensive evaluation is done for ACAT inhibitors that have emerged.

A probabilistic approach to high throughput drug discovery.

A methodology is presented in which high throughput screening experimental data are used to construct a probabilistic QSAR model which is subsequently used to select building blocks for a virtual combinatorial library. The methodology is based upon statistical probability estimation and not regression. The methodology is applied to the construction of two focused virtual combinatorial libraries: one for cyclic GMP phosphodiesterase type V inhibitors and one for acyl-CoA:cholesterol O-acyltransferase inhibitors. The results suggest that the methodology is capable of selecting combinatorial substituents that lead to active compounds starting with binary (pass/fail) activity measurements.

Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 ( S)-2′,3′,5′-trimethyl-4′-hydroxy-α-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4–72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition ( ic 50 values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the ic 50 value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17α-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3′,5′-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 μM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 μM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.

Structure-activity relationships of N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N'-(3,4-dimethylphenyl)piperazine and analogues as inhibitors of acyl-CoA: cholesterol O-acyltransferase.

A novel series of acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors were synthesized from a lead compound, 1-(4-hydroxy-3-methoxyphenyl)-7-phenylhept-1-en-3-one (1, Yakuchinone B) through a modification of three regions (A, B, C) in the molecule. In this study, the compounds prepared were tested for in vitro inhibitory activity on microsomal ACAT from the liver of rats and for in vivo hypocholesterolemic activity in rats given a high cholesterol diet. N-(3,5-Dimethoxy-4-n-octyloxycinnamoyl)-N'-(3,4-dimethylphenyl)piperazine (45), which belongs to the amide compounds, has finally been discovered. Compound 45 inhibited rat hepatic ACAT in a more striking manner than CI-976, an amide compound ACAT inhibitor, and it exhibited a high level of hypocholesterolemic activity in vivo. Since 45 strongly inhibited both microsomal ACAT prepared from HepG2 (a cell line derived from human hepatocarcinoma) and Caco2 (a cell line derived from human colon adenocarcinoma), there is speculation that 45 might have the ability to inhibit ACAT in both the human intestine and liver independent of the difference in the distribution of ACAT isozymes. On the other hand, 45 did not induce adrenotoxicity in subacute toxicity studies in rats. These results suggest that it has promise for development as a new therapeutic agent for hypercholesterolemia and atherosclerosis.

Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.

Effects of naringin and lovastatin on plasma and hepatic lipids in high-fat and high-cholesterol fed rats

The effects of naringin on lipid metabolism were examined in rats that were fed a high-fat ( 15 g lard 100 g ) and high-cholesterol ( 1 g 100 g ) diet. Low- or high-doses ( 0.02 g or 0.05 g 100 g ) of either naringin or lovastatin were supplemented in an experimental diet for 35 d. The concentrations of plasma total cholesterol were significantly lower in the naringin- and lovastatin-supplemented groups than in the control group fed a basal diet. In addition, both doses of lovastatin and a high-dose naringin significantly lowered plasma triglyceride concentrations compared to the control group. All naringin and lovastatin supplements, except for the high-dose lovastatin, significantly lowered the hepatic cholesterol concentration compared to the control group. The concentrations of hepatic triglyceride in the naringin-supplemented groups (0.34±0.06 mmol/g and 0.30±0.06 mmol/g) and control (0.33±0.06 mmol/g) were significantly lower than in the lovastatin-supplemented groups (0.40±0.03 mmol/g and 0.42±0.05 mmol/g). High and low doses of naringin significantly lowered the activities of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase by 54% and 39%, respectively, compared to the control group, while lovastatin did not demonstrate any inhibitory activity on this enzyme. The activities of acyl coenzyme A:cholesterol O-acyltransferase (ACAT) were significantly lower in all experimental groups than in the control. Accordingly, lipid-lowering action of naringin seems to be different from that of lovastatin in a high-fat and high-choleterol fed rats.

Effects of passive smoking on the regulation of rat aortic cholesteryl ester hydrolases by signal transduction.

The effects of exogenous oxidative stress due to passive smoking on cholesteryl ester (CE)-metabolizing enzymes and their regulatory kinases were examined by exposing rats to cigarette smoke (CS) for a 1-h period twice a day for 8, 12, or 20 wk. An oxidatively modified low density lipoprotein (Ox-LDL) with a high lipid peroxide was identified in three CS groups after all three exposure periods. The rat aortic acid and neutral CE hydrolases (ACEH and NCEH) were activated to similar extents by both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the presence of their respective cofactors. The aortic PKC activity in the three CS groups exhibited significant reductions of 72, 84, and 75% as compared with the respective controls, which coincided with the reductions in the ACEH activities (86, 71, and 80%, respectively), whereas the PKA activities increased to 121, 197, and 252% in the three CS groups, respectively. Reflecting the increase of the PKA activity, the NCEH activity exhibited increases of 112% at 8 wk and 140% until 12 wk of exposure and decreased by 50% of the control value at 20 wk of exposure, suggesting inactivation of NCEH itself. The activation of acyl-CoA:cholesterol O-acyltransferase activity was associated with an increase of free cholesterol in aorta. The vitamin E diet prevented the formation of Ox-LDL and the oxidative inactivation of most enzymes, especially PKC, until 12 wk, but was less effective by 20 wk. The oxidative inactivation of PKC, particularly its activated form that translocated to the membrane fraction, was confirmed in the in vitro exposure to active oxygen generators at an optimal concentration; this inactivation was prevented by catalase and superoxide dismutase. These results suggested that the formation of Ox-LDL and alterations in CE-metabolizing enzymes caused by passive smoking could contribute to a twofold increase in the aortic CE content, thereby contributing to one of the mechanisms for atherosclerosis associated with smoking.