Enzymatic formation of cholesteryl ester from cholesterol by gallbladder mucosa

The formation of cholesteryl ester from cholesterol and acyl CoA catalyzed by the enzyme acyl CoA: cholesterol acyltransferase (EC 2.3.1.26) was studied in guinea pig gallbladder mucosa homogenate and the subcellular fractions. The enzymatic activity was enriched in the microsomal fraction. Highest activity was observed with palmitoyl, stearoyl or oleoyl CoA as substrate. Lowest activity was observed with linoleoyl CoA. These data elucidate one mechanism for the formation of cholesteryl ester from cholesterol by the gallbladder wall.     

Regulation by long-chain fatty acids of the expression of cholesteryl ester transfer protein in HepG2 cells

Cholesteryl ester transfer protein (CETP) is an important determinant of lipoprotein function, especially high density lipoprotein (HDL) metabolism, and contributes to the regulation of plasma HDL levels. Since saturated and polyunsaturated fatty acids (FA) appear to influence the CETP activity differently, we decided to investigate the effects of FA on the expression of CETP mRNA in HepG2 cells using an RNA blot hybridization analysis. Long-chain FA (>18 carbons) at a 0.5 mM concentration were added to the medium and incubated with cells for 48 h at 37°C under 5% CO₂. After treatment with 0.5 mM arachidonic (AA), eicosapentaenoic (EPA), and docosahexaenoic acid (DHA), the levels of CETP mRNA were less than 50% of the control levels (AA, P=0.0005; EPA, P<0.01; DHA, P<0.0001), with a corresponding significant decrease in the CETP mass. These results suggest that FA regulate the gene expression of CETP in HepG2 and this effect is dependent upon the degree of unsaturation of the acyl carbon chain in FA.     

Effect of dietary vitamin C on adrenal cholesteryl ester content in the guinea pig

Male guinea pigs fed a vitamin C-deficient diet for 3 weeks had lower concentrations of cholesteryl esters in their adrenals than did control animals fed the recommended intake of the vitamin. Not all esters were affected to the same degree, and the fatty acid profiles of the esters from control and deficient guinea pigs differed; there was proportionately more palmitic and linoleic acids and less docosatetraenoic acid [22∶4 (n−6)] in the deficient guinea pig adrenal esters. [Fatty acids are designated as X∶Y (n−Z), where X and Y are the numbers of carbon atoms and olefinic bonds in the acid and Z is the number of carbon atoms after the terminal olefinic bond.] A 100-fold excess of vitamin C in the diet also resulted in lower concentrations of adrenal cholesteryl esters than did the control diet, but they were not as low as in the deficient animals. Fatty acid profiles were similar for esters from control and excessively supplemented guinea pigs. Vitamin C deficiency apparently imposes a long term stress which results in a depletion of adrenal cholesteryl esters, possibly specific esters, to meet the requirements for glucocorticoid synthesis.     

Effect of detergents on in vitro 7α-hydroxycholesterol formation by rat liver microsomes

Formation of 7α-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7α-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7α-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7α-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (K_s>K_p). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.     

Origin of fatty acids of cholesteryl ester accumulated by Fu5AH cells in culture

The Fu5AH rat hepatoma cell line accumulates cholesteryl ester (CE) upon incubation in medium supplemented with hyperlipemic serum or hyperlipemic serum lipoproteins. This cell line was used to investigate the origin of the fatty acids esterified to cholesterol in intracellular accumulations of CE. The intracellular CE-fatty acid distribution was found to be markedly different from that of the lipoprotein which stimulated the accumulation. Free fatty acids added to the culture medium were found esterified to cholesterol in the cells, demonstrating that cellular esterification contributes to the accumulation of CE. Using a subline of Fu5AH cells containing radioactively labeled intracellular fatty acids, it was found that about one-third of the fatty acid moiety of CE accumulated by the cells during a 24 hr incubation with hyperlipemic serum was derived from endogenous fatty acids. The drug chloroquine was found to inhibit cellular cholesterol esterification, so that only 4% of CE-fatty acids were derived from endogenous fatty acids. Evidence is presented suggesting a major role for cellular esterification in CE accumulation by Fu5AH cells.     

Purification and properties of aortic cholesteryl ester hydrolase

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, 0-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific, esterase by column chromatography. Both synthetic and the hydrolytic activities are apprently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dired powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7 Both activities were found to be fairly nonspecific with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; corprostanol, 24; ergosterol, 20; and β-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic>linoleic>arachidonic>palmitic>stearic>butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate and laurate in decreasing order.     

Cholesterol metabolism in human monocyte-derived macrophages: Stimulation of cholesteryl ester formation and cholesterol excretion by serum lipoproteins

The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages (HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within 24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence of cholesterol acceptors in the medium, but cellular cholesterol content was.     

Temperature sensitivity of cholesteryl ester hydrolases in the rat testis

Cholesteryl ester hydrolase (CEH) (EC 3.1.1.13) activity was assayed in the 104,000×g supernatant (S104) of rat and mouse testes and livers at various temperatures between 27 C and 44 C. The CEH activity in the testis dropped from 44 pmol [4-¹⁴C] cholesteryl oleate hydrolyzed/hr/mg protein to 14 pmol hydrolyzed/hr/mg protein (a 68% decrease) between testicular and abdominal temperatures (32 C and 37 C, respectively) in the rat. This decrease in activity is essentially a reversible phenomenon. CEH from the testis S104 was stabilized in 10 mM EDTA and was purified by HPLC size exclusion. These steps did not alter the temperature effect previously noted. The temperature effect on the testicular CEH was demonstrated in vivo by assaying the enzyme following unilateral cryptorchidism. The HPLC purification yielded 3 peaks of CEH activity from the testicular S104. The 28,000 MW peak was found to be temperature insensitive while the 70,000 and 420,000 MW peaks were temperature labile. The liver CEH of both species remained relatively constant over the range 32—37 C. CEH is a potential regulator of both steroidogenesis and membrane composition in the testis and its temperature lability may suggest a unique regulatory mechanism responsible for impaired spermatogenesis seen with elevated testicular temperatures.     

Changes in cholesteryl ester transfer protein concentration during normal gestation

Background: It has been shown that endogenous estrogen may affect the concentration of cholesteryl ester transfer protein (CETP) and that the gestation in women is accompanied by increased levels of endogenous estrogen. Objectives: To examine the changes in CETP concentration and to investigate the relationship between CETP and estrogen levels during normal pregnancy. Methods: Serum concentrations of CETP, estrogen and lipid traits were determined from 296 healthy women at different times during gestation. Results: Pregnant women exhibited a significant elevation in the concentration of CETP as compared with non‐pregnant women (3.11 ± 1.51 mg/L vs. 2.64 ± 1.25 mg/L, p <0.001). The CETP concentration was highest in the first trimester (3.85 ± 1.33 mg/L, compared with controls, p <0.0001), declined progressively from the first to the third trimester and still remained elevated in the third trimester as compared with non‐pregnant subjects (p <0.05). A negative correlation was noted between serum CETP and estradiol (E₂) levels in pregnant women (r = –0.288, p <0.0001). In addition, significantly negative correlations between the serum CETP concentration and all the lipid traits examined were found in gestating women. Conclusion: CETP concentrations were significantly elevated in pregnancy and decreased progressively from early to late gestation. The results suggest that endogenous E₂ is either not involved or its effect on CETP is dominated by elevated lipid levels or some other factors during mid and late pregnancy.     

The effect of oral contraceptives on mononuclear cell cholesteryl ester hydrolase activity

The influence of sex steroids on mononuclear cell cholesteryl ester hydrolase (CEH) activity in premenopausal women and women on combined estrogen-progestin oral contraceptives has been studied. In addition, plasma and mononuclear cell cholesterol and esters were measured along with plasma estrogen and progesterone levels. Mononuclear cell CEH activity in control women is highest on Day 20 of their menstrual cycle. The control women had significantly higher CEH activities than women on oral contraceptives. Plasma esters were higher in the oral contraceptive group. However, in mononuclear cells free cholesterol but not cholesteryl esters were higher in women on oral contraceptives.     

Effect of phospholipase a upon brain cholesterol ester formation

On addition of snake venom phospholipase A to homogenates of rat brain we have been able to show increased ester formation. Within certain limits, the amount of ester formed was dependent upon the amount of phospholipase added. The fatty acid patterns of free fatty acid released and of cholesteryl ester fatty acid formed during 18 hr incubation were compared. Although fatty acid patterns were different, the data were still consistent with the hypothesis that, in demyelination, a neural phospholipase A may play a role in cholesteryl ester deposition.     

Effects of cholesterol and 25-hydroxycholesterol on smooth muscle cell and endothelial cell growth

Auto-oxidation products of cholesterol may play a role in atherogenesis. In order to determine whether cholesterol or 25-hydroxycholesterol, a cholesterol auto-oxidation product, affected growth of vessel wall cells, sparse and confluent cultures of rabbit thoracic aorta smooth muscle cells and human umbilical vein endothelial cells were exposed to these compounds for 88 hr. The compounds were administered at 10⁻⁴, 10⁻⁵, 10⁻⁶ or 10⁻⁷ M in either ethanol or fetal bovine serum (FBS) vehicle. Cells were counted electronically, and the results were expressed as the percent growth in experimental vs control wells. Cholesterol did not inhibit cell growth under any experimental condition. 25-Hydroxycholesterol had the following effects: inhibited confluent smooth muscle cell growth at 10⁻⁴ M in ethanol vehicle only; inhibited sparse smooth muscle cell growth in a dose-related manner at 10⁻⁴, 10⁻⁵ and 10⁻⁶ M in ethanol vehicle, but in FBS vehicle inhibited at only 10⁻⁴ and 10⁻⁵ M; inhibited confluent human umbilical vein endothelial cells at 10⁻⁴ M in ethanol vehicle only; and inhibited sparse human umbilical vein endothelial cell growth at 10⁻⁴ and 10⁻⁵ M in ethanol vehicle only. Thus, rabbit aortic smooth muscle cell growth was more sensitive to inhibition by 25-hydroxycholesterol than human umbilical vein endothelial cell growth was. Part of this work was presented at the 10th Annual Hugh Lofland Conference on Arterial Wall Metabolism, Boston, MA, in 1985 and at the 70th Annual FASEB Meeting, St. Louis, MO, in 1986. It is published in abstract form (Fed. Proc.45;684, 1986).     

Properties of an acid cholesteryl ester hydrolase inhibitor from rat serum

The inhibitory effect of a protein isolated from rat serum on lysosomal acid cholesteryl ester hydrolase (acid CEH; EC.3.1.1.13) activity was studied. An inhibitor was purified from rat serum following ultracentrifugation and heat treatment using column chromatography on Sephacryl S-200 and ultrafiltration. The purified inhibitor appeared as a single protein band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was 28,000 Daltons as judged by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis. The purified inhibitor was shown to be apolipoprotein A-I (apo A-I), the major apolipoprotein of high-density lipoprotein (HDL), using immunoprecipitation with rat anti-apo A-I immunoglobulin (Ig)G. Inhibition of acid CEH activity by apo A-I was dependent on the concentration of apo A-I. The values of V_max obtained were similar with or without apo A-I. Apo A-I of various other mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. Other apolipoproteins, such as apo A-II and apo B, also showed inhibiting activity. On the other hand, apo A-I had no effect on the activity of other enzymes found in lysosomes, such as cathepsin D, β-glucuronidase and acid phosphatase. The results suggest that apolipoproteins may play a role in the regulation of hydrolysis of cholesteryl esters in lipoproteins, that have been transferred to the liver, and that the inhibition of acid CEH activity by apo A-I may be a characteristic of the lipid-binding protein or be due to changes of the lipid/water interface.     

Characterization of a cytosolic protein inhibiting lysosomal acid cholesteryl ester hydrolase

An inhibitor of lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) was found in the cytosolic fraction of rat liver and various other tissues. The extent of the inhibitory effect was dependent on the concentration of the cytosolic protein. The Acid CEH inhibitor was heat-labile, nondialyzable, and its inhibitory activity significantly decreased by trypsin or chymotrypsin digestion, but not by lipase digestion. The inhibitor had no effect on the activity of cathepsin D, β-glucuronidase and acid phosphatase, which are other enzymes found in lysosomes. The present findings suggest that the inhibitor may be involved in the regulation of the hydrolysis of cholesteryl esters in lipoproteins that have been transferred into the liver.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.     

In vitro toxicity of different-sized ZnO nanoparticles in Caco-2 cells

There has been rapid growth in nanotechnology in both the public and private sectors worldwide, but concern about nanosafety exists. To assess size-dependent cytotoxicity on human cancer cells, we studied the cytotoxic effect of three kinds of zinc oxide nanoparticles (ZnO NPs) on human epithelial colorectal adenocarcinoma (Caco-2) cells. Nanoparticles were first characterized by size, distribution, and intensity. Multiple assays have been adopted to measure the cell activity and oxidative stress. The cytotoxicity of ZnO NPs was time dependent and dose dependent. The 24-h exposure was chosen to confirm the viability and accessibility of the cells and taken as the appropriate time for the following test system. The IC₅₀ value was found at a low concentration. The oxidative stress elicited a significant reduction in glutathione with increase in reactive oxygen species and lactate dehydrogenase. The toxicity resulted in a deletion of cells in the G1 phase and an accumulation of cells in the S and G2/M phases. One type of metallic oxide (ZnO) exerted different cytotoxic effects according to different particle sizes. Data from the previous experiments showed that 26-nm ZnO NPs appeared to have the highest toxicity to Caco-2 cells. The study demonstrated the toxicity of ZnO NPs to Caco-2 cells and the impact of particle size, which could be useful in the medical applications.     

Effects of cholesterol oxidation derivatives on cholesterol esterifying and cholesteryl ester hydrolytic enzyme activity of cultured rabbit aortic smooth muscle cells

The effects of 5 μg/ml of 25-hydroxycholesterol; cholestane-3β, 5α,6β-triol; and cholesterol on acyl CoA cholesterol acyltransferase, acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase was studied in cultured rabbit aortic smooth muscle cells. After 1 hour incubation, 25-hydroxycholesterol resulted in a fourfold stimulation of acyl CoA cholesterol acyltrans-ferase activity. No stimulation by 25-hydroxycholesterol was noted before 15 minutes or after 5 hours of incubation. Neither cholestane-3β,5α,6β-triol nor cholesterol influenced acyl CoA cholesterol acyltransferase activity at any time interval. No significant effects of any of the sterols were noted on acid cholesteryl ester hydrolase or neutral cholesteryl ester hydrolase activity. The imbalance between acyl CoA cholesterol acyl trans-ferase and hydrolase activities induced by 25-hydroxycholesterol could result in cholesteryl ester accumulation by arterial smooth muscle cells, which may be associated with atherosclerosis.     

Characterization of a cytosolic protein in rat liver inhibiting neutral cholesteryl ester hydrolase

Neutral cholesteryl ester hydrolase activity (EC 3.1.1.13) present in microsomes isolated from lactating rat mammary glands was found to be inhibited by a factor (or factors) occurring in the cytosolic fraction of male rat liver. The inhibitor was heat-labile, non-dialyzable, destroyed by proteolysis, and was stable following preparation of an acetone/diethyl ether powder of the cytosolic fraction. The protein also inhibited the activity of hormone-sensitive lipase (HSL) (from bovine adipose tissue) and esterase fromCandida cylindracea, but seemed to be more active against the neutral hydrolase found in rat liver microsomes. For the mammary gland microsomal cholesteryl ester hydrolase, the extent of the inhibitory effect was dependent on the concentration of the cytosolic protein, 50% inhibition being achieved by about 100 μg of cytosolic protein, and on the method of initiating the enzyme assay. Kinetic analysis indicated that, under circumstances where the reaction was initiated by the addition of substrate, the inhibition was characterized as “uncompetitive”. When an inhibitor/substrate complex was allowed to form in the absence of enzyme, an element of “competitive” inhibition was introduced into the reaction. Food withdrawal reducted the activity of the inhibitor in live by 56%, but activity was fully restored by short-term re-feeding. In contrast, feeding a diet high in fat led to a 34% increase in activity. The present findings suggest that the inhibitory factor(s) may be involved in the regulation of the hydrolysis of cholesteryl esters in the liver and also in other cell types.     

A diurnal variation of hepatic acid cholesteryl ester hydrolase activity in the rat

The diurnal variation in lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) has been examined in fed, fasted and adrenalectomized rats. The Acid CEH activity of normal rat liver exhibits a diurnal rhythm with maxima at 06.00 hours and minima at 18.00 hours, but such a rhythm was not observed in spleen and brain. This rhythm was abolished after fasting for two days, and the resulting Acid CEH activity remained constant at the minimum level. However, adrenalectomy did not abolish the diurnal rhythm. These results indicate that the Acid CEH activity varies according to a diurnal rhythm with maxima and minima separated by approximately 12 hr. Further, it is evident that the appearance of this rhythm is dependent upon dietary, but not adrenal hormone influence.     

Preparation,chromatography and mass spectrometry of cholesteryl ester and glycerolipid-bound aldehydes

To identify lipid ester core aldehydes (aldehydes still bound to parent lipid molecules) among the secondary products of peroxidation, we have prepared reference standards of cholesteryl esters, diacylglycerols and the common glycerophospholipids (GPL) containing an aldehyde function. We subjected the unsaturated lipid esters to hydroxylation with osmium tetroxide and to carbon-carbon bond cleavage with periodic acid. The resulting aldehyde esters (mainly 5-oxovalerates and 9-oxononanoates of cholesterol and the glycerolipids) were isolated and purified by thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (HPLC). Liquid chromatography/mass spectrometry (LC/MS) was used to identify the cholesteryl and other neutral lipid ester aldehydes as the dinitrophenylhydrazones (DNPH). The choline (CGPL) and ethanolamine (EGPL) glycerophospholipids containing core aldehydes were identified by fast atom bombardment mass spectrometry (FAB-MS) and by gas chromatography/mass spectrometry (GC/MS) of the aldehyde-containing diacylglycerol moieties following dephosphorylation with phospholipase C and preparation of the methoximes (MOX) and the trimethylsilyl (TMS) ethers. The lipid esters containing the C₅ and C₉ aldehydes are chemically stable compounds with excellent chromatographic properties yielding mass spectra characteristic of the parent compounds. The overall yield of the core aldehydes was 10–20% of the unsaturated starting material. Part of this work was presented at the Annual AOCS Meeting, Chicago, IL, May, 1991 (see ref. 1).