Secretion Of Cholesteryl Esters Research Articles

Hypolipidemia associated with inactivation of TM6SF2 is due to decreased VLDL-lipids secretion

Abstract Background A missense variant in Transmembrane 6 Superfamily Member 2 [TM6SF2 (E167K)] is associated with reduced plasma lipid levels and protection from coronary atherosclerosis. The substitution of lysine for glutamate at residue 167 is associated with a marked decrease in TM6SF2 protein expression, consistent with a loss-of-function mutation. However the biological role of TM6SF2 is not known, and the mechanism(s) responsible for the hypolipidemia associated with mutation gene has not been fully defined. To elucidate the pathological mechanism for the hypolipidemia associated with TM6SF2 deficiency, we inactivated Tm6sf2 in mice and rats. Methods Tm6sf2−/− mice were generated as described previously. Two lines of Tm6sf2−/− rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Primary hepatocytes were isolated from WT and Tm6sf2−/− mice for microscopy. Rats were fasted 16 or 4 hours and tissues were collected on ice for cell fractionation, and in liquid nitrogen for biochemical analyses. Frozen samples were stored at −80°C for subsequent analyses. Result In both mice and rats, inactivation of Tm6sf2 recapitulated the phenotype of humans with the E167K substitution: steatosis, reduced plasma lipid levels, and transaminitis. The phenotype was readily apparent in animals fed chow diets. Both species had reduced secretion of VLDL-TG, as determined by TRITON WR1399 injection, with no decrease in secretion of ApoB. Experiments in isolated perfused livers from WT and Tm6sf2−/− mice confirmed that the decreased TG secretion observed in intact animals reflected reduced TG secretion from the liver. Lipidomic analysis of the liver perfusates by by LC-MS indicated that secretion of cholesteryl esters, and phospholipids was also decreased in the KO animals. Taken together, these findings are consistent with a role for TM6SF2 in lipidation of ApoB-containing lipoproteins. To further elucidate the function of TM6SF2, we used fluorescence microscopy and cell fractionation to determine the subcellular localization of the protein. Microscopic analysis showed that TM6SF2 co-localized with ER and Golgi markers, but cell fractionation studies indicated that the protein is located primarily in the smooth ER. The ratio of TG to ApoB was lower in Golgi fractions from TM6sf2−/− rats than in corresponding fractions from WT animals. Conclusions Since the sequela of TM6SF2 inactivation are already apparent in the Golgi, we speculate that TM6SF2 promotes lipidation of VLDL in a pre-Golgi compartment. We are currently performing additional studies to further define the specific mechanism whereby TM6SF2 promotes lipidation of ApoB-containing lipoproteins. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Institutes of Health

Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

Dietary Taurine Reduces Hepatic Secretion of Cholesteryl Ester and Enhances Fatty Acid Oxidation in Rats Fed a High-Cholesterol Diet

We investigated the fate of exogenous fatty acid in connection with decreased hepatic accumulation and secretion of cholesteryl esters in rats fed diets containing taurine. Providing taurine as 5% of the diet for 14 d significantly decreased concentrations of cholesterol, especially cholesteryl esters in both serum and liver. Ketone body production and incorporation of exogenous [1-(14)C]oleate into ketone bodies in liver perfusate were consistently higher during a 4-h perfusion period in taurine-fed rats than in control rats. The elevation was accompanied by increased activity of liver mitochondrial carnitine palmitoyltransferase, a rate-limiting enzyme for fatty acid oxidation. Dietary taurine significantly reduced hepatic secretion of cholesteryl ester and decreased incorporation of exogenous oleic acid substrate into this lipid molecule. Further, the extent of reduction in hepatic secretion of cholesteryl ester was closely related to its diminished accumulation in the liver. The conversion pattern of exogenous [1-(14)C]oleic acid substrate suggested a decreased esterification-to-oxidation ratio in the taurine group compared with the control. These results suggest that taurine-induced reduction in hepatic accumulation of cholesteryl ester was associated with reduced hepatic secretion of this lipid molecule, and was inversely related to enhanced ketone body production and fatty acid oxidation.

Microsomal Triglyceride Transfer Protein Enhances Cellular Cholesteryl Esterification by Relieving Product Inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.

Overexpression of Apolipoprotein A-IV Enhances Lipid Secretion in IPEC-1 Cells by Increasing Chylomicron Size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Control of ACAT2 liver expression by HNF1

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Yolk sac cholesteryl ester secretion rates can be manipulated in the Golden Syrian hamster: Effect of yolk sac cholesterol concentrations

The yolk sac is one of two extra-embryonic fetal tissues that separates the fetal and maternal circulations. The yolk sac can secrete lipoprotein particles to the vitelline vessels, which supply yolk sac-derived nutrients to the embryo. The amount and composition of lipoproteins secreted from the rat yolk sac can be manipulated by fatty acid content and gestational age. The goals of the current studies were to determine, first, if tissue cholesterol concentration could mediate cholesterol secretion rate from the yolk sac and, second, if some of the secreted cholesterol could be derived from the maternal circulation. Golden Syrian hamsters were fed 2% added cholesterol to increase the yolk sac cholesterol concentration. Yolk sac explants secreted similar amounts of triglyceride and apolipoproteins B and E into the media regardless of yolk sac cholesterol concentration. In contrast, yolk sacs with greater cholesterol concentrations secreted 2.3-fold more cholesterol into the media as compared to control yolk sacs; the increase was found mostly as cholesteryl ester. At least part of the secreted cholesterol was maternally derived. These data demonstrate that yolk sac cholesterol concentration influences cholesterol secretion rates, and that at least some of the cholesterol secreted originates from the maternal circulation.

Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion

The presence of elevated circulating triacylglycerol (TG)-rich very low density lipoprotein (VLDL) and apolipoprotein B-100 (apoB-100) levels represents an independent risk factor for coronary artery disease. Triacylglycerol hydrolase catalyzes the mobilization of cytoplasmic TG stores. To test the hypothesis that the enzyme plays a role in the provision of core lipids for the assembly of VLDL, we inhibited the lipase activity in primary rat hepatocytes and analyzed lipid and apoB synthesis and secretion. Inhibition of lipolysis resulted in a dramatic decrease in secretion of TGs. In addition, secretion of cholesteryl ester and phosphatidylcholine was substantially decreased. Analysis of secreted apolipoproteins indicated that apoB-100 secretion was much more sensitive to lipase inhibition than was apoB-48 secretion, perhaps because of the ability of apoB-48 to be secreted as a relatively lipid-poor particle. The results agreed with those obtained with hepatoma cells transfected with triacylglycerol hydrolase cDNA, in which preferential lipidation of apoB-100 was observed. Together, our findings provide evidence that inhibition of intracellular TG hydrolysis significantly decreases apoB-100 secretion and suggest that triacylglycerol hydrolase may be a suitable pharmacological target in efforts to lower plasma lipid levels.

Cafestol increases serum cholesterol levels in apolipoprotein E*3-Leiden transgenic mice by suppression of bile acid synthesis.

Cafestol, a diterpene present in unfiltered coffee, potently increases serum cholesterol levels in humans. So far, no suitable animal model has been found to study the biochemical background of this effect. We determined the effect of cafestol on serum cholesterol and triglycerides in different mouse strains and subsequently studied its mechanism of action in apolipoprotein (apo) E*3-Leiden transgenic mice. ApoE*3-Leiden, heterozygous low density lipoprotein-receptor (LDLR+/-) knockout, or wild-type (WT) C57BL/6 mice were fed a high- (0.05% wt/wt) or a low- (0.01% wt/wt) cafestol diet or a placebo diet for 8 weeks. Standardized to energy intake, these amounts are equal to 40, 8, or 0 cups of unfiltered coffee per 10 MJ per day in humans. In apoE*3-Leiden mice, serum cholesterol was statistically significantly increased by 33% on the low- and by 61% on the high-cafestol diet. In LDLR+/- and WT mice, the increases were 20% and 24%, respectively, on the low-cafestol diet and 55% and 46%, respectively, on the high-cafestol diet. These increases were mainly due to a rise in very low density lipoprotein (VLDL) and intermediate density lipoprotein cholesterol in all 3 mouse strains. To investigate the mechanism of this effect, apoE*3-Leiden mice were fed a high-cafestol or a placebo diet for 3 weeks. Cafestol suppressed enzyme activity and mRNA levels of cholesterol 7alpha-hydroxylase by 57% and 58%, respectively. mRNA levels of enzymes involved in the alternate pathway of bile acid synthesis, ie, sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase, were reduced by 32% and 48%, respectively. The total fecal bile acid output was decreased by 41%. Cafestol did not affect hepatic free and esterified cholesterol, but it decreased LDLR mRNA levels by 37%. The VLDL apoB and triglyceride production rates, as measured after Triton injection, were 2-fold decreased by cafestol, indicating that the number of particles secreted had declined and that there was no change in the amount of triglycerides present in the VLDL particle during cafestol treatment. However, the VLDL particles contained a 4-times higher amount of cholesteryl esters, resulting in a net 2-fold increased secretion of cholesteryl esters. The decrease in triglyceride production was the result of a reduction in hepatic triglyceride content by 52%. In conclusion, cafestol increases serum cholesterol levels in apoE*3-Leiden mice by suppression of the major regulatory enzymes in the bile acid synthesis pathways, leading to decreased LDLR mRNA levels and increased secretion of hepatic cholesterol esters. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.

Acyl-coenzyme A:cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and in vivo in the rat.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (Cl- 1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and rats fed different diets. Avasimibe dose-dependently decreased ACAT activity in rat hepatocytes in the presence and absence of beta-migrating very low-density lipoproteins (betaVLDL) (by 93% and 75% at 10 micromol/L) and reduced intracellular storage of cholesteryl esters. Avasimibe (3 micromol/L) increased bile acid synthesis (2.9-fold) after preincubation with betaVLDL and cholesterol 7alpha-hydroxylase activity (1.7- and 2.6-fold, with or without betaVLDL), the latter paralleled by a similar induction of its messenger RNA (mRNA). Hepatocytes treated with avasimibe showed a shift from storage and secretion of cholesteryl esters to conversion of cholesterol into bile acids. In rats fed diets containing different amounts of cholesterol and cholate, avasimibe reduced plasma cholesterol (by 52% to 71%) and triglyceride levels (by 28% to 62%). Avasimibe did not further increase cholesterol 7alpha-hydroxylase activity and mRNA in cholesterol-fed rats, but prevented down-regulation by cholate. Avasimibe did not affect sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase, 2 enzymes in the alternative pathway in bile acid synthesis. No increase in the ratio of biliary excreted cholesterol to bile acids was found, indicating that ACAT inhibition does not result in a more lithogenic bile. Avasimibe increases bile acid synthesis in cultured hepatocytes by enhancing the supply of free cholesterol both as substrate and inducer of cholesterol 7alpha-hydroxylase. These effects may partially explain the potent cholesterol-lowering effects of avasimibe in the rat.

International Congress of Biochemistry and Molecular Biology Satellite Conference: Cellular Cholesterol Ester Metabolism

A 1-day satellite conference to the International Congress of Biochemistry and Molecular Biology entitled “Cellular Cholesterol Ester Metabolism” was held at the University of California, San Francisco on August 23, 1997. It was organized to bring together individuals from different disciplines to exchange ideas and information in this important area. Cellular cholesteryl ester metabolism plays a key role in maintenance of cholesterol homeostasis which is vital for normal cell growth and function. The Conference began with an overview by John Dietschy (UT, Southwestern) on the physiological importance of the cholesterol esterification/hydrolysis system. Although acyl coenzyme A:cholesterol acyltransferase (ACAT) often is said to protect cells from a sudden influx of large amounts of cholesterol, this is an uncommon event physiologically. More commonly, ACAT (along with cholesteryl ester hydrolases, CEH) is responsible for maintaining cellular steady-state levels of cholesteryl esters. In vivo, as in vitro, the level of esterification appears to be driven in part by availability of cholesterol and fatty acids. Feeding cholesterol to a new steady state expands both the unesterified and esterified levels of cholesterol, while feeding different fatty acids significantly shifts the ratio between these two components. In humans, as well as in experimental animals, the levels of both dietary cholesterol and fatty acids profoundly affect the steadystate levels of cholesteryl esters in the liver, the level of hepatic low density lipoprotein (LDL) receptor activity and the outflow of cholesterol in VLDL particles. Further, one of the causes of variable responses of the LDL level to cholesterol challenge is mediated by differences in the amount of cholesterol absorbed across the gastrointestinal tract which in turn appears to be dependent on intestinal ACAT levels. Clearly, it is essential that future work include identification of the genes involved and that appropriate studies be undertaken to identify how these enzymes are regulated and whether they are responsible for genetic differences in responses of the plasma LDL level to dietary challenge. The first session, “Physiology/Pathophysiology of Cholesterol Esters,” chaired by Richard Havel (UCSF) and Jane Glick (University of Pennsylvania), opened with a discussion by Larry Rudel (Bowman Gray) of relationships between hepatic production and secretion of cholesteryl esters and atherosclerosis in African green monkeys. Although plasma apoB and LDL cholesterol concentrations were lower in monkeys fed monounsaturated and polyunsaturated fat than in those fed saturated fat, LDL particles in the monounsaturated fat group were remarkably enriched in cholesteryl oleate and the livers of this group had significantly higher cholesteryl ester concentrations. Liver cholesteryl ester content, plasma cholesterol and apoB levels, liver perfusate cholesteryl ester accumulation rate, hepatic ACAT activity, and coronary artery cholesteryl ester concentration were all highly correlated in each group. These data provide the first direct demonstration of the importance of hepatic cholesteryl ester synthesis and secretion in the development of atherosclerosis in primates.

Identification of a Form of Acyl-CoA:Cholesterol Acyltransferase Specific to Liver and Intestine in Nonhuman Primates

The present study demonstrates that two different forms of the intracellular cholesterol esterification enzyme acyl-CoA:cholesterol acyltransferase (ACAT) are present in the nonhuman primate hepatocyte; one is similar to that originally cloned from human genomic DNA, here termed ACAT1, while a second gene product, termed ACAT2, is reported here. The primate ACAT2 gene product was cloned from an African green monkey liver cDNA library. Sequence analysis of an isolated, full-length clone of ACAT2 cDNA identified an open reading frame encoding a 526-amino acid protein with essentially no sequence similarity to the ACAT1 cDNA over the N-terminal 101 amino acids but with 57% identity predicted over the remaining 425 amino acids. Transfection of the cloned ACAT2 cDNA into two different mammalian cell types resulted in the production of abundant ACAT activity which was sensitive to ACAT inhibitors. Northern blot analysis showed that the ACAT2 mRNA was expressed primarily in liver and intestine in monkeys. In contrast, ACAT1 mRNA was expressed in almost all tissues examined. Topologic predictions from the amino acid sequence of ACAT2 indicates that it has seven trans-membrane domains in a configuration that places the putative active site of the enzyme in the lumen of the endoplasmic reticulum. This orientation of ACAT2 in the endoplasmic reticulum membrane, in addition to its expression only in liver and intestine, suggests that this enzyme may have as a primary function, the secretion of cholesteryl esters into apoB-containing lipoproteins.

The formation and secretion of cholesteryl esters in rat hepatocytes are reduced by lovastatin and simvastatin.

Conference Article| February 01 1998 98 The formation and secretion of cholesteryl esters in rat hepatocytes are reduced by lovastatin and simvastatin MARÍA J. MARTÍNEZ; MARÍA J. MARTÍNEZ 1Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, E-48080 Bilbao, Spain Search for other works by this author on: This Site PubMed Google Scholar MARÍA L. HERNÁNDEZ; MARÍA L. HERNÁNDEZ 1Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, E-48080 Bilbao, Spain Search for other works by this author on: This Site PubMed Google Scholar EFRÉN ISUSI; EFRÉN ISUSI 1Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, E-48080 Bilbao, Spain Search for other works by this author on: This Site PubMed Google Scholar PATRICIA ASPICHUETA; PATRICIA ASPICHUETA 1Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, E-48080 Bilbao, Spain Search for other works by this author on: This Site PubMed Google Scholar BEGOÑA OCHOA BEGOÑA OCHOA 1Department of Physiology, University of the Basque Country Medical School, P.O. Box 699, E-48080 Bilbao, Spain Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1998) 26 (1): S82. https://doi.org/10.1042/bst026s082 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation MARÍA J. MARTÍNEZ, MARÍA L. HERNÁNDEZ, EFRÉN ISUSI, PATRICIA ASPICHUETA, BEGOÑA OCHOA; 98 The formation and secretion of cholesteryl esters in rat hepatocytes are reduced by lovastatin and simvastatin. Biochem Soc Trans 1 February 1998; 26 (1): S82. doi: https://doi.org/10.1042/bst026s082 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: CE, cholesteryl ester, FC, free cholesterol, HMG-CoA, 3-hydroxy, 3-methylglutaryl-CoA, LDL, low density lipoprotein, SDS-PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulphate, PL, phospholipids, TG, triglycerides, VLDL, very low density lipoprotein This content is only available as a PDF. © 1998 Biochemical Society1998 Article PDF first page preview Close Modal You do not currently have access to this content.

Effect of micellar beta-sitosterol on cholesterol metabolism in CaCo-2 cells

CaCo-2 cells were used to address the effect of the plant sterol, beta-sitosterol, on cholesterol trafficking, cholesterol metabolism, and apoB secretion. Compared to cells incubated with micelles (5 mM taurocholate and 250 microM oleic acid) containing cholesterol, which caused an increase in the influx of plasma membrane cholesterol to the endoplasmic reticulum and increased the secretion of cholesteryl esters derived from the plasma membrane, beta-sitosterol did not alter cholesterol trafficking or cholesteryl ester secretion. Including beta-sitosterol in the micelle together with cholesterol attenuated the influx of plasma membrane cholesterol and prevented the secretion of cholesteryl esters derived from the plasma membrane. Stigmasterol and campesterol had effects similar to beta-sitosterol, although campesterol did not promote a modest influx of plasma membrane cholesterol. Including beta-sitosterol in the micelle with cholesterol decreased the uptake of cholesterol. Compared to cholesterol, 60% less beta-sitosterol was taken up by CaCo-2 cells. No observable esterification of beta-sitosterol was appreciated and the transport of the plant sterol to the basolateral medium was negligible. Cholesterol synthesis and HMG-CoA reductase activities were decreased in cells incubated with beta-sitosterol. This was associated with a decrease in reductase mass and mRNA levels. Cholesteryl ester synthesis and ACAT activities were unaltered by beta-sitosterol. Both stigmasterol and campesterol decreased reductase activity, but only campesterol increased ACAT activity. beta-sitosterol did not affect the secretion of apoB mass. The results suggest that beta-sitosterol does not promote cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. beta-sitosterol interferes with the uptake of micellar cholesterol causing less plasma membrane cholesterol to influx and less cholesteryl ester to be secreted. Despite its lack of effect on cholesterol trafficking, beta-sitosterol decreases cholesterol synthesis at the level of HMG-CoA reductase gene expression.

Effect of F-1394, a potent and selective inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), on esterification of cholesterol and basolateral secretion of cholesteryl ester in Caco-2 cells

The present study was conducted to investigate the inhibitory effect of F-1394, a potent and selective inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), on incorporation of 14C-oleic acid into cholesteryl ester in cultured Caco-2 cells, a human intestinal cell line, and compare its effect to those of other ACAT inhibitors and hypolipidemic agents. The cholesterol esterification in Caco-2 cells was strongly inhibited by F-1394 in a concentration-dependent manner with the estimated IC50 value of 71 nM. In contrast, the estimated IC50 values of the other ACAT inhibitors such as YM-17E, CI-976, CL-277,082 and DL-melinamide are 121 nM, 702 nM, 21.5 microM and 20.9 microM, respectively. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, also inhibited the ACAT activity in Caco-2 cells with an IC50 value of 22.5 microM, whereas pravastatin Na, probucol and clofibrate did not affect the activity. Furthermore, F-1394 at a concentration of 100 nM inhibited the basolateral secretion of cholesteryl ester by 90% from differentiated Caco-2 cells that were cultured on a membrane filter. These results demonstrate that F-1394 strongly inhibits human intestinal ACAT activity and basolateral secretion of cholesterol from Caco-2 cells. Therefore, F-1394 may have a therapeutic potential for dietary hyperlipidemic subjects.

Effect of ethanol on the synthesis and secretion of apoA-I- and apoB-containing lipoproteins in HepG2 cells

The short- and long-term effects of ethanol on the production of lipids and apolipoproteins (apo) in HepG2 cells were studied. Short-term incubation with 1% ethanol caused a significant 32% increase in the cellular content of both triglycerides and cholesteryl esters. Under these conditions, the net accumulation in the medium of triglycerides, unesterified cholesterol, cholesteryl esters, apoA-I, and apoE was stimulated by 75%, 41%, 43%, 19%, and 39%, respectively. ApoA-I and apoE mRNA levels increased by 15%. The major short-term effect of ethanol was on the net accumulation of apoB in the medium which was stimulated by 56-100% in the presence of 0.1-1.0% ethanol. Under these conditions, apoB mRNA abundance was elevated by 17-26% and LDL receptor activity was unchanged. The increase in apoB accumulation in the medium was predominantly due to augmented secretion of newly synthesized apoB-100 which was evident at 0.05% ethanol. The secretion of newly synthesized apoA-I was not altered by short-term incubation with < or = 0.5% ethanol. The rate of apoB production was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. Addition of Pfizer CP-113,818, an inhibitor of acyl-CoA:cholesterol acyltransferase, caused a 69% reduction in the secretion of cholesteryl esters and a 24% decrease in that of apoB-100. In contrast to the short-term effect of ethanol, long-term incubation with ethanol resulted in a dose-dependent increase in the secretion of newly synthesized apoA-I without significantly affecting that of apoB-100. The increase in apoA-I secretion was evident at 0.05% ethanol and reached a maximum of 77% at 0.5% ethanol. These observations indicate that in HepG2 cells the effect of ethanol on the production of apoA-I- and apoB- containing lipoproteins is both time- and dose-dependent and is different in these two apolipoproteins.

Effect of a coffee lipid (cafestol) on regulation of lipid metabolism in CaCo-2 cells.

The influence of cafestol, a lipid component found in boiled coffee, on low density lipoprotein (LDL) and lipid metabolism was investigated in CaCo-2 cells cultured on filter membranes. The rate of uptake and degradation of 125I-labeled tyramine cellobiose-LDL was increased 50% in CaCo-2 cells incubated with cafestol (20 micrograms/ml, 63 microM) for 24 h, whereas in cells incubated with 25-hydroxycholesterol (10 micrograms/ml, 25 microM) the rate of uptake and degradation showed a 30% decrease. A mixture of kahweol and cafestol, both natural components of coffee beans, modestly enhanced the rate of LDL uptake and degradation, as compared to pure cafestol. Incubation of cafestol with CaCo-2 cells induced a 3-fold up-regulation of LDL receptor mRNA, as compared to control cells. In contrast, incubation of the cells with 25-hydroxycholesterol produced a 30% decrease of LDL receptor expression. CaCo-2 cells were transfected with a promoter region containing the sterol regulatory element-1 (SRE-1) coupled to the reporter gene chloramphenicol acetyltransferase (CAT). When cells transfected with SRE-1 promoter were incubated with cafestol, there was a 20% up-regulation of CAT activity, whereas 25-hydroxycholesterol abolished this activity. Cafestol contributed to a significantly lowered secretion of cholesteryl ester and triacylglycerol, regardless of the radiolabeled precursor used ([2-14C]acetic acid, [1,2,3-3H]glycerol, [3H]water, and [1-14C]oleic acid). This reduction in secretion of lipids was accompanied by an increase in trichloroacetic acid-soluble activity when radiolabeled oleic acid was used as a tracer. We conclude that cafestol promotes an enhanced rate of uptake and degradation of LDL, probably due to an increase in transcription of LDL receptor mRNA and a reduced secretion of cholesteryl ester and triacylglycerol in CaCo-2 cells.

Role of the neutral lipid accessible pool in the regulation of secretion of apoB-100 lipoprotein particles by HepG2 cells.

The rate of secretion of apoB-100-containing lipoprotein particles by HepG2 cells is determined to an important extent by post-translational mechanisms, the mass of neutral lipids clearly playing a role in this process. Our previous data indicated that cholesteryl ester might influence the proportion of newly synthesized apoB-100 molecules that are incorporated into nascent lipoproteins rather than being catabolized intracellularly shortly after they have been synthesized. The present studies, therefore, were designed: 1) to examine in more detail the relationship between the mass of triglyceride and cholesteryl ester in HepG2 cells and the rate of apoB-100 secretion, and 2) to determine whether cholesteryl ester molecules that have been synthesized and stored within these cells must undergo hydrolysis and re-esterification before being secreted with newly synthesized apoB-100 molecules. Changes in apoB-100 secretion in HepG2 cells were assessed in response to changes in intracellular triglyceride and/or cholesteryl ester pool size. This was accomplished through lipid loading of the cells by incubating them overnight with exogenously supplied very low density lipoprotein (VLDL) (with lipoprotein lipase, LPL), low density lipoprotein (LDL), oleate, or LDL + oleate. The medium was changed to fresh serum-free medium and apoB-100 secretion was shown to increase over at least 8 h. After overnight incubation with VLDL, intracellular triglyceride mass increased 6-fold, while intracellular cholesteryl ester mass increased 2-fold. Medium apoB-100 increased up to 3-fold, while apoB-100 mRNA increased by only 12%. Both heparin (10 IU/ml) and lactoferrin (20 microM) independently blocked the VLDL-mediated increases in intracellular cholesteryl ester mass (-56% and -46%) without decreasing triglyceride mass. ApoB-100 secretion was also reduced by 53% and 72%, respectively. Incubation of HepG2 cells with LDL increased intracellular cholesteryl ester mass but triglyceride mass remained unchanged. In this instance, apoB-100 secretion increased 2-fold but there was no change in apoB-100 mRNA. Overall, there was little relationship between the mass of intracellular triglyceride and the rate of apoB-100 secretion (r2 = 0.034, NS) whereas there was a strong correlation between the intracellular mass of cholesteryl ester and apoB-100 secretion (r2 = 0.67, P < 0.0005). To examine the process of cholesteryl ester secretion, intracellular triglyceride and cholesteryl ester mass were increased after incubation with LDL + oleate. The medium was then changed to fresh serum-free medium containing an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor (Sandoz compound 58-035). Although de novo cholesteryl ester synthesis was inhibited up to 89%, cholesteryl ester mass secretion remained constant with up to 15% of total cholesteryl ester mass secreted over the 8-h period. ApoB-100 secretion also remained elevated above control, with 92% of the cholesteryl ester secreted associated with apoB-100 particles (27% with d < 1.006 g/mL particles and 65% with d 1.006-1.063 g/mL particles). Therefore, not only newly synthesized cholesteryl ester but also stored cholesteryl ester can associate with newly synthesized apoB-100 molecules and can be secreted without the necessity of an intracellular hydrolysis/re-esterification step.

ACAT inhibitors CL 283,546 and CL 283,796 reduce LDL cholesterol without affecting cholesterol absorption in African green monkeys.

Previous studies with a number of selective acylcoenzyme A:cholesterol acyltransferase (ACAT) inhibitors in several animal models have demonstrated significant reductions in plasma cholesterol and, in some studies, triglyceride levels. This study was conducted to examine the effects of two ACAT inhibitors, CL 283,546 and CL 283,796, in cholesterol-high fat diet fed African green monkeys, a relevant primate model of hyperlipidemia and coronary artery atherosclerosis. Treatment with CL 283,546 or CL 283,796 resulted in significant reductions (ca. 25-30%) in total plasma cholesterol at both 10 and 30 mg/kg per day doses. This reduction in plasma cholesterol was due almost entirely to reduction in low density lipoprotein (LDL) cholesterol (ca. 45%) without significantly affecting high density lipoprotein (HDL) cholesterol, very low density lipoprotein + intermediate density lipoprotein (VLDL + IDL) cholesterol, or triglyceride concentrations. There were no significant effects on plasma concentrations of apolipoproteins A-I, E, or B and, thus, the reduction seen in LDL cholesterol appears to be due to a diminished cholesterol content of LDL particles. Our studies revealed that treatment with these compounds did not reduce cholesterol absorption, which was somewhat surprising as ACAT inhibitors are generally thought to exert their hypolipidemic effects, at least in part, by inhibition of intestinal cholesterol absorption. Our data are consistent with a principal activity of these drugs on the liver to reduce cholesteryl ester secretion in VLDL, leading to a diminished LDL-cholesterol content, and, presumably, enhanced biliary cholesterol-bile acid excretion.

ACAT inhibitors decrease secretion of cholesteryl esters and apolipoprotein B by perfused livers of African green monkeys

To test the hypothesis that newly synthesized cholesteryl esters are required for hepatic lipoprotein assembly and secretion, isolated livers of African green monkeys were perfused with medium containing an ACAT inhibitor. Three ACAT inhibitors with different structural and chemical properties were used: CI-976 (Parke-Davis), CP-113818 (Pfizer), or PD-138142-15 (Parke-Davis). Each compound produced variable effects on secretion of lipids and apolipoproteins. A significant decrease in the secretion rates of cholesteryl ester and apoB was common to all three inhibitors, indicating that fewer lipoprotein particles were secreted during ACAT inhibition. Triacylglycerol secretion was decreased in the presence of CP-113818 and PD-138142-15 but no decrease in triacylglycerol secretion was observed with CI-976. Particles secreted in the presence of CI-976 were enriched in triacylglycerol relative to apoB. Effects on secretion of other apolipoproteins (apoA-I, apoA-II, and apoE) were variable. When all data were combined, the percent inhibition of cholesteryl ester secretion and apoB secretion in the presence of ACAT inhibitors was positively correlated (r = 0.84), whereas a similar relationship was not observed between triacylglycerol and apoB. While the results demonstrate some lack of specificity for these ACAT inhibitors, the complimentary results using the three different ACAT inhibitors suggest that secretion of cholesteryl ester and apoB are coordinately regulated. The data suggest that newly synthesized cholesteryl esters may participate in and promote the assembly and secretion of hepatic apoB-containing lipoproteins. The availability of triacylglycerol during lipoprotein assembly, while also important, would appear to serve a role separate from that of cholesteryl ester.