Inhibition of acylcoenzyme A:cholesterol acyltransferase activity in CaCo-2 cells results in intracellular triglyceride accumulation

The activity of acylcoenzyme A:cholesterol acyltransferase (ACAT) in CaCo-2 cells was inhibited by the ACAT inhibitor, 58-035. The inhibitory effect of this acylamide was specific for cholesterol esterification catalyzed by ACAT; the rates of triglyceride, phospholipid, and cholesterol synthesis were not inhibited by this agent. Cholesteryl esters were depleted in CaCo-2 cells 24 hr after inhibition of ACAT activity, whereas the unesterified cholesterol content increased by 56% after 96 hr. Moreover, inhibiting ACAT activity with 58-035 resulted in a time-dependent 2.5-fold increase in intracellular triglycerides. This accumulation of triglycerides in CaCo-2 cells was associated with a 37% increase in triglyceride synthesis by 96 hr in the presence of 58-035. Triglyceride-rich lipoprotein secretion (d less than 1.006 g/ml) was not affected by inhibiting ACAT activity for up to 6 hr. However, triglyceride-rich lipoprotein secretion was significantly decreased in CaCo-2 cells that were preincubated with 58-035 for 24 to 96 hr. Lipoproteins of density less than 1.006 g/ml that were isolated from CaCo-2 cells incubated with the ACAT inhibitor were deficient in cholesteryl esters and triglycerides compared to lipoproteins isolated from control cells. The data suggest that triglycerides accumulate in CaCo-2 cells in which ACAT activity has been inhibited by 58-035. This accumulation of triglycerides is associated with a modest increase in triglyceride synthesis and a decrease in triglyceride secretion. Altering intracellular cholesterol pools by regulating ACAT activity in the gut could result in the decrease of triglyceride transport and/or the secretion of triglyceride-rich lipoprotein particles of abnormal composition.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.     

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.     

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.     

Oleic acid decreases the expression of a cholesterol transport-related protein (NPC1L1) by the induction of endoplasmic reticulum stress in CaCo-2 cells

The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25–1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.     

An inhibitor of acylCoA: cholesterol acyltransferase increases expression of ATP-binding cassette transporter A1 and thereby enhances the ApoA-I-mediated release of cholesterol from macrophages

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments.     

Investigation of the role of micellar phospholipid in the preferential uptake of cholesterol over sitosterol by dispersed rat jejunal villus cells

The uptake of radioactive cholesterol and sitosterol by rat jejunal villus cells was examined using mixed micellar solutions containing sodium taurocholate, equimolar mixtures of the two sterols, and a variety of phospholipid types. The addition of phospholipid to the incubation solutions reduced the cellular absorption of both sterols and gave rise to uptake kinetics that were linear with time. In the presence of egg yolk phospholipid, uptake of the sterols by villus cells occurred with a modest preference for cholesterol over sitosterol. The ratio of accumulated cholesterol/sitosterol increased from 1.0 initially to 1.23 +/- 0.04 (n = 18) after a 30-min incubation at 37 degrees C. The selectivity displayed in the villus cells increased significantly as egg phosphatidylethanolamine was added to the egg phosphatidylcholine (PC) preparation in micellar solution. It was markedly decreased when dipalmitoyl PC or the primarily saturated egg yolk sphingomyelin were incorporated into the micelles. In every case examined, phospholipid was taken up by the cells concurrently with the sterols. The selectivity between cholesterol and sitosterol was maintained when the donor species were multilamellar vesicles composed of egg PC and the sterols, but not when the donor particles were albumin-stabilized sterol dispersions or taurocholate solutions in the absence of PC. The results show that the selective absorption of cholesterol over the plant sterol occurs only in the presence of unsaturated phospholipid. The phospholipid may act by influencing the permeability of the cellular membranes to the two sterols or the rate of sterol desorption from the phospholipid-containing micellar or liposomal carriers.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of inhibition of cholesterol esterification on the fate of cholesterol derived from HDL in rat hepatocyte monolayers

Rat HDL2 is known to stimulate bile acid synthesis in rat hepatocyte monolayers. The intracellular fate of the cholesterol derived from the HDL2 was studied using the inhibitor of cholesterol esterification, Sandoz compound 58-035. Rat HDL2 added to rat hepatocyte monolayers caused a stimulation of cholesterol esterification of 32%. This stimulation could be inhibited by 58-035. A small significant increase in bile acid synthesis was also observed in cells in the presence of HDL2, confirming our earlier observations. 58-035 prevented this increase. These observations imply that cholesterol entering the cell from HDL2 is first esterified and can only enter the substrate pool for bile acid synthesis after subsequent intracellular hydrolysis.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.     

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

The Th1-derived cytokine gamma interferon, IFN-gamma, is present within the microenvironment of an atheromatous lesion and likely contributes to lesion progression through macrophage activation. While the inflammatory effects of IFN-gamma are well known, the role of this cytokine in cholesterol metabolism in macrophage derived foam cells is unclear. In the present study, the incubation of foam cells with IFN-gamma resulted in the reduction of HDL(3)-mediated cholesterol efflux. The decrease in cholesterol efflux was not observed with other macrophage-activating factors as colony-stimulating factors failed to demonstrate a similar effect. The reduction in cholesterol efflux was independent of apoE synthesis or SR-BI expression and was associated with a redistribution of intracellular cholesterol with an increase in cholesteryl ester accumulation. The increase in the esterified pool, primarily in cholesterol eicosapentadenoate, docosapentaenoate, arachidonate, and linoleate was associated with a 2-fold increase in acyl-CoA:cholesterol-O-acyltransferase, ACAT, activity and message without any change in neutral cholesteryl ester hydrolase activity. While CD36 message was reduced in IFN-gamma-treated foam cells, the ability to reverse the decrease in efflux by the ACAT inhibitor A58035 in a dose-dependent manner suggests that the IFN-gamma effect on efflux is primarily through the modulation of ACAT expression. Therefore, in addition to its inflammatory effects, IFN-gamma can contribute to the progression of an atherosclerotic lesion by altering the pathway of intracellular cholesterol trafficking in macrophage derived foam cells.     

Inhibition of cholesterol absorption in rats by plant sterols

The extent and site(s) of inhibition of cholesterol absorption by plant sterols, sitosterol and fucosterol, were studied in rats. The intragastric administration of a single emulsified lipid meal containing 25 mg [3H]cholesterol and 25 mg of either sitosterol or fucosterol inhibited the lymphatic absorption of cholesterol by 57% and 41%, respectively, in 24 hr. Less than 2% of each plant sterol was absorbed in the 24-hr period. In contrast, neither plant sterol (50 microM) inhibited cholesterol absorption when co-administered with equimolar amounts of cholesterol in phospholipid-bile salt micelles nor was either absorbed from the micellar solution. A series of in vitro studies was conducted to identify the site(s) of plant sterol inhibition of cholesterol absorption and to account for the difference in inhibitory effectiveness of sitosterol and fucosterol. A comparison of the micellar solubility of each sterol alone and in equimolar binary mixtures (to 2.0 mM) revealed that the solubility of individual sterols decreased in the following order: cholesterol, fucosterol, sitosterol, and that in binary mixtures cholesterol solubility was decreased by sitosterol and, to a lesser extent, by fucosterol relative to its solubility alone. A comparison between micellar-solubilized cholesterol and either sitosterol or fucosterol for binding to isolated brush border membranes, intestinal mucin, or for esterification by either cholesterol esterase or acyl coenzyme A:cholesterol acyltransferase revealed moderate to no competition. The data suggest that plant sterols displace cholesterol from bile salt (taurocholate) micelles and that sitosterol is more effective than fucosterol in this capacity.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification

Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification.     

Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm-acclimated carp. Based on current concepts of cholesterol metabolism, it is concluded that the cold-enhanced expression of hepatic HMG-CoA reductase activity is a homeostatic response directed against and compensating for a cold-induced but not yet characterized deficiency in hepatic cholesterol availability.     

Differences in the release of cholesterol from taurocholate versus taurochenodeoxycholate micellar solutions

The maximal micellar solubility, distribution and apparent monomer activity of cholesterol in taurine-conjugated cholate and chenodeoxycholate micellar solutions were studied to clarify the different modulating effect of these bile salt species on cholesterol uptake in an intestinal lumen. The maximal micellar solubility was significantly greater in taurochenodeoxycholate. The intermicellar cholesterol monomer concentration was not significantly different between the two kinds of micellar solution. However, the apparent cholesterol monomer activity determined using an artificial organic phase (polyethylene disc) was significantly higher in taurocholate than that in taurochenodeoxycholate. A linear relationship between the intermicellar cholesterol concentration and the apparent cholesterol monomer activity was found, with the slope depending upon the bile salt species. It is concluded that the difference in partitioning of cholesterol from taurocholate and taurochenodeoxycholate micelles into a fixed organic phase may contribute in part to the different regulating effects of these bile salts on the uptake of cholesterol in the intraluminal phase.