Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

Regulation of rat hepatic cholesterol metabolism. Effects of lipoprotein composition on acyl coenzyme A:cholesterol acyltransferase in vivo and in the perfused liver and on hepatic cholesterol secretion

Lipoproteins that are removed from the circulation by the liver can deliver both cholesterol and triglycerides to the hepatocyte. Relative proportions of these lipids may vary in lipoproteins and, thus, their uptake may have differing effects on cholesterol homeostasis. To study this, lipoproteins containing the same amounts of cholesterol but different amounts of triglyceride were administered to intact rats or to an isolated perfused rat liver. The responses of acyl coenzyme A:cholesterol acyltransferase (ACAT), very low density lipoprotein (VLDL) triglyceride and cholesterol secretion, and biliary cholesterol content were examined after 2 hr. Administration of triglyceride-rich chylomicrons (average triglyceride:cholesterol = 136.5 by mass) in vivo or their remnants (average triglyceride:cholesterol = 32.7 by mass) to the perfused liver resulted in an 80% decrease in ACAT activity. In the perfused liver system, VLDL cholesterol and triglyceride secretion was increased while biliary cholesterol content decreased. Administration of standard chylomicrons (average triglyceride:cholesterol = 33.9 by mass) or their remnants (average triglyceride:cholesterol = 11.4 by mass) lowered ACAT activity by 24% in vivo, but had no significant effect on any of the parameters measured in the perfused liver system. Administration of cholesterol-rich VLDL (average triglyceride:cholesterol = 0.47 by mass) in vivo increased ACAT activity 1.4-fold, but administration of their remnants (average triglyceride:cholesterol = 0.17 by mass) had little effect on any of the parameters measured in the perfused liver. Thus, the lipid composition of lipoproteins removed by the liver elicited acute responses by parameters important in the maintenance of hepatic cholesterol homeostasis. These responses reflected the net effects of both the cholesterol and the triglyceride contents of the particles.     

Regulation of hepatic cholesterol biosynthesis by berberine during hyperhomocysteinemia

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. We previously demonstrated that hyperhomocysteinemia stimulated hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leading to hepatic lipid accumulation and liver injury. The liver plays an important role in cholesterol biosynthesis and overall homeostasis. HMG-CoA reductase catalyzes the rate-limiting step in cholesterol biosynthesis. Hepatic HMG-CoA reductase is a major target for lowering cholesterol levels in patients with hypercholesterolemia. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on hepatic cholesterol biosynthesis in hyperhomocysteinemic rats and to identify the underlying mechanism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. HMG-CoA reductase activity was markedly elevated in the liver of hyperhomocysteinemic rats, which was accompanied by hepatic lipid accumulation. Activation of HMG-CoA reductase was caused by an increase in its gene expression and a reduction in its phosphorylation (an inactive form of the enzyme). Treatment of hyperhomocysteinemic rats with berberine for 5 days inhibited HMG-CoA reductase activity and reduced hepatic cholesterol content. Such an inhibitory effect was mediated by increased phosphorylation of HMG-CoA reductase. Berberine treatment also improved liver function. These results suggest that berberine regulates hepatic cholesterol biosynthesis via increased phosphorylation of HMG-CoA reductase. Berberine may be therapeutically useful for the management of cholesterol homeostasis.     

Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diminution of blood and hepatic cholesterol induced by an apple-supplemented diet in the hamster. Trials in man]

The effects of an apple-supplemented diet on plasma and liver cholesterol were investigated in two strains of Hamster, a normal one and another which exhibits a spontaneous accumulation of cholesteryl esters in the liver (FEC animals). Adding of apples to the standard diet promoted a 20 % decrease of cholesterolemia in normal animals and normalized the high level of plasma cholesterol in the FEC animals. Similarly, the cholesterol content of the liver was diminished. The levels of cholesteryl esters were strongly lowered, threefold and tenfold respectively in normal and in FEC animals. Incorporation of labelled mevalonate into cholesterol was increased, but esterification of the neosynthesized sterol was markedly reduced in the liver from FEC animals. In human daily ingestion of apples caused a significative reduction (16 %) of cholesterolemia.     

Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.     

Inhibition of hepatic cholesterol synthesis by the alpha 1-adrenoceptor blocker doxazosin in the hypercholesterolemic golden hamster

The effect of treatment with the alpha 1-specific adrenoceptor blocker, Doxazosin, on lipid parameters was studied in male Golden hamsters fed a cholesterol-enriched diet. Within 1 week the Doxazosin-treated animals had a lower plasma (-12%) and hepatic (-30%) cholesterol content than the cholesterol-fed controls. De novo cholesterol synthesis in the liver was lowered by 39% in the Doxazosin-treated animals. These data indicate that the reported beneficial effect of alpha 1-blockade on plasma cholesterol levels may be due to lowering of the hepatic cholesterol synthesis.     

Regulation of hepatic cholesterogenesis by polar steroids accumulated after cholesterol feeding

The incorporation of mevalonate into nonsaponifiable lipids by chick liverin vivo strongly increased between 1–18 days after hatching. Cholesterol feeding (2%) inhibited this. Synthesis of cholesterol was strongly inhibited, whereas the intermediates isolated by TLC accumulated. Most of the polar nonsaponifiable lipids that accumulated in liver 90 minutes after mevalonate administration to 18-day-old cholesterol-fed chicks were identified as lanosterol derivative. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, as well as acetate and mevalonate incorporation into nonsaponifiable lipids, was inhibited by the presence of these compounds. To our knowledge, this is the first report of such inhibition; this confirms the physiological function of polar steroids in the regulation of cholesterogenesisin vivo.To whom correspondence should be addressed.     

Effects of dietary cholesterol and fatty acids on plasma cholesterol level and hepatic lipoprotein metabolism

The effects of dietary cholesterol and fatty acids on the plasma cholesterol level and rates of very low density lipoprotein (VLDL) cholesterol secretion and low density lipoprotein (LDL) transport through LDL receptors in the liver of the hamster were investigated. Increases of plasma VLDL- and LDL-cholesterol levels and VLDL-cholesterol secretion from hepatocytes were observed in animals fed a diet enriched with 0.1% cholesterol for 2 weeks in comparison with animals fed a control diet. The addition of dietary palmitic acid accelerated the effect of dietary cholesterol on plasma VLDL- and LDL-cholesterol levels and VLDL-cholesterol secretion from hepatocytes. Dietary linoleic acid accelerated the effect of dietary cholesterol on VLDL-cholesterol secretion from hepatocytes and diminished the effect on the plasma LDL-cholesterol level. Hepatic LDL receptor activity was considerably suppressed by a control diet containing 0.05% cholesterol and a further small suppression was induced by a diet enriched with 0.1% cholesterol with or without 5% palmitic acid. However, dietary linoleic acid diminished the effect of dietary cholesterol on the suppression of hepatic LDL receptor activity. These results suggest that dietary palmitic acid augments the effect of dietary cholesterol in elevating the plasma LDL-cholesterol level through acceleration of VLDL-cholesterol secretion from the liver, and that dietary linoleic acid diminishes the effect of dietary cholesterol in elevating the plasma LDL-cholesterol level by preventing the suppression of hepatic LDL receptor activity induced by cholesterol.     

Regulation of hepatic lanosterol 14 alpha-demethylase gene expression by dietary cholesterol and cholesterol-lowering agents.

Binding of sterol response element binding protein 1a to sterol response element-1 (SRE-1) in the promoter region of lanosterol 14 alpha-demethylase (14DM) has been demonstrated previously. Decreased 14DM activity has been shown to result in accumulation of the intermediate, 3 beta-hydroxy-lanost-8-en-32-al, a known translational downregulator of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Since it has also been demonstrated that feedback regulation of hepatic HMG-CoA reductase occurs primarily at the level of translation, the effects of dietary cholesterol and cholesterol lowering agents on levels of hepatic 14DM mRNA and immunoreactive protein were investigated. Addition of 1% cholesterol to a chow diet markedly decreased hepatic 14DM mRNA and protein levels in Sprague-Dawley rats. The extent and time course of this decrease in 14DM immunoreactive protein closely paralleled that of HMG-CoA reductase. Supplementation of the diet with the HMG-CoA reductase inhibitor, Lovastatin, to a level of 0.02%, raised 14DM mRNA and protein levels 2- to 3-fold. Addition of 2% Colestipol, a bile acid binding resin, to the chow diet caused smaller increases. The highest level of 14DM protein expression was observed in liver, the major site of feedback regulation of HMG-CoA reductase by cholesterol. Taken together, these observations suggest a critical role for 14DM in the feedback regulation of hepatic HMG-CoA reductase.     

Regulation of guinea pig hepatic acyl-coa:cholesterol acyltransferase activity by dietary fat saturation and cholesterol

We measured the interactive effects of dietary cholesterol and fat on the regulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity and its relationship to hepatic microsomal lipid composition in guinea pigs fed 15 g/100 g (w/w) fat diets (corn oil, olive oil, or lard) with 0.01, 0.08, 0.17, or 0.33 g/100 g (w/w) added cholesterol. Guinea pigs exhibited a dose dependent increase in hepatic microsomal ACAT activity, with increasing levels of cholesterol intake (P < 0.001) in all dietary fat groups. Animals fed monounsaturated olive oil had the highest hepatic ACAT activity with the exception of the 0.33 g/100 g cholesterol diet (P < 0.001). There were no differences in ACAT activity with intake of polyunsaturated corn oil or saturated lard. Dietary cholesterol resulted in increased microsomal free cholesterol (FC) concentrations in a dose dependent manner but had no effects on microsomal phosphatidylcholine (PC) concentrations. Guinea pigs fed olive oil generally had the highest microsomal FC/PC molar ratios, and hepatic ACAT activities correlated significantly with this parameter. After modification of the lipid compositions of the microsomes from guinea pigs fed the 12 test diets with FC/PC liposome treatment, microsomal ACAT activities remained significantly related to the microsomal FC/PC molar ratios, and dietary fat type did not affect this correlation. Our findings do not support the hypothesis that the stimulation of hepatic ACAT activity with cholesterol intake is enhanced by polyunsaturated fat intake. The data demonstrate that although dietary fat type and cholesterol amount have differential effects on hepatic ACAT activity, substrate availability, expressed as microsomal FC/PC molar ratio, is a major regulator of hepatic microsomal ACAT activity.     

Regulation of cholesterol 7 alpha-hydroxylase by hepatic 7 alpha-hydroxylated bile acid flux and newly synthesized cholesterol supply

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.     

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.     

Female sex steroid induced epithelial changes in the gallbladder of the ovariectomized Syrian hamster.

Ovariectomized Syrian hamsters treated by female sex steroids during a 1-month period show gallbladder surface epithelial changes in the fundic area consistent with apical bulging and decapitations of the epithelial cells. These events were detected in the infundibulum and the fundic or body regions of estrogen- and estrogen+progesterone-treated hamsters. In control hamsters, these events were restricted to the region in the vicinity of the bile duct. Following steroid treatment, intraluminal deposits detected resembled Ca-bilirubinate deposits described in previous studies while decapitations are similar to endometrial epithelium changes associated with hormonal physiological changes or treatments. Moreover some small electron-dense deposits are comparable to those found in human cholesterol gallstones. This report indicates that, besides an alteration in bile composition, cell fragments originating from the surface epithelium of the bile duct and/or of the gallbladder mucosal epithelium could participate in gallstone nucleation.