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.     

Normal cholesterol absorption in rats deficient in intestinal acyl coenzyme A:cholesterol acyltransferase activity

Acyl coenzyme A:cholesterol acyl transferase and/or cholesterol esterase may regulate the esterification and absorption of exogenous cholesterol. To assess this, mucosal acyl coenzyme A:cholesterol acyl transferase activity was inhibited selectively with three different drugs [Sandoz #58-035, inhibitor 1; Lederle inhibitor 2 and inhibitor 3] and the effect upon the absorption of a [4-14C]cholesterol meal was studied in the lymph fistula rat. Compared to control rats, ACAT activity measured in mucosal homogenates from the drug-treated rats was reduced 80-90%, 40%, and 30%, respectively, during the predicted time-frame for maximum mucosal esterification of cholesterol (i.e., after cholesterol is fed and before it appears in lymph). In contrast, [14C]cholesterol absorption in the drug-treated animals was unchanged from controls [5.7 +/- 1.2 (inhibitor 1) vs. 5.4 +/- 1.6 mumol/6 hr (control); 6.1 +/- 2.1 (inhibitor 2) and 5.2 +/- 1.5 (inhibitor 3) vs. 4.1 +/- 1.3 mumol/6 hr (control)]. Of the absorbed [14C]cholesterol, approximately 75% was esterified in all groups. Cholesterol esterase activity measured in the drug-treated rats was unchanged compared to controls nor did the drugs inhibit this enzyme in vitro. Under the conditions of this study, drugs causing substantial inhibition of acyl coenzyme A:cholesterol acyl transferase activity had no effect on the absorption of exogenous cholesterol.     

Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035

Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]pro panamide) has been found to inhibit the accumulation of cholesteryl esters in both rat hepatoma (Fu5AH) cells and arterial smooth muscle cells in culture. To explore the specificity of 58-035, we have studied the esterification of cholesterol, retinol, and glycerides by the Fu5AH cell and by isolated membranes. Exposure of Fu5AH to cholesterol/phospholipid dispersions and 58-035 (greater than 100 ng/ml) for 24 h resulted in greater than 95% inhibition of cholesterol esterification while cellular free cholesterol increased slightly. Inhibition was also rapid; incorporation of [3H]oleate into cholesteryl [3H]oleate equaled only 12% of control value after 30 min with 58-035 at 5 micrograms/ml. In contrast, there was no decrease in [3H]oleate incorporation into phospholipids or diglycerides, nor was the esterification of [3H]retinol inhibited by 58-035. In microsomal fractions, acyl-CoA:cholesterol acyltransferase could be inhibited completely by 58-035, while activities of acyl-CoA: retinol acyltransferase and triglyceride synthesis proceeded at 75-100% of control values. These observations that 58-035 is highly selective allow the inference that acyl-CoA:cholesterol acyltransferase is a separate microsomal enzyme whose activity can be modulated independently from acyl-CoA:retinol acyltransferase and other cellular acyltransferases.     

Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.     

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.     

Regulation of protein secretion by crinophagy in perfused rat liver.

We examined the secretion of three serum proteins, albumin (RSA), alpha 2 mu-globulin (alpha 2 mu G), and transferrin (Trf), in the isolated perfused liver. Within 4 h of perfusion, only 20 to 35% of previously synthesized proteins were secreted by the liver into the recirculating medium. Low temperature inhibited the secretion of alpha 2 mu G and Trf, but not RSA. The amount of RSA secreted by the liver increased twofold in the presence of leupeptin, a proteinase inhibitor, or primaquine, a weak base capable of neutralizing acidic compartments. Neither drug affected Trf secretion, while the release of alpha 2 mu G was enhanced threefold by primaquine treatment. Only 55 to 70% of the total amount of these serum proteins present in the liver at the onset of perfusion could be accounted for after 4 h of perfusion. Our evidence suggests that these losses are due to protein degradation. The degradation of RSA and alpha 2 mu G was inhibited at 15 degrees C and by both leupeptin and primaquine. Contrary, RSA degradation was not altered when livers were perfused at 20 degrees C. Morphological techniques combined with immunological probes were utilized to identify possible intracellular sites of RSA degradation. RSA and cathepsin L were colocalized to large vacuoles found near the cell periphery. Entry of RSA into these vacuoles occurred at 20 degrees C but not at 15 degrees C. Our results using perfused rat livers suggest that as much as 40% of hepatic serum proteins are degraded via fusion of secretory vesicles with lysosomes (e.g., crinophagy).     

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)     

Sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase from the corn earworm, Heliothis zea

The enzymatic activity and sterol substrate specificity of acyl coenzyme A:cholesterol acyltransferase (ACAT) were measured in microsomes of cells from Heliothis zea. Under standard assay conditions, the specific enzymatic activity of ACAT was highest in the intestine followed by the fat body and ovary (380.7, 30.7, 8.3 pmol/min per mg, respectively). The structure of the exogenous sterol used in the ACAT assay affected its rate of esterification. The relative rates of esterification of analogs of cholesterol with various modifications of the side chain were: 24-H greater than 24 alpha-CH3 greater than delta 22 greater than delta 24 greater than 24 alpha-C2H5 greater than 24 beta-CH3, delta 22-24 beta-CH3 and delta 22-24 alpha-C2H5. The number and position of double bonds in the B-ring of the sterol nucleus greatly affected the rate of esterification of sterols by ACAT. The average relative rates of esterification of sterols with differences in their B-rings were: delta 7 much greater than delta 8 greater than delta 0 greater than delta 5 greater than delta 5.7. The presence of a 9,14-cyclopropane group and/or methyl groups at the C-4 and 14 positions prevented significant esterification of such sterols. The formation of cholesteryl and lathosteryl esters was partially inhibited in microsomes from the intestine, fat body, and ovary by the addition of the ACAT inhibitor, 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide (Sandoz Compound 58-035).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of production and release of lipoprotein by the perfused rat liver

The relationship between protein and triglyceride release into d < 1.007 lipoprotein was studied in the isolated perfused rat liver. Livers were perfused with a medium either high or low in linoleate content. Perfusion with the linoleate-rich medium resulted in a marked increase in the net release of both d < 1.007 lipoprotein triglyceride and lipoprotein protein, and caused a significant increase in amino acid incorporation into the protein moiety. Amino acid incorporation into d 1.008-1.21 protein was not affected by fatty acid concentration, while incorporation into whole perfusate and tissue proteins was depressed by a perfusate high in fatty acid content. Electron microscopic studies demonstrated that the livers with the higher rate of triglyceride release also produced a greater number of lipoprotein particles. The particles they released were also somewhat larger. These studies suggest that the intracellular concentration of newly esterified triglyceride and (or) some other lipid metabolite can specifically influence the release and perhaps the synthesis of d < 1.007 lipoprotein protein. They also suggest that the liver increases its rate of triglyceride release primarily by producing more lipoprotein particles.     

Regulation of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in the rat adrenal gland.

The activities of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in rat adrenal gland were measured at various time intervals over 24 h. The activity of cholesterol esterase displayed diurnal rhythm, with a major peak at the onset of darkness coinciding with the peak in the diurnal rhythm of plasma corticosterone concentration. The activity of acyl-CoA : cholesterol acyltransferase also exhibited a characteristic diurnal rhythm, with the minimum activity occurring 3 h after the onset of darkness. The profile of the rhythm exhibited by the activity of the esterifying enzyme was similar to the mirror image of the pattern of diurnal rhythm in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Microsomal non-esterified cholesterol showed a gradual decline with a significant decrease in concentration at the onset of darkness, thus suggesting that diurnal removal of cholesterol in the environment of the esterifying enzyme and hydroxymethylglutaryl-CoA reductase leads to such diurnal decrease or increase in the activities of these two enzymes. Acute administration of corticotropin led to a 3-fold increase in the activity of cholesterol esterase, a 50% decrease in the activity of acyl-CoA : cholesterol acyltransferase and a 2-fold increase in the activity of hydroxymethylglutaryl-CoA reductase. Corticotropin administration also resulted in a significant decrease in microsomal non-esterified cholesterol and increase in plasma corticosterone concentration. These observations suggest that corticotropin plays an important part in generating the diurnal rhythm in the activities of the three enzymes.     

Clearance of acetyl low density lipoprotein by rat liver endothelial cells. Implications for hepatic cholesterol metabolism

We have studied the hepatic uptake of human [14C] cholesteryl oleate labeled acetyl low density lipoprotein (LDL). Acetyl-LDL injected intravenously into rats was cleared from the blood with a half-life of about 10 min. About 80% of the injected acetyl-LDL was recovered in the liver after 1 h. Initially, most of the [14C]cholesterol was recovered in liver endothelial cells (about 60%). Some radioactivity (about 15%) was also recovered in the hepatocytes, while the Kupffer cells and stellate cells contained only small amounts of the label (less than 5%). About 1 h after injection, radioactivity started to disappear from endothelial cells and appeared instead in hepatocytes. Radioactivity subsequently declined in hepatocytes as well. After a lag phase of 4 h, significant amounts of radioactivity were recovered in bile. The in vitro uptake and hydrolysis of [14C]cholesteryl oleate-labeled acetyl-LDL were saturable in isolated rat liver endothelial cells. Native LDL does neither affect the uptake nor the hydrolysis of acetyl-LDL. Ammonia and monensin reduced the hydrolysis of acetyl-LDL in isolated liver endothelial cells. Furthermore, monensin at concentrations above 10 microM completely blocked the binding of acetyl-LDL to the liver endothelial cells, suggesting that the receptor for acetyl-LDL is trapped inside the cells. The liver endothelial cells may be involved in the protection against atherogenic lipoproteins, e.g. liver endothelial cells may mediate uptake of cholesterol from plasma and transfer of cholesterol to the hepatocytes for further secretion into the bile.     

Regulation of cholesterol metabolism in the ethionine-induced premalignant rat liver

The early premalignant liver provides a model in which to study metabolic alterations that may be permissive for the development of full malignancy. Although there are biochemical changes in this model, there are no detectable morphological ones when compared with a normal, fully differentiated liver. The maintenance of cholesterol homeostasis, essential for proper functioning of mammalian cells, is known to be altered in malignancy. We used the ethionine-induced premalignant liver model to study the effects of the premalignant state on cellular parameters involved in the maintenance of hepatic cholesterol homeostasis. Cholesterol synthesis was elevated about twofold in the livers of rats treated with ethionine as was the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, its rate limiting enzyme. There was no change in reductase activation state. Acyl coenzyme A:cholesterol acyl-transferase (ACAT) was decreased about 30%, and cholesterol 7 alpha-hydroxylase, about 50%. There was no significant change in neutral cholesteryl ester hydrolase activity, but acid hydrolase activity was decreased. There was little change in low density lipoprotein receptor protein as determined by immunoblotting. Biliary lipid secretion was in the normal range when expressed per gram liver; however, bile flow was doubled. The ethionine-fed animals were mildly hypocholesterolemic and had an altered serum lipoprotein pattern. Cholesterol synthesis and HMG-CoA reductase activity exhibited decreased sensitivities to inhibition by dietary cholesterol when compared to control livers. However, sensitivity to intragastrically administered mevalonolactone was not altered. Although ACAT activity was increased by mevalonolactone administration to levels similar to those in untreated animals, it was not increased in the ethionine-fed animals by feeding cholesterol. The ethionine-induced premalignant liver responded to ethinyl estradiol treatment in a manner similar to that of the control, i.e., profound hypolipidemia, increased low density lipoprotein receptors, decreased reductase activity, and increased cholesterol esterification. Thus, these livers retained their estrogen responsiveness. Taken together, the data demonstrate that the major elements involved in maintaining hepatic cholesterol homeostasis are present in the premalignant liver, although in some cases at levels that are different from the control. However, the susceptibility to regulation was altered in these livers to suggest markedly decreased availability of cholesterol of exogenous origin to the regulatory compartment(s). Further, coupling of the different elements involved in maintenance of hepatic cholesterol homeostasis appeared to have been changed.