Application of 2-hydroxypropyl-β-cyclodextrin in the assay of acyl-CoA: cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.     

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

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

Cholesterol ester hydrolase activity in mammary tissue of the lactating rat

Cholesterol ester hydrolase activity has been studied in mammary glands of rats. Subcellular fractionation of the glands obtained in mid-lactation indicated that around 80% of the recovered activity was associated with particulate fractions. Two distinct cholesterol ester hydrolase activities were identified, one with an optimum pH of 7.5–9.0 and the second (approximately 5% of the total activity) with a more acidic pH optimum. Although the neutral cholesterol ester hydrolase had some properties in common with the lipoprotein lipase in mammary tissue, it was shown to be a separate entity by several criteria. Its activity could be increased following treatment with Mg-ATP and cAMP-dependent protein kinase, suggesting identity with the hormone sensitive lipase of adipose tissue. The cholesterol ester hydrolase activity in mammary glands just after parturition was greater than in glands obtained either from late-pregnant or midlactating animals. The subcellular distribution of the neutral cholesterol ester hydrolase suggested that it may have a different function to the neutral cholesterol ester hydrolase of adrenals and other tissues. Nevertheless the fact that the activity of the enzyme can be modulated by cAMP-dependent protein kinase suggests the possibility that hormonal control of this enzyme may be involved in the regulation of cholesterol metabolism in the mammary gland.     

Characterization of a cytosolic protein inhibiting lysosomal acid cholesteryl ester hydrolase

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

Effect of cupric ions on serum and liver cholesterol metabolism

Cupric ions were administered subcutaneously to male Sprague-Dawley rats at a single dose of 200 μmol/kg. At 24 hr after administration, a remarkable increase of total and free cholesterol was seen in the rat serum. Also, when lecithin-cholesterol acyltransferase (LCAT) (E.C. 2.3.1.43) activity was expressed as the percentage of the total serum that free cholesterol esterified, the acyltransferase activity in rats treated with cupric ions showed a slight decrease while the triglyceride content in rat serum and liver decreased by 54% and 61%, respectively. However, the content of hepatic cholesterol in rats treated with cupric ions did not show such a marked change. On the other hand, acid cholesteryl ester hydrolase activity (Acid CEH) (E.C. 3.1.1.14) in liver lysosomes of rats treated with cupric ions showed a marked decrease with increasing cupric ion concentration both in vivo and in vitro. Furthermore, cupric ions caused a marked release of the lysosomal enzymes cathepsin D and β-glucuronidase into the cytosolic fraction. The changes in acid cholesteryl ester hydrolase activity induced by cupric ions appear to be a direct effect of cupric ions on the enzyme. These results suggest that excessive cupric ion concentrations could cause various disorders in lipid metabolism.     

The inhibition of neutral cholesteryl ester hydrolase by a cytosolic protein factor in female rat liver: The influence of varying hormonal and nutritional conditions on the inhibitory activity

A cytosolic protein, that is inhibitory to neutral cholesteryl ester hydrolase, has been investigated in the livers of female rats using microsomes isolated from the mammary gland of lactating rats as an enzyme source. To facilitate comparisons, inhibitory activity is expressed in terms of the amount (μg) of cytosolic protein required to reduce esterase activity by 50% and is compared to the hepatic content of both cholesterol and cholesteryl esters. The experiments revealed a sexual difference in the level of inhibitory activity, with the livers of both suckling and mature male animals containing less of the material than the corresponding females. Alterations in the physiological status of the females, such as pregnancy and lactation, led to a decrease in the activity of the protein. This was reversed by blocking lactation with a combination of an antiserum to rat growth hormone and the anti-prolactin drug, bromcoriptine, but not by premature weaning of the animals. Food withdrawal for 24 hr also had the effect of increasing inhibitory activity. In general the cholesteryl ester content of the livers correlated with the level of inhibitory activity. Thus the activity of the cytosolic inhibitor of neutral cholesteryl ester hydrolase responded to changes in both the hormonal and the nutritional status of the female animal. It is suggested that the presence of the greater cholesteryl ester hydrolase inhibitory activity in the female liver may help to explain the lower risk of coronary heart disease in premenopausal females by facilitating increased hepatic storage of the sterol in the form of the ester.     

Hepatic zonation of the formation and hydrolysis of cholestery esters in periportal and perivenous parenchymal cells

The periportal (PP) and perivenous (PV) zones of the liver acinus differ in enzyme complements and capacities for cholesterol and bile acid synthesis and other metabolic processes. The aim of this investigation was to determine the acinar distribution of the catalytic activity of the enzymes governing the formation and hydrolysis of cholesteryl esters using PP and PV hepatocytes from normal or cholestyramine-fed rats. The hepatocyte subpopulations were isolated by centrifugal elutriation, characterized according to the distribution pattern of a number of cell parameters and marker enzymes, and assayed for acyl-CoA: cholesterol acyltransferase (ACAT) and lysosomal, cytosolic and microsomal cholesteryl ester hydrolase (CEH). In normally fed rats, no zonation was found in the activity of lysosomal CEH and ACAT, and the activity of both cytosolic and microsomal CEH zonated toward the PV zone of the acinus. Concentrations of free and esterified cholesterol in homogenates, cytosol, and microsomes of PP and PV cells were, however, similar. Cholestyramine raised significantly the PV/PP ratio of ACAT because of an exclusive PP reduction of activity and abolished the heterogeneity in microsomal CEH because of a greater inhibitory PV response, whereas the PV dominance of cytosolic CEH and the homogeneous distribution of lysosomal CEH were unaffected. These results demonstrated homogeneity within the liver acinus for the enzymatic degradation of endocyted lipoprotein-derived cholesteryl esters, a structural zonation of the cytosolic CEH and a dynamic zonation of ACAT and the microsomal CEH, with a PV dominance of the enzymatic capacity for the degradation of stored cholesteryl esters in normal livers.     

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

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

The effects of clofibrate and bezafibrate on cholesterol metabolism in the liver of the male rat

Fibric acid derivatives are used to treat hyperlipidemia and have wide ranging effects on lipid metabolism. The action of these compounds on cholesterol esterification, catalyzed by acyl-coenzyme A:cholesterol acyltransferase (ACAT), has been quite widely studied, but their effect on cholesteryl ester hydrolysis and the enzyme neutral cholesteryl ester hydrolase (nCEH) has been largely ignored. Male rats were therefore fed for 10 d on a standard chow diet supplemented with either clofibrate or bezafibrate, to study their effects on plasma lipid levels and hepatic cholesterol metabolism. Plasma triacyglycerols were not significantly altered by these diets, but bezafibrate significantly lowered plasma cholesterol levels (29.7%,P<0.01). When expressed per unit weight of DNA, both fibrates reduced the hepatic content of triacylglycerol, cholesterol and cholesteryl esters (40, 18.7, 16.5 and 66.7, 28.6, 34.2% for clofibrate and bezafibrate, respectively). ACAT activity was significantly reduced by both drugs, but clofibrate (65% inhibition) was more effective than bezafibrate (35% inhibition). The most dramatic effect of the diets was a marked increase in the activity of both the microsomal and the cytosolic nCEH. When expressed on a whole liver basis, the effect of bezafibrate on the cytosolic enzyme (13.6-fold increase in activity) was much greater than that of clofibrate (4.8-fold increase). Increases in the activity of a cytosolic protein that inhibits the activity of nCEH were also noted, but these changes were relatively small. The results suggest that the activation of nCEH, in combination with the inhibition in ACAT activity, contributes to a decrease in the cholesteryl ester content of the liver which may influence the secretion of very low density lipoprotein.     

Acyl-CoA:cholesterol acyltransferase activity in the rat mammary gland: Variation during pregnancy and lactation

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) has been studied in microsomes isolated from the mammary glands of rats in late pregnancy, in early and mid-lactation, and following premature weaning. The mammary glands were freeze-clamped and the microsomes prepared in the presence of phosphatase inhibitors to preserve the phosphorylation status of the enzyme. Optimal conditions were established for the assay of enzyme activity in the presence of endogenous cholesterol. Supplementation of the microsomes with exogenous cholesterol as a dispersion in Triton WR-1339 was shown to lead to an increase in enzyme activity. Incubation of microsomes with MgATP led to an increase in ACAT activity which could be reversed by treatment of the microsomes with a phosphoprotein phosphatase preparation from rat liver. The results suggested that ACAT activity in the mammary gland was activated by phosphorylation in a similar way to that observed for the hepatic enzyme. The mammary glands from pregnant animals contained a higher level of ACAT activity than did the glands of the lactating animals and this correlated with the higher cholesteryl ester content of the pregnant glands. The highest level of ACAT activity was found in the weaned animals but the cholesteryl ester content of the microsomes was lower than expected. The influence of progesterone levels and changes in feeding patterns during gestation were considered as factors in these variations in ACAT activity.     

Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion

To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary. bile acid, phospholipid, and cholesterol and in increased cholesterol 7α-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, ∼45%; apoB-100, ∼32%) and lipids (∼55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol out-put pathways in hepatocytes.     

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl₂ and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca²⁺/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca²⁺, Ca²⁺/calmodulin, Mg²⁺ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca²⁺/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca²⁺/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.     

The activity and properties of a hepatic acid cholesteryl ester hydrolase obtained from rats of different age groups

The activity of lysosomal acid cholesteryl ester hydrolase (acid CEH, EC 3.1.1.13) in rat liver was determined at 3, 5, 7, 10 and 20 wk following birth. The levels of acid CEH activity showed a marked decrease as rats grew older, whereas those of other lysosomal marker enzymes, such as acid phosphatase, β-glucuronidase and cathepsin B and D, showed only a slight decrease. On the other hand, acid CEH activity was detected in all subcellular fractions obtained from rat liver, but the enzyme activity in these fractions did not show the age-related decrease observed in the lysosomal fraction. The results presented here suggest that the marked alteration of lysosomal acid CEH activity that accompanies aging may be related to its possible involvement in the regulation of cholesterol concentration in rat liver.     

Inhibition of neutral cholesteryl ester hydrolase by the glycolytic enzyme enolase. Is this a secondary function of enolase?

There is an accumulation of the glycolytic enzyme enolase and of cholesteryl esters in macrophages that have been converted into “foam” cells. In this study, we questioned whether enolase could be involved in this accumulation of cholesteryl esters by inhibiting the activity of neutral cholesteryl ester hydrolases. Enolase from both yeast and rabbit muscle were incubated with three different cholesteryl ester hydrolases and were shown to inhibit the hydrolysis of cholesteryl esters. Inhibition was dependent on the concentration of enolase and appeared to occur through binding of the enolase to the cholesteryl ester. Nevertheless, the yeast and rabbit muscle enolases differed in their efficiency of inhibition and in their mechanism of action. Purification of commercial enolase preparations by gel-filtration yielded single proteins with the same inhibitory activities as the originals, indicating that the inhibition was not due to the presence of an impurity. Partially purified αα-and γγ-isoforms of the enzyme from rat brain also appear to have inhibitory effects on cholesteryl ester hydrolysis. Negative control of the hydrolytic phase of the cholesterol/cholesteryl ester cycle may be a secondary function of enolases which correlates with the accumulation of cholesteryl esters in a number of neuro-degenerative and demyelinating diseases.     

The effects of simvastatin and cholestyramine,alone and in combination,on hepatic cholesterol metabolism in the male rat

The influence of dietary simvastatin, cholestyramine, and the combination of simvastatin plus cholestyramine on hepatic cholesterol metabolism has been investigated in male rats. Recovery from the effects of the drugs was also investigated by refeeding normal chow for 24 h. Both drugs, alone and in combination, increased 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activityin vitro, but activity returned toward control values, after drug withdrawal. Acyl-CoA:cholesterol acyltransferase (ACAT) was significantly reduced (P<0.001) by simvastatin (−75%), cholestyramine (−71%), and by the drug combination (−81%), due both to a decrease in microsomal cholesterol and to nonsubstrate-dependent modulation of enzyme activity. Refeeding control diet increased ACAT activity but not to control levels. The enhanced activity arose partly from higher microsomal cholesterol and partly from increases in total enzyme activity. Cytosolic neutral cholesteryl ester hydrolase (CEH) activity was substantially elevated by simvastatin (3-fold) and by the drug combination (6-fold), whereas the effect of cholestyramine was smaller (1.5-fold). Normal chow for 24 h only partially returned cytosolic CEH activity to control values. Microsomal CEH activity was increased by simvastatin, alone and in combination with cholestyramine (1.4 to 1.7-fold), and was also enhanced, in the cholestyramine-treated animals, following drug withdrawal. Removal of simvastatin did not allow recovery of this enzyme activity, while withdrawal of the drug combination led to values 29% below controls. The results indicate that in the rat, simvastatin and cholestyramine alter both ACAT and CEH activity, as well as inhibiting HMG-CoA reductase activity.     

Cholesteryl ester hydrolase activity in human symptomatic atherosclerosis

Acid cholesteryl ester hydrolase (CEH) activity was assayed in mononuclear cells of patients with symptomatic atherosclerosis (transient ischemic attacks, TIA) and in age-matched controls showing no evidence of atherosclerosis. The acid CEH level of TIA patients was significantly lower than that of controls (1074±128 vs 2113±255 pmol/mg P/hr, mean±SE). Neither mononuclear cell nor plasma cholesterol and cholesteryl ester concentrations differed significantly between atherosclerotic and control groups. TIA women had lower mononuclear cell concentrations of free sholesterol than men.     

The esterification of cholesterol in the yolk sac membrane of the chick embryo

The uptake of lipid from the yolk by the yolk sac membrane of the chick embryo is accompanied by the rapid esterification of a large proportion of the yolk cholesterol. This could arise from enhanced acyl-CoA:cholesterol acyltransferase (ACAT) activity and/or inhibition of cholesteryl ester hydrolase (CEH) activity. The activity of ACAT was therefore measured in microsomes obtained from yolk sac membranes at various stages of development. A high level of activity (up to 929 pmol of cholesteryl oleate formed per min per mg protein) was found during the second half of this period. Supplementation with exogenous cholesterol stimulated ACAT activity in microsomes obtained from the tissue at the earlier, but not at the later, stages of development suggesting that the enzyme became saturated with microsomal cholesterol as development proceeded. Correlating with this, the concentration of cholesterol in the microsomes increased 4-fold between 9 and 20 d of development. The activity of CEH was very low in the microsomes and could not be detected in the cytosolic fraction. The activity of a protein, which has been shown to function as an inhibitor of CEH, was found to be present at all stages of development. The high activity of ACAT, together with the low activity of CEH and an active CEH inhibitor protein is a combination well suited to promote an essentially unidirectional conversion of cholesterol to cholesteryl ester. This process may be a major determinant of the rate of lipid transfer from the yolk to the embryo.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

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

Stimulation of microsomal cholesterol ester hydrolase by glucagon,cyclic AMP analogues,and vasopressin in isolated rat hepatocytes

Short-term activation of microsomal cholesterol ester hydrolase by glucagon, cAMP analogues, and vasopressin in isolated rat hepatocytes is described. Glucagon led to a dose-and time-dependent activation of cholesteryl oleate hydrolysis, but values returned to basal levels within 120 min. Exposure of isolated hepatocytes to 0.5 mM concentrations of dibutyryl-cAMP or 8-[4-chlorophenylthio]-cAMP, or 25 μM forskolin caused persistent activation of cholesterol ester hydrolase activity after a lag period of 30 min. The three agents resulted in early marked intracellular accumulation of cAMP that declined progressively, and moderate and sustained reductions in the diacylglycerol content. The actions of glucagon on hepatocytes were inhibited by pretreatment of cells with 10 nM [8-arginine] vasopressin. Vasopressin elicited a consistent and sustained increase in cholesterol ester hydrolase activity and diacylglycerol without affecting cAMP while reducing the effect of glucagon on cAMP. Furthermore, the effects of glucagon and vasopressin on the activation of cholesterol ester hydrolase were not additive despite the similarity of their stimulation of diacylglycerol formation. Blockade of vasopressin-mediated activation of cholesterol ester hydrolase and diacylglycerol content were induced by excess prazosin. These data suggest that stimulation of microsomal cholesterol ester hydrolase in isolated liver cells may involve at least two signal transduction systems.     

Cholesteryl ester hydrolysis in rat liver cytosol. Modulation by female sex hormones

The regulation of cholesterol ester hydrolase activity by female sex hormones was studied in cytosolic preparations from female rat liver. The investigation was undertaken in order to determine whether a reduction in the enzyme activity might be responsible for the increased content of esterified cholesterol found in rat liver after estradiol or progesterone treatments. The single injection of estradiol (0.75 mg/100 g) or progesterone (1.50mg/100g) produced respectively significant decreases and increases in sterol hydrolase activity. Both opposite effects were noted after a similar lag period of 3–4 hr and were of short duration. No alterations were observed in rats receiving short-term treatments. When hormones were added to the incubation medium, the activity of cholesterol ester hydrolase decreased progressively with increasing concentrations of hormones. Kinetic studies demonstrate that both estradiol and progesterone compete with the substrate (cholesteryl oleate) for the active center. The findings of the present paper exclude a direct relationship between hepatic hydrolytic activity and lipid deposition. However, they provide evidences that female sex hormones act as modulatory agents of the hydrolysis of cholesteryl esters in rat liver cytosol and suggest that other factors besides competitive inhibition are involved in such regulatory effects.