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.     

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase,triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 ×g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the three activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.     

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.     

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.     

Purification of lysosomal cholesteryl ester hydrolase from rat liver by preparative isoelectric focusing

Ion-exchange chromatography and preparative isoelectric focusing (PIEF) were compared to produce a stable rat liver lysosomal cholesteryl ester hydrolase of high specific activity. The PIEF purification method proved to be more rapid and easier to perform. PIEF purification involved the following steps: i) osmotic shock of the lysosome fraction, ii) (NH₄)₂ SO₄ precipitation (10–70%, w/v), iii) Sepharose CL-6B gel filtration, and iv) PIEF. The enzyme was purified 60–120-fold with a yield of 2–4%. The activity of the purified enzyme was best restored by stabilizing with a 0.5% (w/v) albumin solution. The purified enzyme produced one major band on SDS-polyacrylamide gel electrophoresis having a MW of 58,500 daltons. Gel filtration showed a MW of 58,000 daltons. The optimum pH of the enzyme was 4.5, and the isoelectric point was 6.0–6.2. The specific activity of hydrolysis of cholesteryl oleate and triolein increased by similar rates during purification.     

Evidence that cholesteryl ester hydrolase and triglyceride lipase are different enzymes in rat liver

Studies on intracellular cholesteryl ester hydrolase (CEH) and triglyceride lipase (TGL) from rat adipose tissue and adrenal cortex have suggested that a single protein is responsible for both activities. To determine whether one hepatic protein catalyzes both reactions, we studied several properties of CEH and TGL in rat liver. During liver perfusion with heparin, perfusate peaks of TGL and CEH did not consistently coincide, and TGL activity was considerably higher and less heat-stable than that of CEH. Significant TGL, but not CEH, activity was released during incubation of isolated hepatocytes. Although microsomes isolated from hepatocytes contained both activities, the specific activities of CEH and TGL in cytosol from hepatocytes were 95% and 3%, respectively, of those found in cytosol from whole liver. Preincubation of liver cytosol with 5 mM Mg²⁺ decreased CEH, but not TGL, activity. Intracellular CEH and TGL activities were completely separated by prep-disc gel electrophoresis. Finally, both cytosolic and microsomal TGL, but not CEH, activities were inhibited by antiserum against rat hepatic TGL. We conclude that extracellular TGL does not have CEH activity and intracellular CEH differs from TGL.     

Properties of an acid cholesteryl ester hydrolase inhibitor from rat serum

The inhibitory effect of a protein isolated from rat serum on lysosomal acid cholesteryl ester hydrolase (acid CEH; EC.3.1.1.13) activity was studied. An inhibitor was purified from rat serum following ultracentrifugation and heat treatment using column chromatography on Sephacryl S-200 and ultrafiltration. The purified inhibitor appeared as a single protein band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was 28,000 Daltons as judged by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis. The purified inhibitor was shown to be apolipoprotein A-I (apo A-I), the major apolipoprotein of high-density lipoprotein (HDL), using immunoprecipitation with rat anti-apo A-I immunoglobulin (Ig)G. Inhibition of acid CEH activity by apo A-I was dependent on the concentration of apo A-I. The values of V_max obtained were similar with or without apo A-I. Apo A-I of various other mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. Other apolipoproteins, such as apo A-II and apo B, also showed inhibiting activity. On the other hand, apo A-I had no effect on the activity of other enzymes found in lysosomes, such as cathepsin D, β-glucuronidase and acid phosphatase. The results suggest that apolipoproteins may play a role in the regulation of hydrolysis of cholesteryl esters in lipoproteins, that have been transferred to the liver, and that the inhibition of acid CEH activity by apo A-I may be a characteristic of the lipid-binding protein or be due to changes of the lipid/water interface.     

Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme

A rat liver cytosolic cholesteryl ester hydrolase (CEH) was purified 12,600-fold by ammonium sulfate precipitation, cation exchange chromatography and gel permeation high-performance liquid chromatography, with an overall yield of 20%. Its properties are compared to those of pancreatic CEH, with which it has sometimes been identified. Liver CEH exhibited a single silver stained band following SDS-polyacrylamide gel electrophoresis (M_r=66 kDa), was activated by 0.5–10 mM taurocholate but was strongly inhibited by higher levels of taurocholate, which activate pancreatic CEH. Whereas bile salts are known to induce formation of a hexamer of pancreatic CEH, in the current study, 0.5 mM taurocholate dissociated a multimeric form of liver CEH to monomer. Liver CEH did not coelute with pancreatic CEH from cation exchange and chromatofocusing columns, exhibited no immunoreactivity with anti-rat pancreatic CEH IgG in Western blots, was not inhibited by anti-rat pancreatic CEH IgG and had a different amino acid composition from pancreatic CEH. In contrast to liver CEH, which is known to be activated by protein kinases A and C, pancreatic CEH was unaffected by cofactors for protein kinase A and was inhibited by cofactors for protein kinase C.     

Temperature lability and cAMP-dependent protein kinase activation of cholesteryl ester hydrolase as a function of age in developing rat testis

Cholesteryl ester hydrolase (CEH) was measured at 32°C and 37°C, and with and without cofactors for stimulation of cyclic AMP-dependent protein kinase, in 104,000×g supernatants from rats aged 14–365 days. Activity at the two temperatures was also partially resolved by cation exchange FPLC. Total specific activity of CEH was relatively constant, with or without addition of cofactors, from 14 to 47 days, during which time temperature labile CEH was a very small fraction of total CEH activity. At later times, 51–150 days, activity was increased as much as two-fold, both with and without cofactors, with most of the increase occurring in the temperature labile fraction. Activation of temperature stable and temperature labile activities, where present, by protein kinase cofactors could be demonstrated in all age groups, but was highly variable as a function of age and protein concentration used in the assay. Apparent induction of temperature labile activity over the interval 47–51 days coincides with reported increases in testosterone synthesis and first appearance of spermatozoa in the testis. This and other lines of evidence suggest unique roles for these enzymes in regulation of availability of free cholesterol for testosterone and membrane synthesis, respectively.     

A diurnal variation of hepatic acid cholesteryl ester hydrolase activity in the rat

The diurnal variation in lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) has been examined in fed, fasted and adrenalectomized rats. The Acid CEH activity of normal rat liver exhibits a diurnal rhythm with maxima at 06.00 hours and minima at 18.00 hours, but such a rhythm was not observed in spleen and brain. This rhythm was abolished after fasting for two days, and the resulting Acid CEH activity remained constant at the minimum level. However, adrenalectomy did not abolish the diurnal rhythm. These results indicate that the Acid CEH activity varies according to a diurnal rhythm with maxima and minima separated by approximately 12 hr. Further, it is evident that the appearance of this rhythm is dependent upon dietary, but not adrenal hormone influence.     

Purification and properties of aortic cholesteryl ester hydrolase

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, 0-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific, esterase by column chromatography. Both synthetic and the hydrolytic activities are apprently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dired powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7 Both activities were found to be fairly nonspecific with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; corprostanol, 24; ergosterol, 20; and β-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic>linoleic>arachidonic>palmitic>stearic>butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate and laurate in decreasing order.     

Purification and characterization of a 28 kDa cytosolic inhibitor of cholesteryl ester hydrolases in rat testis

A 28 kDa inhibitory protein was purified from ratestis cytosol by sequential 40–65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. The heat-stable, trypsin-labile protein exhibited nonenzymatic, concentration-dependent inhibition of testicular and pancreatic cholesteryl ester hydrolases at all stages of putification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection of excised electrophoretic bands cross-reacted with both proteins on western blots, immunoprecipitated both proteins, and neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS-polyacrylamide gels were different from those of other surface-active proteins of similar molecular weights. Both proteins exhibited identical pl of 4.8 on chromatofocusing columns and two-dimensional gel electrophoresis. Although the subcellular distribution of the 28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8×10⁻⁹ mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required forin vitro inhibition (10⁻⁹ mols/L), consistent with a physiological role for this protein.     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

Cholesterol metabolism in the rat lactating mammary gland: The role of cholesteryl ester hydrolase

An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme was strongly inhibited by Cu²⁺, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione (GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid, lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition, a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for secretion into milk.     

Over-expression of hepatic neutral cytosolic cholesteryl ester hydrolase in mice increases free cholesterol and reduces expression of HMG-CoAR,CYP27, and CYP7A1

Hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) is a key enzyme in the regulation of hepatic free cholesterol (FC). In examining the effects of over-expression of this enzyme on cholesterol homeostasis, mice were infected with a recombinant adenovirus construct (AdCEH) of the rat hncCEH cDNA driven by the human cytomegalovirus promoter. Cholesteryl esterase and p-nitrophenylcaprylate (PNPC) esterase activities were measured in liver postmitochondrial supernatants at 1, 3, 7, and 11 d after infection with AdCEH or a control virus expressing β-galactosidase (AdβGAL). The PNPC esterase activity of AdCEH mice peaked threefold higher than controls on day 2, declining on subsequent days. In contrast, cholesteryl esterase peaked eightfold higher than controls on day 3, indicating a shift in substrate selectivity of hncCEH. Hepatic FC peaked at 144% of controls, 7 d postinfection. The mRNAs for cholesterol 7α-hydroxylase, sterol 27-hydroxylase, and HMG-CoA reductase decreased to 47, 46, and 58% of controls, respectively, on day 7, coinciding with peak FC concentrations. Coiniding with increased cholesteryl esterase activity, hepatic esterified cholesterol dropped precipitously from day 3 onward, to 11% of controls by day 11. Hepatic TAG levels also declined, consistent with the reported TAG lipase activity of hncCEH. These results demonstrate elevation of FC and depletion of cholesteryl esters by over-expression of hncCEH, which were resistant to compensatory responses by other enzymes of cholesterol homeostasis.     

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.     

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 effect of oral contraceptives on mononuclear cell cholesteryl ester hydrolase activity

The influence of sex steroids on mononuclear cell cholesteryl ester hydrolase (CEH) activity in premenopausal women and women on combined estrogen-progestin oral contraceptives has been studied. In addition, plasma and mononuclear cell cholesterol and esters were measured along with plasma estrogen and progesterone levels. Mononuclear cell CEH activity in control women is highest on Day 20 of their menstrual cycle. The control women had significantly higher CEH activities than women on oral contraceptives. Plasma esters were higher in the oral contraceptive group. However, in mononuclear cells free cholesterol but not cholesteryl esters were higher in women on oral contraceptives.     

Cupric ion-dependent inhibition of lysosomal acid cholesteryl ester hydrolase in the presence of hydroxylamine

In the presence of hydroxylamine or ascorbic acid, the inhibitory effects of Cu²⁺ on lysosomal acid cholesteryl ester hydrolase (acid CEH) partially purified from rat liver were studied. Hydroxylamine stimulated the inhibition of acid CEH activity by Cu²⁺ but not that by Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺ and Hg²⁺. This Cu²⁺-dependent inhibition of acid cholesterol ester hydrolase (CEH) activity was completely prevented by ethylenediamine tetraacetic acid (EDTA), EGTA and o-phenanthroline, a chelator with a stability constant for Cu²⁺, and also by sulfhydryl agents and cytoplasmic reducing agents such as cysteine, glutathione and mercaptoethanol. In addition, the stimulative effects of hydroxylamine on Cu²⁺-dependent inhibition were maintained even after preincubation of Cu²⁺ with hydroxylamine. On the other hand, ascorbic acid was found to replace the stimulation by hydroxylamine of the Cu²⁺-dependent inhibition of acid CEH activity but the effects of ascorbic acid progressively became smaller with prolongation of the preincubation time. Moreover, addition of chemical radical scavengers to the reaction mixture did not prevent the Cu²⁺-dependent inhibition of acid CEH activity in the presence of ascorbic acid. These results suggest that Cu²⁺ causes inhibition of lysosomal acid CEH activity through the formation of Cu¹⁺ in a reductive medium.     

Lipid-lowering effects of WAY-121,898, an inhibitor of pancreatic cholesteryl ester hydrolase

WAY-121,898 is an inhibitor of pancreatic cholesteryl ester hydrolase (pCEH). After confirming its in vitro potency and relative lack of a major effect on acyl-CoA:cholesterol acyltransferase (ACAT), it was found that this compound lowers plasma cholesterol in cholesterol-fed, but not chow-fed, rats. Measures of liver cholesteryl ester content and the direct determination of cholesterol absorption (lymph-fistula model) show that inhibition of cholesterol absorption is at least one mechanism for the observed cholesterol lowering. However, WAY-121,898 was also active when administered parenterally to cholesterol-fed rats, and in cholesterol-fed hamsters cholesterol-lowering occurred with oral dosing despite no change in cholesterol absorption, suggesting other modes of action possibly relating to inhibition of liver CEH. Combination treatment in cholesterol-fed rats with the ACAT inhibitor CI-976 resulted in a greater-than-additive reduction in plasma cholesterol, imlying that both pCEH and ACAT may play a role in cholesterol absorption in this species. In rabbits, WAY-121,898 prevented the rise in plasma cholesterol due to the feeding of cholesteryl ester but not in rabbits fed (free) cholesterol. In guinea pigs, the compound induced an increase in adrenal cholesteryl ester mass. Taken together, the overall profile in these animal models suggests that WAY-121,898 inhibits more than just the intestinal (lumenal) pCEH, and that the role of this enzyme in cholesterol metabolism may be different within and across species, the former depending upon the dietary cholesterol load.