Cholesteryl Esterase Activity Research Articles

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding

Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by ∼6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.

Purification and characterisation of a novel steryl esterase from Melanocarpus albomyces

A novel steryl esterase from the ascomycete Melanocarpus albomyces was purified using hydrophobic interaction chromatography and anion exchange chromatography, and the enzyme was biochemically characterised. The enzyme has a tetrameric structure with a molecular weight of 238 kDa. Native gel electrophoresis indicated an isoelectric point of 4.5. The enzyme had broad substrate specificity for different steryl esters, p-nitrophenyl esters and triglycerides with long-chain fatty acids. The pH optimum was dependent on the substrate. The pH optimum of lipase activity was at pH 7, whereas cholesteryl esterase and carboxyl esterase activities had optima at pH 5.5 and 5, respectively. The steryl esterase was more stable at lower pH than at values above pH 7. The enzyme retained over 70% of its activity after 5 h incubation at 50 °C. Activity on p-nitrophenyl butyrate was clearly increased by low detergent concentrations (≤0.1%). M. albomyces steryl esterase is considered to be an interesting tool for the enzymatic modifications of wood pitch due to its broad substrate specificity. Treatment of a model pitch simulating the TMP resin showed that both steryl esters and triglycerides were hydrolysed in the presence of a detergent, whereas only triglycerides were degraded in the absence of detergent.

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 beta-galactosidase (AdbetaGAL). 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 7alpha-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. Coinciding 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.

Expression of human hormone-sensitive lipase (HSL) in postmeiotic germ cells confers normal fertility to HSL-deficient mice.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase that is essential for male fertility and spermatogenesis and that also plays important roles in the function of adipocytes, pancreatic beta-cells, and adrenal cortical cells. Gene-targeted HSL-deficient (HSL-/-) male mice are infertile, have a 2-fold reduction in testicular mass, a 2-fold elevation of the ratio of esterified to free cholesterol in testis, and unique morphological abnormalities in round and elongating spermatids. Postmeiotic germ cells in the testis express a specific HSL isoform. We created transgenic mice expressing a normal human testicular HSL cDNA from the mouse protamine-1 promoter, which mediates expression specifically in postmeiotic germ cells. Testicular cholesteryl esterase activity was undetectable in HSL-/- mice, but in HSL-/- males expressing the testicular transgene, activity was 2-fold greater than normal. HSL transgene mRNA became detectable in testes between 19 and 25 days of age, coinciding with the first wave of postmeiotic transcription in round spermatids. In contrast to nontransgenic HSL-/- mice, HSL-/- males expressing the testicular transgene were normal with respect to fertility, testicular mass, testicular esterified/free cholesterol ratio, and testicular histology. Their cauda epididymides contained abundant, normal-appearing spermatozoa. We conclude that human testicular HSL is functional in mouse testis and that the mechanism of infertility in HSL-deficient males is cell autonomous and resides in postmeiotic germ cells, because HSL expression in these cells is in itself sufficient to restore normal fertility.

Mutation of Residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) Confer Cholesteryl Esterase Activity on Rat Lung Carboxylesterase: SER-506 IS REQUIRED FOR ACTIVATION BY cAMP-DEPENDENT PROTEIN KINASE

Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.

The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.

To directly ascertain the physiological roles in adipocytes of hormone-sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. Homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild-type and deficient littermates. HSL-deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL-/- mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL-/- adipocytes, lipolysis is not significantly increased by beta3-adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL-/- adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48-hour period at 4 degrees C was similar in HSL-/- mice and controls. Overnight fasting was well-tolerated clinically by HSL-/- mice, but after fasting, liver triglyceride content was significantly lower in HSL-/- mice compared with wild-type controls. In isolated fat cells, the lipolytic rate after beta-adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL-/- adipocytes suggests that HSL-independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.

Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.

Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity.

Human lysosomal acid lipase (LAL) catalyses the deacylation of triacylglycerol and cholesteryl esters in the acidic lysosomal compartment. Treatment of LAL with the reducing agent dithiothreitol affected the triacylglycerol and cholesteryl esterase activities differentially, suggesting the involvement of cysteine residues in determining substrate specificity. To identify the residues involved, human LAL cDNA, under the control of the T7 promoter and tagged with a herpes simplex virus coding epitope, was specifically mutated in order to introduce single amino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity towards cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towards the two substrates to a variable degree, depending on the side chain of the amino acid introduced. The replacement of Cys-41 or Cys-188 did not result in the preferential cleavage of either one of the two substrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-244 play a crucial role in determining LAL substrate specificity. We propose that these cysteine residues are involved in the hydrolysis of cholesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalytic activity is never completely abolished in cysteine mutants demonstrates that LAL is not a thiol enzyme.

Catalytic Properties of the Purified Rat Hepatic Cytosolic Cholesteryl Ester Hydrolase

The purified enzyme hydrolyzed cholesteryl oleate, cholesteryl linoleate, and triolein at similar rates over a broad range of concentrations. Hydrolytic activity was relatively low withp-nitrophenyl acetate, but much higher with PNP-esters of the more lipophilic C4-C18fatty acids, in sharp contrast to microsomal esterases which hydrolyze PNP-acetate more efficiently. Zn2+, Cu2+, Cd2+, Hg2+, and phenylmethylsulfonyl fluoride inhibited, whereas N-ethyl maleimide and iodoacetamide stimulated activity of the pure enzyme. Limited trypsin digestion selectively inhibited cholesteryl esterase activity with retention of activity toward PNP-octanoate, suggesting involvement of a trypsin-labile loop in the lipophilic substrate binding pocket.

Oleic acid inhibits cholesteryl esterase and cholesterol utilization for testosterone synthesis in mouse leydig cells

We have observed that nonesterified fatty acids (NEFA) inhibit testosterone synthesis in response to luteinizing hormone (LH) in mouse Leydig cells, possibly by affecting cholesterol utilization or endogenous concentrations. We have now studied the influence of oleic acid (OA) on the cellular content of cholesterol, hydrolysis of cholesterol esters, and steroidogenesis in isolated mouse Leydig cells. OA (700 μmol/L added with fatty acid-free [FAF] albumin, 3 g/dL) significantly ( P < .025) reduced testosterone production in response to LH (10 ng/mL), total cholesterol concentrations of Leydig cells and the culture medium, and cholesteryl esterase activity in the cytosol and mitochondria. We also studied the effects of OA on steroidogenesis and cellular cholesterol concentrations after treatments to increase cellular cholesterol. OA at lower concentrations (5 μmol/L with albumin, 0.1 g/dL) or low-density lipoprotein ([LDL] 4 μg protein/mL) increased cellular cholesterol ( P < .01) without affecting basal steroidogenesis. These treatments failed to reverse the inhibitory ( P < .05) effect of OA on testosterone synthesis following LH stimulation, but did significantly ( P < .01) increase cellular cholesterol. In summary, OA appears to inhibit testosterone synthesis by inhibiting cholesteryl esterase activity.

Tobacco Smoke Exposure Alters Cholesteryl Esterase Activities and Causes Accumulation of Cholesteryl Esters in the Rat Aorta.

The mechanism responsible for the atherogenic effects of passive smoking was studied. Four weeks and 12 weeks of exposure of rats to tobacco sidestream smoke resulted in an increase in lipid peroxide contents, expressed as thiobarbituric-acid reactive substances, in low-density lipoprotein (LDL) and in aortic tissue. The LDL isolated from plasma exhibited elevations of 136, 155, and 170% over the control values for TBA, cholesterol, esterified cholesterol, respectively, whereas the corresponding increases in the aorta were 118, 148, and 254%, respectively. Electrophoretic examinations of LDL revealed a typical denatured LDL. Among esterified cholesterol-metabolizing enzymes in the aorta, lysosomal acid cholesteryl ester hydrolase, microsomal neutral cholesteryl ester hydrolase, and acyl coenzyme A: cholesterol acyltransferase were significantly suppressed by the 4 weeks' exposure; and further suppression of the acid cholesteryl ester hydrolase activity along with increases in the neutral cholesteryl ester hydrolase and acyl coenzyme A: cholesterol acyltransferase activities was observed after 12 weeks' exposure. A possible mechanism of the observed lipid accumulation in the aorta was discussed in connection with the effect of lipid peroxides in oxidatively modified-LDL on esterified cholesterol-metabolizing enzymes.

Age-related decline in the steroidogenic capacity of isolated rat leydig cells: A defect in cholesterol mobilization and processing

This study was designed to evaluate the effects of aging on steroidogenesis and intracellular cholesterol processing in rat Leydig cells. Maximum gonadotropin-induced testosterone secretion was significantly reduced in Leydig cells from 18 to 27-month-old rats compared to 2 to 5-month-old rats. The decreased production of testosterone in older groups persisted after incubation with cAMP analogs or other non-specific stimulatory agents. This age-related loss in testosterone response was not due to changes in gonadotropin receptor concentration, cAMP concentration, protein kinase A activation or the activity of key steroidogenic enzymes. The content of cellular cholesteryl esters doubled as rats aged from 5 to 18 months, and this high cholesteryl esters level remained constant through 27 months. The ability of hCG to mobilize (hydrolyze) stored cholesteryl ester for testosterone production was significantly reduced (65–75%) in cells from the older rats. This change could be accounted for by the decline in activity of neutral cholesteryl esterase in Leydig cells from 18-month-old rats. In contrast, the activity of a non-specific lysosomal acidic cholesteryl esterase did not change with age. The activity of HMG CoA reductase, the rate limiting enzyme in cholesterol biosynthesis decreased about 70% between 5 and 18 months and fell slightly further as the rats aged to 27 months. Also, [ 14C]acetate or [ 3H]H 2O incorporation into cellular sterols showed a similar decline. Cyanoketone plus hCG stimulated pregnenolone production was reduced about 70–80% in old as compared to young cells. Leydig cells from young rats responded to hCG with increased accumulation of mitochondrial cholesterol in the presence and absence of steroidogenic inhibitors. On the other hand, old cells responded poorly to hCG and mitochondrial cholesterol levels were little affected by hCG plus cycloheximide or aminoglutethimide. Together, these data indicate that alterations in the intracellular processing and metabolism of cholesteryl esters occur in Leydig cells of aging rats, and we suggest they may be responsible for the observed age-related changes in testosterone production.

Metabolism of LDL in mast cells recovering from degranulation. Description of a novel intracellular pathway leading to proteolytic modification of the lipoprotein.

Rat serosal mast cells contain cytoplasmic secretory granules composed of a proteoglycan matrix in which histamine and neutral proteases are embedded. On stimulation, these granules are exocytosed, but some of them remain in the degranulation channels where on exposure to the extracellular fluid, they lose their histamine and a fraction of their proteoglycans. In vitro, such granule remnants efficiently bind low density lipoprotein (LDL) present in the incubation medium. After a lag period of about 10 minutes, the granule remnants, still within the channels and coated with LDL particles, are internalized by the parent mast cells. During subsequent recovery from degranulation, the apolipoprotein B of the intracellularly located remnant-bound LDL becomes efficiently (up to 70%) degraded by the proteolytic enzymes of the granule remnants. Since the granule remnants lack cholesteryl esterase activity, no LDL cholesterol is made available for cellular nutrition. Instead, selective proteolytic degradation of the bound LDL leads to formation of LDL particles enlarged by fusion on the granule remnant surface. In response to restimulation of the mast cells, about 50% of the fused LDL particles are exocytosed with the granule remnants. Of these, about one in five are expelled into the incubation medium. The granule remnants that again remain in the degranulation channels bind and internalize more LDL. This "round trip" of LDL in mast cells exposed to repeated stimulation constitutes a hitherto-unknown intracellular pathway for modification of LDL.

Hydrolysis of lipid droplets in acid cholesteryl-esterase-deficient fibroblasts.

The mechanisms of hydrolysis and accumulation of cholesteryl oleate-lipid droplets prepared in vitro were studied in acid cholesteryl-esterase-deficient fibroblasts (GM00863, GM03111). Acid cholesteryl esterase activity was reduced in both GM00863 and GM03111 (8.9% and 17.4% of the normal level, respectively), while neutral cholesteryl esterase activity was highly stimulated in GM03111. The hydrolysis of [14C]-cholesteryl oleate-lipid droplets in GM00863 was almost as efficient as in normal cells, while that in GM03111 was highly stimulated. When viewed by polarized microscopy the lipid droplets which had accumulated in the mutant cells showed anisotropic liquid crystalline structures. As in normal cells, some of these lipid droplets were observed by transmission electron microscopy as membrane-free lipid inclusion bodies in the cytoplasm. These results suggest that lipid droplets internalized into phagolysosomes of these mutant cells transferred to the cytoplasm, and were hydrolyzed there probably by neutral cholesteryl esterase.

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.

CDNA cloning of carboxyl ester lipase from human pancreas reveals a unique proline-rich repeat unit.

We report the isolation and nucleotide sequence of the cDNA for carboxyl ester lipase (CEL) from human pancreas. CEL was purified from human pancreas and microsequence analysis was performed on the amino-terminal and internal peptides. Peptide sequence was used to design oligonucleotide probes for screening a human pancreas cDNA library. Partial length cDNAs for CEL were isolated from the library, and the 5' portion of the cDNA was obtained using the anchored polymerase chain reaction. The deduced amino acid sequence indicates that mature CEL contains 722 amino acids and is synthesized with a 20 amino acid leader peptide. The amino acid sequence is rich in proline (12.2%), with 68% of the proline residues occurring within the final 25% of protein length. This is due to the occurrence of a series of proline-rich tandem repeat units near the carboxyl terminus, and accounts for the previously observed species variation in CEL size and amino acid composition. The primary sequence of CEL shows strong similarity to members of the serine esterase family, including the identical G-E-S-A-G motif at the putative active site. A striking homology also occurs between CEL and acetylcholinesterase and cholinesterase, essential enzymes of the nervous system. Proteins with cholesteryl esterase activity have been detected in extra-pancreatic tissues including liver, intestine, kidney, aorta, macrophage, and in the milk of some species (human, gorilla, cat, dog), but not others (rat, cow). To clarify the structural relationships between these various esterases and CEL, we used the CEL cDNA to study expression in pancreas and liver. CEL mRNA was abundant in pancreas of human and rat, with the human CEL mRNA approximately 300 nucleotides larger than that from rat. CEL mRNA was not detected in human adult or fetal liver, nor in rat liver. These results indicate that CEL is not synthesized in significant amounts in liver, and suggest that the cholesterol esterase activity that has been described in liver may be due to a distinct enzyme, or may be derived from pancreas, as has been proposed for the cholesterol esterase activity in intestine.

Comparison of enzymic activities of tissues lining portals of drug absorption, using the rat as a model

Abstract Leucine aminopeptidase and cholesteryl esterase activities of five different tissues from the rat are investigated. The tissues from the intestines, the buccal cavity, the nose, the rectum and the skin were chosen, as they represent barriers to absorption for most commonly used dosage forms. It is shown that the intestinal tissue is the most active in both enzymes. Dermal tissues are the second richest in cholesteryl esterase activity, whereas rectal tissue is the least active. Aminopeptidase activity is weakest in dermal tissues. When adjusted for protein content, dermal tissues show the highest specific esterase activity. Rectal tissue remains the least active. Based on the known relatively high permeability of buccal and nasal tissues, the low aminopeptidase and cholesterol esterase activities would suggest that those routes offer better possibilities for delivering peptides and esters. However, it should be noted that enzymic degradation is only one of the barriers to peptide absorption, and for most peptide drugs an absorption enhancer or a specific delivery system is still likely to be required for successful trans-nasal or buccal absorption.

The influence of estradiol on cholesterol processing by the corpus luteum.

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.

Cholesteryl esterase activities in ventricles, isolated heart cells and aorta of the rat.

Cholesteryl esterase activities were determined in homogenates of rat heart (ventricles), isolated, calcium-tolerant, cardiac myocytes and aortic tissue and were compared with acid and neutral triglyceride lipase activities in these fractions. Using cholesteryl oleate/phosphatidylcholine/taurocholate emulsions and digitonin pretreatment of the enzyme fractions, acid and neutral cholesteryl esterase activities were measured in all tissue preparations. In contrast to the acid and neutral triglyceridase and acid cholesteryl esterase activity, the neutral cholesteryl esterase activity was subject to substrate inhibition. Upon isolation of cardiac myocytes, and in contrast with the recovery of neutral triglyceride lipase activity, only a small portion of the neutral cholesteryl esterase (6%) was recovered, suggesting that nonmyocyte neutral cholesteryl esterase activity markedly contributes to the relatively high activity detectable in whole ventricular homogenates. The recovery of large amounts of neutral cholesteryl esterase activity in the supernatant of collagenase-digested heart tissue, obtained during the isolation of myocytes, which is also markedly enriched in activities of two endothelial marker enzymes (5'-nucleotidase and angiotensine-converting enzyme) may indicate the predominant contribution of neutral cholesteryl esterase activity from coronary endothelial cells to this activity detectable in ventricular homogenates. Relative to the activity in ventricular and myocyte homogenates, aorta homogenates possessed the highest specific neutral cholesteryl esterase activity. We propose that in addition to coronary endothelium, smooth muscle cells also contribute to the neutral cholesteryl esterase activity in ventricular homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)