Relationship of human pancreatic cholesterol esterase gene structure with lipid phenotypes.

Pancreatic cholesterol esterase is one of the enzymes that plays a pivotal role in cholesterol absorption. Differences in the genotype of this enzyme could affect the susceptibility of individuals to dyslipidemia and/or cardiovascular disease. We undertook this study to investigate if any correlation exists between restriction fragment length polymorphism in the human pancreatic cholesterol esterase gene and serum lipid levels. DNA from 96 healthy adults was restricted with Stu I, Southern blotted, and probed with cDNA of human pancreatic cholesterol esterase. Results revealed six distinct patterns which were classified as A, B, C, D, E, and F which had a population frequency of 1%, 34.5%, 49%, 12.5%, 1% and 2% respectively. Correlation of the distribution of lipid and lipoprotein levels by pattern and sex revealed a significant interaction between pattern type and HDL (p=0.03) in the most common group (group C) for males. Male patients of pattern C tended to have a lower LDL cholesterol than non-pattern C males (p=0.07); in addition, 80% of all males in the study population with LDL cholesterol under 100 mg/dl were found in pattern C. Thus, the most common Stu I RFLP genotype is associated with a favorable lipid phenotype. This report shows an association between the human pancreatic cholesterol esterase genotype and serum lipid levels. Further analysis of a larger study group with Stu I and alternative polymorphic restriction enzymes is warranted, to confirm this biologically plausible result.

Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.

Percent cholesterol absorption was measured in 94 normal subjects aged 17- 80 years while consuming diets generally low in cholesterol (mean intake = 226 +/- 126 mg/day). A new dual stable isotope method was used where a cholesterol tracer containing 6 extra mass units was given intravenously and another tracer with 5 extra mass units was given orally during a standard test meal. The ratio of tracers in plasma was determined by negative ion mass spectrometry of pentafluorobenzoyl sterol esters. Absorption values ranged widely from 29.0% to 80.1% with mean 56.2 +/- 12.1 (SD) %. Cholesterol absorption was significantly increased in African-Americans (63.4 +/- 11.8% vs. 55.1 +/- 11.9%, P = 0.027) but was similar for women (53.3 +/- 11.9%) and men (57.6 +/- 12.1%). It was not related to plasma lipoproteins, age, apoE3/E3 or E3/E4 genotype, or chronic dietary intake of energy, fat, or cholesterol quantitated from 7- day food records. However, dietary cholesterol intake was positively related to plasma cholesterol (P = 0.036) and triglycerides (P = 0.026). The milligram amount of dietary cholesterol absorbed (but not percent absorption) was positively correlated with fasting plasma insulin (r = 0.525, P < 0.0001), C-peptide (r = 0.367, P = 0.0003) and glucagon (r = 0.421, P < 0.0001) independent of gender, body fat percent and age.The efficiency of intestinal cholesterol absorption and the milligram amount of dietary cholesterol absorbed were not related to plasma cholesterol or LDL cholesterol in individuals consuming a low-cholesterol low-fat diet. The dominant factor determining dietary cholesterol absorption was intake rather than absorption efficiency. Dietary cholesterol and fat were strongly and independently related to hormonal measures of insulin resistance.-Bosner, M. S., L. G. Lange, W. F. Stenson, and R. E. Ostlund, Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.

Structure of bovine pancreatic cholesterol esterase at 1.6 A: novel structural features involved in lipase activation.

The structure of pancreatic cholesterol esterase, an enzyme that hydrolyzes a wide variety of dietary lipids, mediates the absorption of cholesterol esters, and is dependent on bile salts for optimal activity, is determined to 1.6 A resolution. A full-length construct, mutated to eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show that the purified, recombinant, mutant enzyme has activity identical to that of the native, glycosylated enzyme purified from bovine pancreas. The mutant enzyme is monomeric and exhibits improved homogeneity which aided in the growth of well-diffracting crystals. Crystals of the mutant enzyme grew in space group C2, with the following cell dimensions: a = 100.42 A, b = 54.25 A, c = 106.34 A, and beta = 104.12 degrees, with a monomer in the asymmetric unit. The high-resolution crystal structure of bovine pancreatic cholesterol esterase (Rcryst = 21.1%; Rfree = 25.0% to 1.6 A resolution) shows an alpha-beta hydrolase fold with an unusual active site environment around the catalytic triad. The hydrophobic C terminus of the protein is lodged in the active site, diverting the oxyanion hole away from the productive binding site and the catalytic Ser194. The amphipathic, helical lid found in other triglyceride lipases is truncated in the structure of cholesterol esterase and therefore is not a salient feature of activation of this lipase. These two structural features, along with the bile salt-dependent activity of the enzyme, implicate a new mode of lipase activation.

Dietary induction of pancreatic cholesterol esterase: a regulatory cycle for the intestinal absorption of cholesterol.

Atherosclerosis has a strong dietary basis without a proven molecular mechanism for cholesterol absorption. To investigate the potential role of pancreas in this process and its interaction with the two dietary forms of cholesterol (free and esterified), we undertook to study the role of pancreatic cholesterol esterase in cholesterol absorption. The results showed that (i) cholesterol esters contribute a disproportionately high fraction of absorbed dietary cholesterol, (ii) rates of intestinal cholesterol absorption are related to pancreatic cholesterol esterase activity, (iii) mRNA specific for pancreatic cholesterol esterase is induced 15-fold by dietary sterol esters and 10-fold by free sterol, (iv) the induction of cholesterol esterase mRNA is reversible, and (v) free cholesterol transport into cultured human intestinal cells is enhanced 300% by pancreatic cholesterol esterase. These data implicate pancreatic cholesterol esterase as pivotal in a metabolic loop under positive feedback control for the absorption of dietary cholesterol, whether free or esterified.

Identification of a species specific regulatory site in human pancreatic cholesterol esterase.

All mammalian pancreatic cholesterol esterases (CEase) bind to membrane-associated heparin at a single site on the intestinal brush border membrane with a dissociation constant of 100 nM. While the enzyme is bound to the membrane, the activity of the human and bovine enzymes is enhanced 2-fold when compared to the activity of the enzyme in solution. On the other hand, soluble heparin potently inhibits the human CEase-catalyzed hydrolysis of cholesterol oleate with an IC50 of 2 x 10(-4) mg/mL, a value that is about 10(4) times more potent than that found with the bovine enzyme. The C-terminal portion of the human enzyme contains 16 proline-rich repeats of 11 amino acids each, while that from other species contains only a few of these repeat units. To determine if the unique human C-terminus is responsible for this inhibition, two chimeras containing either the human N-terminus (residues 1-445) and the bovine C-terminus (residues 446-557), HB, or the bovine N-terminus (residues 1-445) and the human C-terminus (residues 446-722), BH, were prepared. The cholesterol oleate hydrolytic activity of these chimeras was similar to that for the recombinant human and bovine enzymes. Importantly, each chimera was inhibited by heparin with IC50 values of 0.03 and 0.1 mg/mL for HB and BH, respectively. These intermediate IC50 values indicate that human CEase has two structural regions that contribute to is unique inhibition by this sulfated glycosaminoglycan, and these could regulate cholesterol uptake in humans.

Cardioprotective effects of alcohol: mediation by human vascular alcohol dehydrogenase.

Numerous studies have shown that moderate drinking protects against coronary disease, but no mechanism for this effect has been established. In the present study we show that the B1 isoenzyme of alcohol dehydrogenase (ADH) is expressed in human blood vessels. Polymerase chain reaction (PCR) employing total human aortic cDNA as a template detected a 0.6 kb band, the nucleotide sequence of which is an identical match to the low Km (50 microM) B1 ADH isoenzyme nucleotide sequence. Immunoblot of vascular homogenates shows a 40 KDa band, i.e., the size of the B1 ADH subunit, and immunohistochemical studies of vessel sections demonstrate high density staining with anti-human ADH (Class I) but not control sera. These studies identify within blood vessels the existence of a metabolic pathway sensitive to low substrate concentrations and capable of producing a reductive (NADH) environment that could antagonize lipoprotein oxidation and hence could account for a protective effect of ethanol on atherosclerosis.

Immune mechanisms of cardiac disease.

It is evident that cellular infiltration can affect cardiac structure and function in a variety of disease states. Myocardial contractility can be impaired by cell-mediated injury or local release of cytokines. The study of immune cardiac disease has entered a period of rapid expansion that should be characterized by delineation of the mechanisms by which immune cells and factors localize in the myocardium, modulate myocyte function, and remodel myocardial architecture (Fig. 2). This new knowledge should result in the ability to target specifically both the pathways by which cardiac contractility is impaired by chronic inflammation and the sustained immune reactivity to cardiac antigens that underlies chronic myocardial inflammation. Nonspecific therapeutic interventions directed at congestive heart failure, currently the only acceptable approach to the treatment of immune myocarditis, should then serve a more ancillary function in the context of the use of rationally designed drugs. Such drugs could, for example, be specifically targeted to inhibiting the trafficking of leukocytes into the heart or the effects of their subsequent activation within the myocardium.

Cholesterol transport function of pancreatic cholesterol esterase: directed sterol uptake and esterification in enterocytes.

We have recently hypothesized that neutral lipids can, in part, move across biological membranes via a mechanism involving enzymes anchored to membrane proteoglycans such as those found in the brush border of the enterocyte [Bosner, M. S., Gulick, T., Riley, D. J. S., Spilburg, C. A., & Lange, L. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7438-7442]. Present results now show a subsequent, essential protein-mediated sorting of neutral lipids for further intracellular metabolism. Thus, in the absence of enzyme, 0.002 pmol of cellular ester appeared after 2 h, and its level increased only 3.5-fold after 12 h. However, in the presence of cholesterol esterase, the level of cholesterol ester increased 39-fold in the same time period, indicating that the enzyme-mediated uptake accounts for 10-fold greater ester synthesis than that from basal absorption. Kinetic analysis reveals that both enzyme-mediated and background absorption depend on taurocholate concentration and are second-order reactions more likely dependent on collision than diffusion. Other lipid-recognizing proteins such as pancreatic triglyceride lipase and the intestinal fatty acid binding protein are not stimulatory to intracellular cholesterol processing. Taken together, these data suggest that pancreatic cholesterol esterase and possibly other proteoglycan-binding extracellular enzymes of neutral lipid metabolism may facilitate movement of neutral lipids into the plasma membrane and direct them into functional intracellular sites.

Assessment of percent cholesterol absorption in humans with stable isotopes.

Dietary cholesterol restriction is a general recommendation for the medical community and emphasizes the importance of intestinal cholesterol absorption and metabolism in humans. However, several methods that may accurately quantify cholesterol absorption utilize radioactive isotopes that are undesirable for younger individuals, women, children, and normal subjects. To eliminate this hazard, we have developed a procedure for measurement of percent cholesterol absorption, based on that of Zilversmit (1972. Proc. Soc. Exp. Med. Biol. 140: 862-865), using stable nonradioactive isotopic tracers of cholesterol. [26,26,26,27,27,27-2H]cholesterol (30 mg) was administered orally and [23,24,25,26,27-13C]cholesterol (15 mg) was administered intravenously on day 0 and percent cholesterol absorption was calculated as the plasma ratio of oral/intravenous isotopic tracer on day 3 as determined by gas chromatography-mass spectrometry with selected ion monitoring. Tracer cholesterol given orally peaked in plasma on day 2 and then slowly declined in parallel with the intravenous tracer. Cholesterol absorption in 16 healthy subjects (on no medication and not ingesting alcohol) consuming a Step One Diet was 53.5% +/- 8.5 SD%. Five subjects underwent repeat testing after 4-6 weeks with excellent replication (SD of difference between tests = 2.8%). No differences in the metabolism of [13C5]cholesterol, [2H6]cholesterol, and [14C]cholesterol were observed. The use of stable isotopes for the study of percent cholesterol absorption is precise and safe, allowing repeated measurements in normal individuals and thus facilitating clinical investigation of this key component of human cholesterol metabolism.

Molecular mechanism of ethanol metabolism by human brain to fatty acid ethyl esters.

Ethanol metabolism in the human brain has been documented to occur with the formation of fatty acid ethyl esters. These neutral lipids can disorder membranes and interrupt mitochondrial function. Their formation is under the control of three synthases, localized to grey matter and purified to homogeneity. cDNA cloning demonstrates two of these enzymes to be GSH S-transferases and has enabled initiation of genetic studies of alcohol-induced CNS injury.

Purification and characterization of fatty acid ethyl ester synthase-II from human myocardium.

Fatty acid ethyl ester synthases metabolize ethanol nonoxidatively in those extrahepatic organs most commonly damaged by alcohol abuse. This study was designed to isolate and purify human myocardial synthase-II, one of the enzymes responsible for catalyzing the formation of fatty acid ethyl esters. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase-I, synthase-II, and synthase-III activities, eluting at conductivities of 5, 7, and 11 mS, respectively. From this elution profile, fatty acid ethyl ester synthase-II accounts for up to 50% of total synthesis in the human heart. This enzyme species was purified over 2200-fold to homogeneity after chromatography over hydroxylapatite, CM-cellulose, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this homogeneous species showed a single band at 65 kDa which corresponded to its molecular weight determined by gel filtration. This molecular weight and its lack of glutathione transferase activity indicate that this species is not related to synthase-I and -III. Homogeneous synthase-II has a Vmax for palmitate, stearate, oleate, and linoleate of 70, 80, 140, and 120 nmol/mg/h, respectively. The Km for palmitate, stearate, oleate, and linoleate is 0.19, 0.12, 0.10, and 0.18 mM, respectively. The substrate specificity with respect to alcohol chain length was also investigated in the presence of 0.65 mM [14C]oleic acid. The Vmax for methanol, ethanol, propanol, and butanol was 180, 100, 280, and 410 nmol/mg/h, respectively. The Km for methanol, ethanol, propanol, and butanol was 1.16, 1.04, 0.58, and 0.33 M, respectively. The N-terminal 17-amino acid sequence of human synthase-II does not correspond to any known N-terminal amino acid sequence, indicating that this may be a novel protein. However, it has over 70% homology to a sequence close to the C terminus of rabbit cytochrome P-450IIC1 and over 50% homology to a sequence of human hemopexin starting at residue 16. Synthase-II does not cross-react with human hemopexin antibody and rat cytochrome P-450C antibody. Thus, this study provides evidence that synthase-II is a novel protein, distinct from synthase-I and -III, and it also provides a foundation for subsequent cloning and genetic studies of fatty acid ethyl ester synthase-II in man.

Structure of the human pancreatic cholesterol esterase gene.

The gene for human pancreatic cholesterol esterase consists of 11 exons and 10 introns and is 9.2 kb in length. The last and longest exon (841 nucleotides) is unique to the human gene. Functional amino acids are encoded on separate exons. The leader sequence is encoded by a single exon which carries two additional N-terminal amino acids of the mature functional protein. A positive TATA element is identified 43 nucleotides from the start codon. Pulse-field gel electrophoresis and hybridization with various cDNA probes and direct sequence data revealed the existence of a CEase-like gene. Partial sequence analysis of this gene from a human cosmid library and human genomic DNA showed a premature stop signal in exon 10, shortly after the codon for the active-site histidine. Both the functional gene and the CEase-like gene have a polyadenylation signal in the 3'-untranslated region. Thus, the complex gene structure for this intestinally active enzyme may provide in part a potential molecular explanation for the well-known heterogeneity of the intestinal absorption of cholesterol.

Nonoxidative ethanol metabolism: expression of fatty acid ethyl ester synthase-III in cultured neural cells.

Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines--N1E115 cells, PC12 cells, and SK-N-MC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is potentially related to the toxic effect of ethanol on the human brain.

Site-specific mutagenesis of two histidine residues in fatty acid ethyl ester synthase-III.

Human myocardial fatty acid ethyl ester synthase-III is a newly described acidic glutathione S-transferase that metabolizes both ethanol and carcinogens. Structure-function studies have not been performed relating these two distinct enzymatic activities. Since there are only two histidine residues in fatty acid ethyl ester synthase-III (His 72 and His 163), the role of each was examined by site-specific mutagenesis. Fatty acid ethyl ester synthase-III mutagenized at position 72 to contain either Gln, Pro or Ala had less than 5% of control glutathione S-transferase activity but retained fatty acid ethyl ester synthase activity under standard assay conditions. In contrast, substitution of histidine 163 with proline had no effect on glutathione S-transferase activity, but it slightly increased synthase activity. Thus, this study indicates that histidine plays a differential role in fatty acid ethyl ester synthase III depending on the nucleophilic substrate.

Immune cytokines and cardiac disease.

In conditions such as idiopathic dilated congestive cardiomyopathy associated with lymphocytic myocarditis and cardiac allograft rejection, the immune system can reversibly impair cardiac function. Cytokines interleukin-1 and tumor necrosis factor disrupt ß-adrenergic signal transduction and agonist stimulation of contractility. Identification of this reversible effect potentially offers a novel pathophysiologic mechanism for producing cardiac injury.

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA.

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.

A new method for assessment of cultured cardiac myocyte contractility detects immune factor-mediated inhibition of beta-adrenergic responses.

BACKGROUND: Potentially reversible congestive heart failure accompanies disease states associated with an immune cell myocardial infiltrate such as cardiac allograft rejection and inflammatory myocarditis. We therefore examined the hypothesis that immune cells can produce noncytotoxic alterations in cardiac function. METHODS AND RESULTS: A novel system to evaluate cultured cardiac myocyte contractility was developed using neonatal rat cardiocytes grown on human amniotic membrane segments. Spontaneous synchronous cell beating produced macroscopic distortion of these membranes. Movement of free-floating membranes anchored within a perfusion chamber was visualized under low-power microscopy and measured from recordings of the rhythmic displacement of membrane-adherent markers. Additions of graded concentrations of isoproterenol to the perfusate produced up to threefold increases in the initial contractile phase velocity (contractile index), with an EC50 of 10(-7) M. When the extracellular Ca2+ concentration was increased from 0.9 to 3.6 mM, 2.43-fold increases in this index occurred. Myocytes incubated for 72 hours in the presence of dilutions of medium conditioned by activated rat splenic macrophages and lymphocytes exhibited an isoproterenol contractile index inhibited by 62% compared with control cells. In contrast, responses of supernatant-exposed and control cells to increased extracellular Ca2+ concentrations were not significantly different. Parallel studies of increases in myocyte intracellular adenosine 3':5'-cyclic monophosphate concentrations in response to isoproterenol stimulation demonstrated correlative inhibition that was specific for exposure to medium conditioned by immune cells. CONCLUSION: Thus, a new method of in vitro cardiac contractility assessment that has significant advantages over existing systems has been developed and characterized. This new method has enabled description of an inhibitor of cardiac contractile function produced by activated immune cells.