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The group 1B phospholipase A2 (PLA2G1B) is a secreted phospholipase that catalyzes the hydrolytic removal of the sn-2 fatty acyl moiety from phospholipids. This enzyme is synthesized most abundantly in the pancreas and is also expressed in the lung. The first part of this review article focuses on the role of pancreatic-derived PLA2G1B in mediating lipid absorption and discusses how the PLA2G1B-derived metabolic product contributes to cardiometabolic diseases, including obesity, hyperinsulinemia, hyperlipidemia, and atherosclerosis. The anti-helminth properties of PLA2G1B will also be discussed. The second part of this review will focus on PLA2G1B expressed in the lung, and in vitro data suggest that how this enzyme may modulate lung inflammation via both hydrolytic activity-dependent and -dependent mechanisms. Finally, recent studies revealing a relationship between PLA2G1B and cancer will also be discussed. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

Objective: Obesity and obesity-related metabolic disorders are major health problems worldwide. The most effective obesity intervention is bariatric surgery. This study tested the hypothesis that bariatric surgery alters phospholipid metabolism in the gastrointestinal tract to favor a metabolically healthy gut microbiota profile and therapeutic intervention of phospholipid metabolism in the gastrointestinal may have similar metabolic benefits. Methods: The first study compared plasma levels of the bioactive lipid metabolites lysophospholipid and trimethylamine N-oxide (TMAO) as well as gut microbiota profile in high fat/carbohydrate (HFHC) diet-fed C57BL/6 mice with or without vertical sleeve gastrectomy (VSG) and in Pla2g1b−/− mice with group 1B phospholipase A2 gene inactivation. The second study examined the effectiveness of the non-absorbable secretory phospholipase A2 inhibitor methyl indoxam to reverse hyperglycemia and hyperlipidemia in HFHC diet-fed C57BL/6 mice after diabetes onset. Results: Both bariatric surgery and PLA2G1B inactivation were shown to reduce lysophospholipid content in the gastrointestinal tract, resulting in resistance to HFHC diet-induced alterations of the gut microbiota, reduction of the cardiovascular risk factors hyperlipidemia and TMAO, decreased adiposity, and prevention of HFHC diet-induced diabetes. Importantly, treatment of wild type mice with methyl indoxam after HFHC diet-induced onset of hyperlipidemia and hyperglycemia effectively restored normal plasma lipid and glucose levels and replicated the metabolic benefits of VSG surgery with diabetes remission and TMAO reduction. Conclusion: These results provided pre-clinical evidence that PLA2G1B inhibition in the digestive tract may be a viable alternative option to bariatric surgery for obesity and obesity-related cardiometabolic disorder intervention. Keywords: Phospholipase A2, Gut microbiota, Cardiometabolic disease, Bariatric surgery, Lysophospholipid

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.

Objective Previous studies have shown that inactivation of the group 1B phospholipase A2 (Pla2g1b) suppresses diet-induced obesity, hyperglycemia, insulin resistance, and hyperlipidemia in C57BL/6 mice. A possible influence of Pla2g1b inactivation on atherosclerosis has not been addressed previously. The current study utilized LDL receptor-deficient (Ldlr−/−) mice with plasma lipid levels and distribution similar to hyperlipidemic human subjects as a preclinical animal model to test the effectiveness of Pla2g1b inactivation on atherosclerosis.

Apolipoprotein (apo) E4 is a major genetic risk factor for a wide spectrum of inflammatory metabolic diseases, including atherosclerosis, diabetes, and Alzheimer disease. This study compared diet-induced adipose tissue inflammation as well as functional properties of macrophages isolated from human APOE3 and APOE4 mice to identify the mechanism responsible for the association between apoE4 and inflammatory metabolic diseases. The initial study confirmed previous reports that APOE4 gene replacement mice were less sensitive than APOE3 mice to diet-induced body weight gain but exhibited hyperinsulinemia, and their adipose tissues were similarly inflamed as those in APOE3 mice. Peritoneal macrophages isolated from APOE4 mice were defective in efferocytosis compared with APOE3 macrophages. Increased cell death was also observed in APOE4 macrophages when stimulated with LPS or oxidized LDL. Western blot analysis of cell lysates revealed that APOE4 macrophages displayed elevated JNK phosphorylation indicative of cell stress even under basal culturing conditions. Significantly higher cell stress due mainly to potentiation of endoplasmic reticulum (ER) stress signaling was also observed in APOE4 macrophages after LPS and oxidized LDL activation. The defect in efferocytosis and elevated apoptosis sensitivity of APOE4 macrophages was ameliorated by treatment with the ER chaperone tauroursodeoxycholic acid. Taken together, these results showed that apoE4 expression causes macrophage dysfunction and promotes apoptosis via ER stress induction. The reduction of ER stress in macrophages may be a viable option to reduce inflammation and inflammation-related metabolic disorders associated with the apoE4 polymorphism.

ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted copper-accepting enzymes. This ATPase has attracted significant attention since the discovery of its mutation leading to human Menkes disease and the demonstration of its distribution in various tissues. Recently, we reported that ATP7A is expressed in the human vasculature. In the present study, we investigated the cellular expression of ATP7A in atherosclerotic lesions of LDL receptor (-/-) mice. Subsequently, we examined the role of ATP7A in regulating the oxidation of LDL in a macrophage cell model. We observed that ATP7A is expressed in atherosclerotic murine aorta and colocalizes with macrophages. To investigate the function of ATP7A, we downregulated ATP7A expression in THP-1 derived macrophages using small interfering RNA (siRNA). ATP7A downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ATP7A resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), a key intracellular enzyme involved in cell-mediated LDL oxidation. In addition, cPLA(2)alpha promoter activity was decreased after downregulation of ATP7A, suggesting that ATP7A transcriptionally regulates cPLA(2)alpha expression. Finally, cPLA(2)alpha overexpression increased LDL oxidation, which was blocked by coadministration of ATP7A siRNA oligonucleotides. These findings suggest a novel mechanism linking ATP7A to cPLA(2)alpha and LDL oxidation, suggesting that this copper transporter could play a previously unrecognized role in the pathogenesis of atherosclerosis.

Previous studies have shown apolipoprotein E (apoE) recruitment to medial layers of carotid arteries after vascular injury in vivo and apoE activation of inducible nitric oxide synthase (iNOS) in smooth muscle cells in vitro. This investigation explored the relationship between medial apoE recruitment and iNOS activation in protection against neointimal hyperplasia. ApoE was present in both neointimal-resistant C57BL/6 mice and neointimal-susceptible FVB/N mice 24 h after carotid denudation, but iNOS expression was observed only in the neointimal-resistant C57BL/6 mice. However, iNOS was not observed in apoE-defective C57BL/6 mice. In contrast, overexpression of apoE in FVB/N mice activated iNOS expression in the injured vessels, resulting in protection against neointimal hyperplasia. ApoE and iNOS were colocalized in the medial layer of neointimal-resistant mouse strains. Endothelial denudation of carotid arteries in the iNOS-deficient NOS2(-/-) mice did not increase neointimal hyperplasia but significantly increased medial thickness and area. The iNOS-specific inhibitor also abrogated the apoE protective effects on vascular response to injury in apoE-overexpressing FVB/N mice. Thus, injury-induced activation of iNOS requires apoE recruitment. Moreover, both apoE and iNOS are necessary for the suppression of cell proliferation, and apoE recruitment without iNOS expression resulted in medial hyperplasia without cell migration to the intima.

Many enzymes and transport proteins participate in cholesterol absorption. This review summarizes recent results on several proteins that are important for each step of the cholesterol absorption pathway, including the important roles of: (i) pancreatic triglyceride lipase (PTL), carboxyl ester lipase (CEL), and ileal bile acid transporter in determining the rate of cholesterol absorption; (ii) ATP binding cassette (ABC) transporters and the Niemann-Pick C-1 like-1 (NPC1L1) protein as intestinal membrane gatekeepers for cholesterol efflux and influx; and (iii) intracellular membrane vesicles and transport proteins in lipid trafficking through intracellular compartments prior to lipoprotein assembly and secretion to plasma circulation.

Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [(3)H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters approximately 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway.

This study compared the physiological process of cholesterol absorption in different strains of inbred mice with the goal of identifying novel mechanism(s) by which cholesterol absorption can be controlled. The rate and amount of cholesterol absorption were evaluated based on [14C]cholesterol appearance in plasma after feeding a meal containing [14C]cholesterol and by the percentage of [14C]-cholesterol absorbed over a 24 h period. Results showed that the rate of [14C]cholesterol appearance in plasma was slower in 129P3/J mice than in SJL/J mice. However, more dietary cholesterol was absorbed over a 24 h period by 129P3/J mice than by SJL/J mice. In both strains of mice, cholesterol delivered with medium-chain triglyceride was absorbed less efficiently than cholesterol delivered with olive oil. The strain- and vehicle-dependent differences in cholesterol absorption efficiency correlated negatively with stomach-emptying rates. Furthermore, inhibition of gastric emptying with nitric oxide synthase inhibitor increased cholesterol absorption efficiency in SJL/J mice. These results document that stomach-emptying rate contributes directly to the rate of dietary cholesterol absorption, which is inversely correlated with the total amount of cholesterol absorbed from a single meal. Additionally, genetic factor(s) that influence gastric emptying may be an important determinant of cholesterol absorption efficiency.

The importance of the cholesterol transporter Niemann-Pick C1 like protein 1 (NPC1L1)in the small intestine for efficient absorption of cholesterol is well documented and its physiological importance is clear (1). Similarly, the drug ezetimibe (Zetia™), which blocks function of this transporter, reduces plasma cholesterol, primarily LDL, by dramatically reducing intestinal absorption of dietary and biliary cholesterol (2). There are controversies about the exact mechanism by which NPC1L1 functions: whether the protein is on the apical cell membrane or intracellular vesicles or both (3–5), and whether ezetimibe directly blocks NPC1L1 or interrupts its association with other proteins in the sterol absorption and transport pathway (6–8). Nevertheless, it is now accepted that this protein is the key player in sterol absorption, although other transporters may play ancillary roles (9, 10).

Carboxyl ester lipase (C33347), previously named cholesterol esterase or bile salt-stimulated (or dependent) lipase, is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, tri-, di-, and mono-acylglycerols, phospholipids, lysophospholipids, and ceramide. The active site catalytic triad of serine-histidine-aspartate is centrally located within the enzyme structure and is partially covered by a surface loop. The carboxyl terminus of the protein regulates enzymatic activity by forming hydrogen bonds with the surface loop to partially shield the active site. Bile salt binding to the loop domain frees the active site for accessibility by water-insoluble substrates. CEL is synthesized primarily in the pancreas and lactating mammary gland, but the enzyme is also expressed in liver, macrophages, and in the vessel wall. In the gastrointestinal tract, CEL serves as a compensatory protein to other lipolytic enzymes for complete digestion and absorption of lipid nutrients. Importantly, CEL also participates in chylomicron assembly and secretion, in a mechanism mediated through its ceramide hydrolytic activity. Cell culture studies suggest a role for CEL in lipoprotein metabolism and oxidized LDL-induced atherosclerosis.Thus, this enzyme, which has a wide substrate reactivity and diffuse anatomic distribution, may have multiple functions in lipid and lipoprotein metabolism, and atherosclerosis.

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.

Bile salt-stimulated carboxyl ester lipase (CEL), also called cholesterol esterase, is one of the major proteins secreted by the pancreas. The physiological role of CEL was originally thought to be its mediation of dietary cholesterol absorption. However, recent studies showed no difference between wild type and CEL knockout mice in the total amount of cholesterol absorbed in a single meal. The current study tests the hypothesis that CEL in the intestinal lumen may influence the type of lipoproteins produced. A lipid emulsion containing 4 mm phospholipid, 13.33 mm[3H]triolein, and 2.6 mm[14C]cholesterol in 19 mm taurocholate was infused into the duodenum of lymph fistula CEL(+/+) and CEL(−/−) mice at a rate of 0.3 ml/h. Results showed no difference between CEL(+/+) and CEL(−/−) mice in the rate of cholesterol and triglyceride transport from the intestinal lumen to the lymph. However, CEL(−/−) mice produced predominantly smaller lipoproteins, whereas the CEL(+/+) mice produced primarily large chylomicrons and very low density lipoprotein. The proximal intestine of CEL(−/−) mice was also found to possess significantly less ceramide hydrolytic activity than that present in CEL(+/+) mice. By using Caco2 cells grown on Transwell membranes as a model, sphingomyelinase treatment inhibited the secretion of larger chylomicron-like lipoproteins without affecting total cholesterol secretion. In contrast, the addition of CEL to the apical medium increased the amount of large lipoproteins produced and alleviated the inhibition induced by sphingomyelinase. Taken together, this study identified a novel and physiologically significant role for CEL, namely the promotion of large chylomicron production in the intestine. The mechanism appears to be mediated through CEL hydrolysis of ceramide generated during the lipid absorption process.

Background & Aims: Numerous studies have suggested phospholipid inhibition of dietary cholesterol absorption through the gastrointestinal tract. This study addressed the importance of luminal phospholipid hydrolysis in this process. Methods: The effect of phospholipase inhibition on cholesterol transport from intestinal lumen to the lymphatics was evaluated in lymph fistula rats. Cholesterol and phospholipid absorption efficiency in intact animals was evaluated in control and phospholipase A 2 (PLA 2 ) gene–targeted mice. Results: The PLA 2 inhibitor FPL 67047XX retarded cholesterol absorption in a lymph fistula rat model. Under basal chow-fed dietary conditions, cholesterol absorption efficiency from a single bolus meal, and plasma lipid levels, were similar among PLA 2 (+/+), PLA 2 (+/−), and PLA 2 (−/−) mice. Interestingly, the nonhydrolyzable phospholipid dioleoyl ether phosphatidylcholine suppressed cholesterol absorption by 10% to 18% in mice without regard to their PLA 2 genotype. When 1-palmitoyl-2-[ 14 C]oleoyl-phosphatidylcholine was used as the substrate, the radiolabeled phospholipid was found to be hydrolyzed and absorbed with equal efficiency in PLA 2 (+/+), PLA 2 (+/−), and PLA 2 (−/−) mice. Conclusions: These results suggested that although phospholipid digestion in the intestinal lumen is a prerequisite for efficient cholesterol absorption, additional enzyme(s) can compensate for pancreatic PLA 2 in catalyzing phospholipid digestion and facilitating cholesterol absorption in PLA 2 knockout mice. GASTROENTEROLOGY 2001;120:1193-1202

This study showed that synthetic peptides containing either a single copy or tandem repeat of the receptor binding domain sequence of apolipoprotein (apo) E, or a peptide containing its C-terminal heparin binding domain, apoE-(211-243), were all effective inhibitors of platelet-derived growth factor (PDGF)-stimulated smooth muscle cell proliferation. In contrast, only the peptide containing a tandem repeating unit of the receptor binding domain sequence of apoE, apoE-(141-155)(2), was capable of inhibiting PDGF-directed smooth muscle cell migration. Peptide containing only a single unit of this sequence, apoE-(141-155), or the apoE-(211-243) peptide were ineffective in inhibiting PDGF-directed smooth muscle cell migration. Additional experiments showed that reductively methylated apoE, which is incapable of receptor binding yet retains its heparin binding capability, was equally effective as apoE in inhibiting PDGF-stimulated smooth muscle cell proliferation. However, reductively methylated apoE was unable to inhibit smooth muscle cell migration toward PDGF. Additionally, the receptor binding domain-specific apoE antibody 1D7 also mitigated the anti-migratory properties of apoE on smooth muscle cells. Finally, pretreatment of cells with heparinase failed to abolish apoE inhibition of smooth muscle cell migration. Taken together, these data documented that apoE inhibition of PDGF-stimulated smooth muscle cell proliferation is mediated by its binding to heparan sulfate proteoglycans, while its inhibition of cell migration is mediated through apoE binding to cell surface receptors.

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.

Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages. Oligonucleotide primers for bile salt-stimulated cholesterol esterase yielded positive reactions with RNA isolated from human peripheral blood monocytes, monocyte-derived macrophages, the human monocytic THP-1 cells, and phorbol ester-induced THP-1 macrophages. In contrast, oligonucleotide primers for hormone-sensitive lipase yielded positive reactions only with RNA isolated from non-differentiated human THP-1 monocytic cells and peripheral blood monocytes, but not those obtained from differentiated THP-1 macrophages or monocyte-derived macrophages. Thus, while human monocytes were capable of synthesizing both enzymes, human macrophages synthesized only bile salt-stimulated cholesterol esterase and not the hormone-sensitive lipase. The synthesis of bile salt-stimulated cholesterol esterase by human macrophages was confirmed by detection of bile salt-stimulated cholesteryl ester hydrolytic activity in conditioned media of differentiated THP-1 cells and human peripheral blood monocyte-derived macrophages. Moreover, incubating human macrophages with oxidized low density lipoprotein (LDL) or acetylated LDL increased bile salt-stimulated cholesterol esterase activity in the conditioned media of these cells. These results with human macrophages were contrasted with results of studies with mouse macrophages, which showed the presence of hormone-sensitive lipase mRNA but not the bile salt-stimulated cholesterol esterase mRNA. Taken together, these results demonstrated species-specific differences in expression of cholesteryl ester hydrolytic enzymes in macrophages. The expression of bile salt-stimulated cholesterol esterase by human macrophages, in a process inducible by modified LDL, suggests a role of this protein in atherogenesis.

This study used the rat pancreatoma AR42J cells as a model to determine the effects of cholesterol on cholesterol esterase biosynthesis. Incubation of AR42J cells with low-density lipoproteins (LDL) or with the cholesterol-induced β-very-low-density lipoproteins did not result in changes in cellular cholesterol levels. These cholesterol-rich lipoproteins also had no effect on cholesterol esterase biosynthesis by the AR42J cells. Cellular cholesterol level was found to increase by approx. 2-fold after incubating the AR42J cells with cationized-LDL. The increase in cellular cholesterol level resulted in a higher level of cholesterol esterase secreted into the culture medium. The increased cellular cholesterol also resulted in higher amounts of cholesterol esterase detected in the AR42J cell lysate. The increase in cholesterol esterase level corresponded to a cholesterol-induced increase in steady-state level of cholesterol esterase mRNA. The results of this study, and our previous observation of post-transcriptional activation of cholesterol esterase induced by intestinal hormones (Huang, Y. and Hui, D.Y. (1991) J. Biol. Chem. 266, 6720–6725), suggested that cholesterol esterase biosynthesis may be regulated by transcriptional and translational mechanisms in response to hormonal and nutrient stimulation of the pancreatic acinar cells. Additionally, the regulation of cholesterol esterase biosynthesis by cholesterol loading of the pancreatic cells suggested a possible role of this enzyme in cholesterol metabolism.

The acidic amino acid residue required for the catalytic activity of rat pancreatic cholesterol esterase has been identified in this study by sequence comparison with other serine esterases and by site-directed mutagenesis experiments. The sequence comparison studies identified 3 acidic residues in homologous domains between cholesterol esterase, acetylcholinesterase, cholinesterase, and Geotrichum candida lipase that may potentially be the catalytic acidic residue in these proteins. The role of Glu78, Asp79, and Asp320 in the catalytic activity of rat cholesterol esterase was then addressed by mutagenesis and expression of the cDNA. Results showed that replacement of Glu78 or Asp79 with alanine has no effect on the ability of the cholesterol esterase to hydrolyze the artificial water-soluble substrate p-nitrophenyl butyrate. In contrast, the Asp320-->Ala320 substitution abolished the enzyme activity of the cholesterol esterase. The specific requirement of Asp320 for optimal enzyme activity was demonstrated by substitution of the aspartic acid with glutamic acid, thus retaining the charge unit at this position. The Asp320-->Glu320 substitution resulted in an enzyme that displayed normal interaction with bile salt. However, catalytic activity of this mutagenized protein was reduced by approximately 50%. These results strongly suggested that aspartic acid 320 is an important component of the catalytic triad of pancreatic cholesterol esterase. The specific requirement of aspartic acid, instead of glutamic acid, for optimal activity is different from that of other members of the serine esterase gene family.

This study used the rat pancreatoma AR42J as a model system to investigate the possible regulation of cholesterol esterase biosynthesis in pancreas. Initial experiments were performed to verify the synthesis of pancreatic cholesterol esterase by the AR42J cells. Results indicated that this pancreatoma cell line synthesized two forms of cholesterol esterase (Mr = 71,000 and 74,000). The 74-kDa protein is most likely the precursor protein, and the 71-kDa protein is the matured enzyme secreted by the AR42J cells. The synthesis and secretion of cholesterol esterase were stimulated 2-5-fold by incubating the cells with 0.5-4 nM of cholecystokinin or 0.5-2 nM of secretin. Analysis of the RNA isolated from the hormone-stimulated cells revealed that stimulation of cholesterol esterase biosynthesis was not due to an increase in cholesterol esterase mRNA. Furthermore, inhibition of transcription with actinomycin D has no effect on the hormone-induced cholesterol esterase biosynthesis. Therefore, the mechanism of hormone stimulation was not dependent on de novo RNA synthesis. In vitro translation studies showed that the cholesterol esterase mRNA isolated from stimulated AR42J cells were translated more efficiently than those from control cells. Thus, the increased cholesterol esterase biosynthesis induced by gastric hormones was most likely mediated by posttranscriptional modification of the cholesterol esterase mRNA.

The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.

Bile salt-stimulated cholesterol esterase is synthesized in the pancreatic acinar cells and is released into the intestinal lumen where it catalyzes cholesterol absorption. In the current study, Caco-2 cells were used as an in vitro model to study the interaction between the pancreatic cholesterol esterase with intestinal cells. Results showed that addition of increasing concentrations of cholesterol esterase in the incubation medium increased the uptake of micellar cholesteryl oleate by Caco-2 cells. The cholesterol esterase also increased the cellular uptake of the nonhydrolyzable cholesteryl linoleoyl ether. However, maximum uptake of the cholesteryl ether analog was 50% of that for cholesteryl oleate. The initial interaction of cholesterol esterase with Caco-2 cells was mediated by binding of the protein to a low affinity and high capacity binding site on the cell surface. Cholesterol esterase bound to the cell surface could be internalized via a monensin-sensitive mechanism. The cholesterol esterase taken up by the cells had a short residence time and was either degraded or was rapidly re-secreted from the cells. Chloroquine had no effect on the degradation or re-secretion of cholesterol esterase by Caco-2 cells, indicating that lysosomes were not involved with these processes. The cholesterol esterase taken up by the cells was not available to mediate further cholesterol uptake. These results indicated that the bile salt-stimulated cholesterol esterase secreted from pancreas could facilitate intestinal lipid absorption only transiently. The data suggest that the regulation of cholesterol esterase synthesis and secretion by the pancreas may be important for regulation of cholesterol absorption.

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.

The molecular basis of cholesteryl ester transfer protein (CETP) deficiency was investigated in 4 unrelated CETP-deficient families. The high density lipoprotein-cholesterol levels of the probands exceeded 150 mg/dl. The plasma of the probands was totally deficient in CETP activity and mass. The genomic DNA of the patients was amplified by polymerase chain reaction, using two oligonucleotide primers located in the intron 12 and 14 of the CETP gene, and the amplified products were directly sequenced. Two patients were homozygous for a G-to-A change at the 5'-splice donor site of the intron 14. The G-to-A change would cause impaired splicing of pre-messenger RNA. The other two probands were heterozygous for the mutation, but totally lacked CETP. Their lipoprotein patterns were also similar to those of the two homozygotes. Thus, other genetic defects or metabolic factors influencing CETP expression are implicated. The data suggest that the G-to-A mutation may be common in human plasma CETP deficiency. Furthermore, there could be compound heterozygotes who totally lack plasma CETP and have lipoprotein profiles similar to those of homozygotes.

The lung is composed of a series of branching conducting airways that terminate in grape-like clusters of delicate gas-exchanging airspaces called pulmonary alveoli. Maintenance of alveolar patency at end expiration requires pulmonary surfactant, a mixture of phospholipids and proteins that coats the epithelial surface and reduces surface tension. The surfactant lining is exposed to the highest ambient oxygen tension of any internal interface and encounters a variety of oxidizing toxicants including ozone and trace metals contained within the 10 kl of air that is respired daily. The pathophysiological consequences of surfactant oxidation in humans and experimental animals include airspace collapse, reduced lung compliance, and impaired gas exchange. We now report that the hydrophilic surfactant proteins A (SP-A) and D (SP-D) directly protect surfactant phospholipids and macrophages from oxidative damage. Both proteins block accumulation of thiobarbituric acid-reactive substances and conjugated dienes during copper-induced oxidation of surfactant lipids or low density lipoprotein particles by a mechanism that does not involve metal chelation or oxidative modification of the proteins. Low density lipoprotein oxidation is instantaneously arrested upon SP-A or SP-D addition, suggesting direct interference with free radical formation or propagation. The antioxidant activity of SP-A maps to the carboxyl-terminal domain of the protein, which, like SP-D, contains a C-type lectin carbohydrate recognition domain. These results indicate that SP-A and SP-D, which are ubiquitous among air breathing organisms, could contribute to the protection of the lung from oxidative stresses due to atmospheric or supplemental oxygen, air pollutants, and lung inflammation.

This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 × 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL2 and HDL3, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.

The ability of apolipoprotein (apo-) B48 to interact with lipoprotein receptors was investigated using three different types of lipoproteins. First, canine chylomicron remnants, which contained apo-B48 as their primary apoprotein constituent, were generated by the hydrolysis of chylomicrons with milk lipoprotein lipase. These apo-B48-containing chylomicron remnants are deficient in apo-E and reacted very poorly with apo-E receptors on adult dog liver membranes and the low density lipoprotein (apo-B,E) receptors on human fibroblasts. Addition of normal human apo-E3 restored the receptor binding activity of these lipoproteins. Second, beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs were subfractionated into distinct classes containing apo-E along with either apo-B48 or apo-B100. Both classes bound to the apo-B,E and apo-E receptors. Their binding was almost completely mediated by apo-E, as evidenced by the ability of the anti-apo-E to inhibit the receptor interaction. Third, beta-VLDL from type III hyperlipoproteinemic patients were subfractionated by immunoaffinity chromatography into lipoproteins containing apo-E plus either apo-B48 or apo-B100. Both subfractions bound poorly to apo-B,E and apo-E receptors due to the presence of defective apo-E2. However, the residual binding of the apo-B48-containing and apo-B100-containing human beta-VLDL was inhibited by the anti-apo-E. After lipase hydrolysis, apo-B100 became a more prominant determinant responsible for mediating receptor binding to the apo-B,E receptor. By contrast, lipase hydrolysis did not increase the binding activity of the apo-B48-containing beta-VLDL. These results indicate that apo-B48 does not play a direct role in mediating the interaction of lipoproteins with receptors on fibroblasts or liver membranes.

Apolipoprotein (apo-) E2 and beta-migrating very low density lipoproteins (beta-VLDL) (which were isolated from type III hyperlipoproteinemic subjects) both demonstrated defective binding to apo-E and apo-B,E receptors on dog liver membranes and to apo-B,E low density lipoproteins (LDL) receptors on fibroblasts. The defective binding activity of the apo-E2 and beta-VLDL varied from very poor to nearly normal. The ability of the beta-VLDL to interact with hepatic apo-E receptors was enhanced by the addition of normal apo-E3 to the beta-VLDL. Furthermore, cysteamine treatment of the apo-E2 in beta-VLDL enhanced binding of the beta-VLDL to both apo-E and apo-B,E receptors. The importance of apo-E in mediating the receptor binding of beta-VLDL to these receptors was confirmed by using monoclonal antibodies. The residual binding activity of beta-VLDL to apo-E and apo-B,E receptors was inhibited by greater than 90% with anti-apo-E, while the addition of anti-apo-B had little effect. The apo-B in the beta-VLDL was capable of binding to apo-B,E receptors after the hydrolysis of the beta-VLDL triglycerides with milk lipoprotein lipase. Lipase treatment yielded, two subfractions of beta-VLDL. One fraction (d = 1.02 to 1.03 g/ml) was enriched with apo-B100; the other fraction (d less than 1.006 g/ml) was enriched with apo-B48 and apo-E2. Significantly increased amounts of the apo-B100-enriched fraction bound to apo-B,E receptors. Inhibition of this binding caused by the addition of anti-apo-B indicated that the binding activity of this subfraction was mediated by apo-B100. The apo-B48-enriched fraction did not show a significant increase in receptor binding, suggesting that apo-B48 does not bind to these receptors. In a control experiment, it was shown that triglyceride-rich VLDL, which contain normal apo-E3 and apo-B100, bind significantly to both liver apo-E receptors and fibroblast apo-B,E receptors. This binding activity was inhibited by greater than 90% with anti-apo-E. Lipase hydrolysis of the VLDL did not further enhance their receptor-binding activity. These results demonstrate that apo-E, and not apo-B, is the major determinant mediating the receptor-binding activity of cholesterol-rich beta-VLDL and triglyceride-rich VLDL.

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105 000 × g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67 000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.

Plasma lipoproteins containing either the B (apo-B) or E (apo-E) apoproteins, e.g. apo-B low density lipoproteins (LDL) isolated from normolipemic humans and apo-E-containing cholesterol-induced high density lipoproteins (apo-E HDLc) isolated from cholesterol-fed dogs, suppress phytohemagglutinin (PHA)-induced lymphocyte activation and inhibit early events such as mitogen-enhanced 45Ca2+ accumulation and late events such as enhanced DNA synthesis. On a molar basis, apo-E HDLc are 3.5 times more effective than apo-B LDL in inhibiting 45Ca2+ accumulation by 50% and 3.8 times more effective than apo-B LDL in suppressing DNA synthesis by 50%. Both lipoproteins bind to the lymphocyte surface, and in competitive binding assays apo-E HDLc and apo-B LDL bind to the same receptors. These receptors, termed immunoregulatory receptors, comprise a homogeneous class of binding sites which do not act cooperatively. The equilibrium dissociation constant (Kd) for apo-B LDL is 2.0 X 10(-7) M at 4 degrees C and 37 degrees C; the Kd for apo-E HDLc is 9.3 X 10(-8) M at 4 degrees C and 7.3 X 10(-8) M at 37 degrees C. At saturation, 16,000 and 20,000 LDL particles are bound/cell at 4 degrees C and 37 degrees C, respectively. The corresponding values for apo-E HDLc are 6,700 at 4 degrees C and 5,500 at 37 degrees C. The increased effectiveness of apo-E HDLc in supp]ressing the PHA-induced Ca2+ accumulation and DNA synthesis is due to the multiple receptor binding of apo-E HDLc. At 37 degrees C, each apo-E HDLc particle occupies multiple receptors at a ratio of 3.6:1 relative to apo-B LDL. The enhanced affinity of apo-E HDLc most likely results from the multiple interactions of this lipoprotein with the receptors.

Hepatic membranes from adult dog livers have receptors which bind to lipoproteins containing the E apoprotein (the apo-E HDLc) but lack specific receptors for the apo-B-containing low density lipoproteins (LDL). Scatchard analysis of direct binding data for 125I-apo-E-HDLc revealed nonlinearity of the binding which could be resolved into two components, suggesting the presence of two separate binding sites. The binding site for apo-E HDLc that possessed the highest affinity (Kd = 0.23 x 10(-9) M) was calcium-dependent and was sensitive to proteolytic digestion with pronase. The lower affinity (Kd = 20 x 10(-9) M) binding site for apo-E HDLc did not require calcium and was resistant to pronase digestion. Chemical modification of the arginyl or lysyl residues of the apo-E HDLc prevented the HDLc from binding to the higher affinity receptor but had no effect on their binding to the lower affinity site. Adult canine liver membranes also bound canine 125I-HDL. However, the binding of HDL was of lower affinity (Kd = 8.2 x 10(-8) M), did not require calcium, was not blocked by modification of the lysyl or arginyl residues, and may not be of physiologic significance. Although the liver membranes from normal chow-fed adult dogs did not bind canine LDL, it was possible to demonstrate specific high affinity binding of LDL under certain metabolic conditions in dogs. When adult dogs were treated with the hypocholesterolemic agent cholestyramine, the liver membranes from these animals readily bound canine LDL. The Kd for canine LDL binding to these liver membranes was 15 x 10(-9) M. Furthermore, it was possible to demonstrate high affinity binding of LDL to the liver membranes from young rapidly growing puppies (Kd = 11 x 10(-9) M). The binding of the 125I-LDL to the liver membranes from the adult cholestyramine-treated dogs or from the puppies appeared to be mediated by apo-B,E receptors which resembled the LDL receptor of human skin fibroblasts. The 125I-LDL binding of these liver membranes was competitively inhibited by the addition of unlabeled LDL or apo-E HDLc. On the other hand, 125I-apo-E HDLc, capable of binding to the apo-B,E or to the apo-E receptors, were only partially displaced by the addition of unlabeled LDL but were totally displaced by apo-E HDLc. In summary, the adult dog liver possessed only the apo-E receptor. An apo-B,E receptor capable of binding LDL and HDLc could be induced by treatment of adult dogs with cholestyramine. Similarly, the liver membranes of young growing puppies possessed the apo-E and po-B,E receptors and were capable of binding both apo-E HDLc and LDL. The mechanism responsible for the control of the expression of the hepatic apo-B,E and/or apo-E receptors remains to be determined. These data indicate that a unique receptor capable of interacting specifically with apo-E-containing lipoproteins, and not with apo-B-containing lipoproteins (LDL), exists in the adult canine liver.