Macrophage Cholesteryl Ester Research Articles

Glucose-Dependent Insulinotropic Polypeptide Suppresses Foam Cell Formation of Macrophages through Inhibition of the Cyclin-Dependent Kinase 5-CD36 Pathway.

Glucose-dependent insulinotropic polypeptide (GIP) has been reported to have an atheroprotective property in animal models. However, the effect of GIP on macrophage foam cell formation, a crucial step of atherosclerosis, remains largely unknown. We investigated the effects of GIP on foam cell formation of, and CD36 expression in, macrophages extracted from GIP receptor-deficient (Gipr−/−) and Gipr+/+ mice and cultured human U937 macrophages by using an agonist for GIP receptor, [D-Ala2]GIP(1–42). Foam cell formation evaluated by esterification of free cholesterol to cholesteryl ester and CD36 gene expression in macrophages isolated from Gipr+/+ mice infused subcutaneously with [D-Ala2]GIP(1–42) were significantly suppressed compared with vehicle-treated mice, while these beneficial effects were not observed in macrophages isolated from Gipr−/− mice infused with [D-Ala2]GIP(1–42). When macrophages were isolated from Gipr+/+ and Gipr−/− mice, and then exposed to [D-Ala2]GIP(1–42), similar results were obtained. [D-Ala2]GIP(1–42) attenuated ox-LDL uptake of, and CD36 gene expression in, human U937 macrophages as well. Gene expression level of cyclin-dependent kinase 5 (Cdk5) was also suppressed by [D-Ala2]GIP(1–42) in U937 cells, which was corelated with that of CD36. A selective inhibitor of Cdk5, (R)-DRF053 mimicked the effects of [D-Ala2]GIP(1–42) in U937 cells. The present study suggests that GIP could inhibit foam cell formation of macrophages by suppressing the Cdk5-CD36 pathway via GIP receptor.

Rab proteins implicated in lipid storage and mobilization.

Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol (TG) leads to fatty liver disease encompassing steatosis, steatohepatitis and fibrosis. This places individuals at risk of developing cirrhosis, hepatocellular carcinoma or hepatic decompensation and also contributes to the emergence of insulin resistance and dyslipidemias affecting many other organs. Excessive accumulation of TG in adipose tissue contributes to insulin resistance as well as to the release of cytokines attracting leucocytes leading to a pro-inflammatory state. Pathological accumulation of cholesteryl ester (CE) in macrophages in the arterial wall is the progenitor of atherosclerotic plaques and heart disease. Overconsumption of dietary fat, cholesterol and carbohydrates explains why these diseases are on the increase yet offers few clues for how to prevent or treat individuals. Dietary regimes have proven futile and barring surgery, no realistic alternatives are at hand as effective drugs are few and not without side effects. Overweight and obesity-related diseases are no longer restricted to the developed world and as such, constitute a global problem. Development of new drugs and treatment strategies are a priority yet requires as a first step, elucidation of the molecular pathophysiology underlying each associated disease state. The lipid droplet (LD), an up to now overlooked intracellular organelle, appears at the heart of each pathophysiology linking key regulatory and metabolic processes as well as constituting the site of storage of both TGs and CEs. As the molecular machinery and mechanisms of LDs of each cell type are being elucidated, regulatory proteins used to control various cellular processes are emerging. Of these and the subject of this review, small GTPases belonging to the Rab protein family appear as important molecular switches used in the regulation of the intracellular trafficking and storage of lipids.

Abstract 18763: Characteristics and Anti-Atherosclerotic Properties of (-)-Epigallocatechin Gallate Loaded Lipid Nanoparticles in vitro

BACKGROUND: Atherosclerotic cardiovascular disease is the leading cause of death and disability in the United States and other developed countries. Macrophages are major cells responsible for atherosclerotic lesion development. (-)-Epigallocatechin gallate (EGCG), a natural compound found in green tea, is valuable to prevent atherosclerosis. But EGCG’s low level of stability, bioavailability and targeting specificity in the body makes administering it in therapeutic doses unrealistic. METHODS AND RESULTS: We have successfully synthesized EGCG loaded lipid nanoparticles (ENP), which are composed of biocompatible and biodegradable lipids, surfactants and EGCG. The size of nanoparticles was less than 100 nm in diameter measured using a dynamic light scattering method and a transmission electron microscope. The encapsulation efficiency was around 90%. As compared to native EGCG, ENP significantly increased EGCG stability and macrophage EGCG content at 37°C. Remarkably, ENP significantly decreased human macrophage cholesteryl ester content measured using a high performance liquid chromatography (HPLC) system. ENP also significantly lowered both gene expression and protein secretion of monocyte chemoattractant protein 1 (MCP-1) in/from human macrophages measured using real-time PCR and ELISA assays, respectively. We also incorporated target ligands on the surface of ENP. The target ligands further increased the binding and uptake of those nanoparticles in human macrophages. CONCLUSIONS: ENP significantly enhanced EGCG stability, increased its cellular bioavailability, decreased cholesteryl ester content and MCP-1 expression in macrophages. Target ligands increased the target specificity of those nanoparticles to human macrophages. ENP with target ligands have a potential for preventing atherosclerotic lesion development through enhancing EGCG uptake by macrophages and decreasing macrophage cholesterol accumulation and inflammatory responses. Funding support: The project described was supported by Grant Number R15AT007013 from NIH NCCAM.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

Nanoencapsulation Enhances Epigallocatechin-3-gallate Stability and Its Antiatherogenic Bioactivities in Macrophages

We have successfully synthesized (-)-epigallocatechin-3-gallate (EGCG) encapsulated nanostructured lipid carriers (NLCE) and chitosan-coated NLCE (CSNLCE) using natural lipids, surfactant, chitosan, and EGCG. Nanoencapsulation dramatically improved EGCG stability. CSNLCE significantly increased EGCG content in THP-1-derived macrophages compared with nonencapsulated EGCG. As compared to 10 μM nonencapsulated EGCG, both NLCE and CSNLCE at the same concentration significantly decreased macrophage cholesteryl ester content. NLCE and CSNLCE significantly decreased mRNA levels and protein secretion of monocyte chemoattractant protein-1 (MCP-1) levels in macrophages, respectively. These data suggest that nanoencapsulated EGCG may have a potential to inhibit atherosclerotic lesion development through decreasing macrophage cholesterol content and MCP-1 expression.

The Mechanism of Action of Bufalin in Inhibition of Lipid Droplet Accumulation in Mouse Macrophages

A cardiotonic glycoside, bufalin, originally isolated from the dried white venom of Chinese toad Bufo gargarizans, was found to inhibit lipid droplet accumulation in mouse macrophages. Bufalin selectively inhibited synthesis of [C]cholesteryl ester (CE), a main component of lipid droplet, from [C]oleic acid and [C]cholesterol with IC values of 8.6 µM and 10 µM, respectively. The postlysosomal metabolism of cholesterol to CE in macrophages was also inhibited by the compound with a similar IC value of 13.2 µM. However, the compound exhibited almost no effect on acyl-CoA : cholesterol acyltransferase, a key enzyme in CE synthesis localized in the endoplasmic reticulum (ER). From the fluorescent microscopic observation of cellular lipids, bufalin-treated macrophages increased the accumulation of free cholesterol in lysosomes and caused to enlarge the shape and volume of lysosomes as well as pregnenolone-treated macrophages. These findings suggest that bufalin inhibited the postlysosomal metabolism of cholesterol, leading to a reduction of lipid droplets in mouse macrophages without cytotoxicity.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Lipid droplet formation on opposing sides of the endoplasmic reticulum

In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders.

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.

Akt3 Deficiency in Macrophages Promotes Foam Cell Formation and Atherosclerosis in Mice

Akt, a serine-threonine protein kinase, exists as three isoforms. The Akt signaling pathway controls multiple cellular functions in the cardiovascular system, and the atheroprotective endothelial cell-dependent role of Akt1 has been recently demonstrated. The role of Akt3 isoform in cardiovascular pathophysiology is not known. We explored the role of Akt3 in atherosclerosis using mice with a genetic ablation of the Akt3 gene. Using hyperlipidemic ApoE(-/-) mice, we demonstrated a macrophage-dependent, atheroprotective role for Akt3. In vitro experiments demonstrated differential subcellular localization of Akt1 and Akt3 in macrophages and showed that Akt3 specifically inhibits macrophage cholesteryl ester accumulation and foam cell formation, a critical early event in atherogenesis. Mechanistically, Akt3 suppresses foam cell formation by reducing lipoprotein uptake and promoting ACAT-1 degradation via the ubiquitin-proteasome pathway. These studies demonstrate the nonredundant atheroprotective role for Akt3 exerted via the previously unknown link between the Akt signaling pathway and cholesterol metabolism.

Abstract 23: Identification of a Novel Intracellular Cholesteryl Ester Hydrolase (CES3) in Human Macrophages: Compensatory Increase Following CES1 Silencing

Removal of free cholesterol (FC) from lesion-associated macrophage foam cells represents the major mechanism for reducing the lipid burden of atherosclerotic plaques and intracellular cholesteryl ester (CE) hydrolysis is recognized as the rate-limiting step in this process. We have earlier reported cloning of human macrophage CE hydrolase (gene symbol CES1) that associated with cytoplasmic lipid droplets and enhanced cellular CE mobilization. Macrophage-specific transgenic expression of CES1 attenuated diet-induced atherosclerosis and lesion necrosis in ldlr -/- mice. Several enzymes have since been identified as intracellular CE hydrolases in human macrophages and the objective of the present study was to determine the relative contribution of CES1 to the total CE hydrolytic activity in human macrophages. Immune-precipitation by CES1-specific antibody resulted in 70-80% decrease in enzyme activity indicating that CES1 is responsible for >70% of the total CE hydrolytic activity (Panel A). To examine the effects of CES1 depletion/knockdown on FC efflux, human monocyte/macrophages (THP1) were stably transfected with 4 different CES1-specific shRNA vectors (p81-p84) to generate THP1-shRNA cells; cells transfected with pRSL vector were used as controls. Despite significant reduction in CES1 expression both at mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel carboxylesterase, CES3 (Panel B). Human macrophage CES3 was identical to the reported CES3 except it lacked exon 8 and, in wild type THP1 cells it was only expressed at 29.2±6.1 percent of CES1. Transient over-expression of this novel variant of CES3 led to an increase in CE hydrolytic activity (>4 fold), mobilization of intracellular lipid droplets and reduction in cellular CE content (108.1±7.65 vs 160.9±7.00, P=0.0005), establishing CES3 as a bona fide CE hydrolase. Reciprocal backup circuits, where deficiency of a gene leads to an increase in another gene with similar function, have been described and this study provides first evidence of functional compensation in intracellular CE hydrolysis whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. To gain insight into the mechanisms underlying the observed increase in CES3 following CES1 deficiency in silico analyses of CES3 promoter was performed and several NF-kB binding sites were identified. Based on our earlier demonstration of a decrease in NF-kB activation by CES1 over-expression, it is speculated that conversely, deficiency of CES1 increases NF-kB activation leading to compensatory increase in CES3 expression.

Early steps in reverse cholesterol transport

Several controversies exist related to the molecular identity and subcellular localization of the enzyme catalyzing macrophage cholesteryl ester hydrolysis. Some of these issues have been reviewed earlier and this review summarizes new developments that describe effects of overexpression or gene ablation. The main objective is to highlight the disagreement between lack of gene expression and incomplete abolition of macrophage cholesteryl ester hydrolytic activity and to emphasize the importance of redundancy. New information resulting from the continuing characterization of the various cholesteryl ester hydrolases (hormone-sensitive lipase, HSL; cholesteryl ester hydrolase, CEH; and KIAA1363/NCEH1) is reviewed. Whereas CEH overexpression leads to beneficial effects such as decreased inflammation, improved glucose tolerance/insulin sensitivity, and attenuation of atherosclerotic lesion progression, deficiency/ablation of HSL or KIAA1363/NCEH1 results in incomplete loss of macrophage cholesteryl ester hydrolysis/turnover. New paradigms challenging the classical view of cytoplasmic cholesteryl ester hydrolysis and reverse cholesterol transport are also presented. The observed beneficial effects of CEH overexpression identify macrophage cholesteryl ester hydrolysis as an important therapeutic target and future studies will determine whether similar effects are obtained with overexpression of HSL or KIAA1363/NCEH1. It is imperative that, for clinical benefit, mechanisms to enhance endogenous cholesteryl ester hydrolase(s) are established.

Regulation of Lipid Droplet Cholesterol Efflux From Macrophage Foam Cells

Cholesterol efflux from macrophages is the first and potentially most important step in reverse cholesterol transport, a process especially relevant to atherosclerosis and to the regression of atherosclerotic plaques. Increasingly, lipid droplet (LD) cholesteryl ester (CE) hydrolysis is being recognized as a rate-limiting step in cholesterol efflux. The traditional view on macrophage CE hydrolysis is that this pathway is entirely dependent on the action of neutral hydrolases, and numerous candidate CE hydrolases have been proposed to play a role in lipid hydrolysis in macrophages and atherogenesis. Although the exact identity of macrophage-specific CE hydrolases remains to be clarified, a common point to all of these studies is that enhancing LD-associated CE hydrolysis increases cholesterol efflux and is antiatherogenic. Understanding how cholesterol is mobilized from LDs offers new steps for modulating cholesterol efflux, and recently a role for autophagy and lysosomal acid lipase in macrophage lipolysis has emerged. Autophagy and lysosomal acid lipase thus represent novel therapeutic targets to enhance macrophage reverse cholesterol transport. This review discusses our current understanding of the relationship between macrophage LDs and atherosclerosis and presents recent insights into the mechanisms for LD CE hydrolysis in macrophage foam cells.

Atherosclerotic lesion progression is attenuated by reconstitution with bone marrow from macrophage-specific cholesteryl ester hydrolase transgenic mice

Accumulation of cholesteryl ester (CE)-enriched macrophage foam cells is central to the development of atherosclerotic lesions. Intracellular CE hydrolysis is the rate-limiting step in the removal of free cholesterol from macrophage foam cells. Enhancing this process by transgenic overexpression of CE hydrolase (CEH) resulted in a significant decrease in diet-induced atherosclerosis in LDL receptor-deficient (LDLR-/-) mice. However, for development of this step as an antiatherosclerotic target it is imperative to demonstrate that increase in CE hydrolysis after initiation of lesion formation will also attenuate further lesion progression. The objective of the present study was to directly address this issue using an animal model. LDLR-/- mice were fed a high-fat high-cholesterol diet (Western Diet) for 8 wk to initiate lesion formation and were then divided into three groups. Group 1 mice were killed to determine baseline lesion development. Mice in groups 2 and 3 were irradiated and transplanted with either LDLR-/- or LDLR-/-CEH transgenic bone marrow and maintained on Western Diet. Atherosclerotic lesion progression was assessed after 12 wk. While a more than fourfold increase in total lesions (compared to group 1) was seen in group 2 receiving LDLR-/- marrow, a significantly lower increase (<2-fold) was noted in mice reconstituted with CEH transgenic marrow (group 3). Lesions in group 3 mice were also more cellular with smaller necrotic cores. Lesion progression is associated with a switch in macrophage phenotype from anti-inflammatory M2 to proinflammatory M1 phenotype and is consistent with reduced lesion progression. Aortas from group 3 mice contained a significantly higher percentage of macrophages in M2 phenotype (Ly6C(lo)). These data demonstrate for the first time that enhancing macrophage CE hydrolysis even after lesion initiation can still attenuate further lesion progression and also switches the phenotype of lesion-associated macrophages to anti-inflammatory M2 phenotype establishing intracellular CE hydrolysis as an anti-atherosclerotic as well as anti-inflammatory target.

Volume-Sensitive Chloride Current in Macrophages is Regulated by High-Cholesterol Diet, Cholesteryl Ester Hydrolase and ROS

Cholesteryl ester (CE) accumulation in macrophages activates proinflammatory mediators and underlies atherosclerotic plaque formation. We tested whether dietary cholesterol regulates volume-sensitive Cl- current (ICl,swell) via reactive oxygen species (ROS) and determined whether depleting CE by macrophage-specific transgenic over-expression of human cholesteryl ester hydrolase (CEHTg) suppresses ICl,swell. ICl,swell was recorded in peritoneal macrophages from LDL receptor-null (Ldlr-/-) and Ldlr-/-CEHTg mice feed chow or a Western-type high-fat, high-cholesterol diet (16 wk). In macrophages from non-fasting Ldlr-/- on chow, ICl,swell at +60 mV was 6.7 ± 1.4 pA/pF under isosmotic control conditions, increased to 112.6 ± 11.4 pA/pF on exposure to 100-μM H2O2 and to 102.9 ± 13.1 pA/pF upon osmotic swelling (0.85T), and was fully blocked by the ICl,swell-inhibitor DCPIB (10 μM). CEHTg expression did not alter ICl,swell under control conditions, but significantly reduced ICl,swell elicited by H2O2 to 33.7 ± 5.0 pA/pF and by 0.85T to 24.7 ± 6.3 pA/pF; ICl,swell remained DCPIB-sensitive. By contrast, ICl,swell already was strongly activated, 113.9 ± 24.1 pA/pF, under isosmotic control conditions in macrophages from Ldlr-/- fed a high-cholesterol diet; H2O2 did not elicit additional current, 126.8 ± 37.7 pA/pF, and DCPIB fully blocked ICl,swell. High-cholesterol-induced ICl,swell was ROS-dependent. Both ebselen (15 μM), a ROS scavenger, and rotenone (10 μM), a mitochondrial e- transport blocker, fully suppressed ICl,swell. Furthermore, CEHTg expression reversed basal ICl,swell activation by high-cholesterol diet. ICl,swell in Ldlr-/-CEHTg macrophages was 2.7 ± 0.8 pA/pF under control conditions, was increased to only 27.4 ± 3.7 and 23.1 ± 3.6 pA/pF by H2O2 and 0.85T, and was fully blocked by DCPIB. These data suggest that macrophage CE regulate both ICl,swell under control conditions and its response to stimulation. Furthermore, mitochondrial ROS are required for cholesterol-dependent ICl,swell activation.

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Improved Insulin Sensitivity in High Fat- and High Cholesterol-fed Ldlr−/− Mice with Macrophage-specific Transgenic Expression of Cholesteryl Ester Hydrolase: ROLE OF MACROPHAGE INFLAMMATION AND INFILTRATION INTO ADIPOSE TISSUE

Cellular cholesterol balance induces changes in the inflammatory status of macrophages, and low grade chronic inflammation is increasingly being recognized as one of the key steps in the development of atherosclerosis as well as insulin resistance. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of intracellular stored cholesteryl esters (CEs) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have earlier reported reduced atherosclerosis and lesion necrosis in macrophage-specific CEH transgenic mice on a Ldlr(-/-) background. In the present study, we tested the hypothesis that reduced intracellular accumulation of CE in macrophages from CEH transgenic mice will attenuate expression of proinflammatory mediators, thereby reducing infiltration into adipose tissue, alleviating inflammation, and resulting in improved insulin sensitivity. Western diet fed Ldlr(-/-)CEH transgenic mice showed improved insulin sensitivity as assessed by glucose and insulin tolerance tests. Macrophages from CEH transgenic mice expressed significantly lower levels of proinflammatory cytokines (interleukin-1beta and interleukin-6) and chemokine (MCP-1; monocyte chemoattractant protein). Attenuation of NF-kappaB- and AP-1-driven gene expression was determined to be the underlying mechanism. Infiltration of macrophages into the adipose tissue that increases inflammation and impairs insulin signaling was also significantly reduced in Ldlr(-/-)CEH transgenic mice. In the OP-9 adipocyte peritoneal macrophage co-culture system, macrophages from CEH transgenic mice had a significantly reduced effect on insulin signaling as measured by Akt phosphorylation compared with nontransgenic macrophages. Taken together, these studies demonstrate that macrophage-specific overexpression of CEH decreases expression of proinflammatory mediators and attenuates macrophage infiltration into the adipose tissue, resulting in decreased circulating cytokines and improved insulin sensitivity.

Lipoprotein lipase: from gene to obesity

Lipoprotein lipase (LPL) is a multifunctional enzyme produced by many tissues, including adipose tissue, cardiac and skeletal muscle, islets, and macrophages. LPL is the rate-limiting enzyme for the hydrolysis of the triglyceride (TG) core of circulating TG-rich lipoproteins, chylomicrons, and very low-density lipoproteins (VLDL). LPL-catalyzed reaction products, fatty acids, and monoacylglycerol are in part taken up by the tissues locally and processed differentially; e.g., they are stored as neutral lipids in adipose tissue, oxidized, or stored in skeletal and cardiac muscle or as cholesteryl ester and TG in macrophages. LPL is regulated at transcriptional, posttranscriptional, and posttranslational levels in a tissue-specific manner. Nutrient states and hormonal levels all have divergent effects on the regulation of LPL, and a variety of proteins that interact with LPL to regulate its tissue-specific activity have also been identified. To examine this divergent regulation further, transgenic and knockout murine models of tissue-specific LPL expression have been developed. Mice with overexpression of LPL in skeletal muscle accumulate TG in muscle, develop insulin resistance, are protected from excessive weight gain, and increase their metabolic rate in the cold. Mice with LPL deletion in skeletal muscle have reduced TG accumulation and increased insulin action on glucose transport in muscle. Ultimately, this leads to increased lipid partitioning to other tissues, insulin resistance, and obesity. Mice with LPL deletion in the heart develop hypertriglyceridemia and cardiac dysfunction. The fact that the heart depends increasingly on glucose implies that free fatty acids are not a sufficient fuel for optimal cardiac function. Overall, LPL is a fascinating enzyme that contributes in a pronounced way to normal lipoprotein metabolism, tissue-specific substrate delivery and utilization, and the many aspects of obesity and other metabolic disorders that relate to energy balance, insulin action, and body weight regulation.

Pentacecilides, new inhibitors of lipid droplet formation in mouse macrophages, produced by Penicillium cecidicola FKI-3765-1: I. Taxonomy, fermentation, isolation and biological properties

New compounds designated pentacecilides A to C were isolated from the fermentation broth of Penicillium cecidicola FKI-3765-1 by solvent extraction, silica gel column chromatography and preparative HPLC. Pentacecilides A and B dose-dependently inhibited lipid droplet formation in mouse macrophages. Furthermore, pentacecilides A and B were found to inhibit the synthesis of cholesteryl ester in mouse macrophages with respective IC(50) values of 3.65 and 4.76 microM without any cytotoxic effect, but pentacecilide C showed almost no activity. The study of the mechanism of action strongly suggested that pentacecilides A and B inhibit acyl-CoA: cholesterol acyltransferase activity in macrophages.

Abdominal localization of Tangier disease mimicking a pancreatic neoplasm

Tangier disease is a rare genetic disorder of lipid metabolism, characterized by severe deficiency of plasma high-density lipoprotein cholesterol, low-plasma total cholesterol, and accumulation of cholesteryl ester in macrophages. Abdominal manifestation of Tangier disease is extremely rare, being reported only once in the English literature. We describe a 55-year-old patient with this condition, who underwent splenectomy 37 years before, because of splenomegaly with thrombocytopenia, and subsequently presented with abdominal pain and pancreatic mass, simulating a pancreatic tumor. The preoperative diagnosis of endocrine or solid-cystic neoplasm was made, and the tumor was successfully resected with distal pancreatectomy. Histological examination showed that the mass was composed of histiocytic cells containing lipids; some aggregates of giant polynucleated histiocytes with intracytoplasmic cholesterol crystals. To our knowledge, this is the first report of pancreatic manifestation of Tangier disease. As suggested earlier, splenectomy in these patients seems to predispose to deposition of lipids and infiltration of the abdomen by inflammatory cells.