Cholesteryl Ester Content Research Articles

Inhibition of ileal apical but not basolateral bile acid transport reduces atherosclerosis in apoE−/− mice

ObjectiveInterruption of the enterohepatic circulation of bile acids induces hepatic bile acid synthesis, increases hepatic cholesterol demand, and increases clearance of apoB-containing lipoproteins in plasma. Based on these effects, bile acid sequestrants have been used for many years to treat hypercholesterolemia and the associated atherosclerosis. The objective of this study was to determine the effect of blocking ileal apical versus basolateral membrane bile acid transport on the development of hypercholesterolemia and atherosclerosis in mouse models. Methods and resultsApoE−/− and Ldlr−/− mice deficient in the apical sodium-dependent bile acid transporter (Asbt) or apoE−/− mice deficient in the basolateral bile acid transporter (Ostα) were fed an atherogenic diet for 16 weeks. Bile acid metabolism, cholesterol metabolism, gene expression, and development of atherosclerosis were examined. Mice deficient in Asbt exhibited the classic response to interruption of the enterohepatic circulation of bile acids, including significant reductions in hepatic and plasma cholesterol levels, and reduced aortic cholesteryl ester content. Ileal Fibroblast Growth Factor-15 (FGF15) expression was significantly reduced in Asbt−/−apoE−/− mice and was inversely correlated with expression of hepatic cholesterol 7-hydroxylase (Cyp7a1). Ileal FGF15 expression was directly correlated with plasma cholesterol levels and aortic cholesterol content. In contrast, plasma and hepatic cholesterol levels and atherosclerosis development were not reduced in apoE−/− mice deficient in Ostα. ConclusionsDecreases in ileal FGF15, with subsequent increases in hepatic Cyp7a1 expression and bile acid synthesis appear to be necessary for the plasma cholesterol-lowering and atheroprotective effects associated with blocking intestinal bile acid absorption.

Abstract 108: Probucol Rescued Reproduction of LCAT Null Male Mice

Low plasma HDL mice, such as lecitin:cholesterol acyltransferase null mice (Lcat-null), Abca1- null mice and ABCA1 inactivator, probucol-fed mice have significantly low cholesterol content in their adrenal glands and ovaries. Cholesterylester content in brain of male LCAT null mice was significantly reduced while in female the content was not change compare to wild type mice. The reproduction of Lcat-null male pups was reduced to 31% of that expected by Mendelian genetics. Gender ratio in reproduction of wild type, ABCA1 null, and ABCG1null mice were 1 to1 in the same mating condition. Supplement of vitamin E in feeding chow increased vitamin E level in plasma and recovered reproduction defect of male LCAT null mice. Thus, plasma HDL is important lipoprotein to carry hydrophobic vitamin to steroidgenetic cells. Furthermore, although 0.2% probucol-fed mice reduced its plasma HDL only to 5% within 2 weeks, probucol-fed mice reproduced normally. This result suggesting some beneficial function may occur in this chow treatment. So that, we fed 0.2% probucol containing chow to LCAT (+/-) mice during their mating and found recovery of gender ratio in the weaned mice. HMGCoA reductase expression was increased in adrenal gland of probucol fed mice indicate cholesterol de novo synthesis may increased to support cholesterol requirement for steroid synthesis during reproduction. Probucol incorporated HDL (1.00 ug probucol / ug HDL-protein) derived probucol into NCI-H295 human adrenal cortex cell (0.73±0.12 ug probucol / mg cellular protein) and the expression of HMGCoA reductase was significantly increased compared to βactin housekeeping gene expression. Both FC and CE contents in the brain of probucol fed mice were significantly increased. Effect of probucol on cholesterol homeostasis in steroidgenic cells and neuronal cells will be examined in the future study.

Abstract 124: Increased Selective Clearance of Cholesteryl-Ester in Patients with Rheumatoid Arthritis is Reversed by Treatment with Tofacitinib

Inflammation is associated with dysfunctional HDL. Rheumatoid arthritis (RA) is a chronic inflammatory disease with significantly elevated cardiovascular risk in spite of low LDL and HDL cholesterol (C) levels. Using stable isotope methods, we examined underlying metabolic mechanisms for the reduced cholesterol levels in active RA and for the changes with tofacitinib (tofa) treatment. Apolipoprotein and cholesterol kinetics were assessed in 36 RA patients at baseline and following 6 weeks of tofa treatment compared to baseline values in 33 non-RA controls matched for age, gender, and menopausal status. All subjects received constant IV infusions of [3,4] 13 C 2 cholesterol and U- 13 C 6 leucine with collection of fasting blood samples hourly for 22 hours. Whole-body cholesterol efflux and cholesteryl ester (CE) and apolipoprotein production and fractional catabolic rates (FCR) were determined by modeling isotope enrichments. At baseline, total-C, HDL-C, LDL-C, CE and apoA-I concentrations were lower and CE FCR was elevated in RA. No differences were seen in CE production rate, whole body cholesterol efflux or HDL-apoA-I and LDL-apoB production and FCRs. RA patients had elevated serum HDL-SAA, myeloperoxidase and reduced in vitro LCAT activity and mass. Following 6 weeks of tofa therapy, cholesterol and apoAI approached levels of controls and apoB concentrations increased. Tofa treatment reduced CE FCR, increased HDL-apoA-I production rate with no changes in LDL-apoB production rates or LDL-apoB and HDL-apoA-I FCRs. Increased CE FCR in the absence of changes in HDL-apoA-I or LDL-apoB particle clearance suggest increased selective CE uptake, presumably by SR-B1, which was normalized by tofa. Change in CE FCR with treatment was negatively correlated with changes in HDL size and concentration of large HDL particles, but not with LDL size or composition. Change in CE production rate with treatment was positively correlated with change in LCAT mass. This is the first study to evaluate the effect of chronic inflammation on in vivo cholesterol and lipoprotein kinetics in humans. Inflammation increases selective CE uptake explaining the reduced cholesterol levels in RA. Reducing inflammation with tofa restored selective CE uptake to normal rates.

Extracellular Acidosis Is a Novel Danger Signal Alerting Innate Immunity via the NLRP3 Inflammasome

Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages. Acidic pH represents a novel danger signal alerting the innate immunity. Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Abstract 396: Acute Hepatic ACAT2 Knockdown Transiently Increases Plasma and Hepatic Free Cholesterol and Fecal Neutral Sterol Excretion

In spite of the advent of statins, atherosclerotic coronary vascular disease (ASCVD) remains the number one killer of Americans. A way to reduce LDL cholesterol, the primary risk factor of ASCVD, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both biliary and non-biliary pathways. The lipoprotein that delivers cholesterol from the liver through the plasma to the small intestine for transintestinal cholesterol excretion (TICE) is not yet known. We have previously shown that chronic knockdown in mice of hepatic acyl-CoA cholesterol acyltransferase 2 (ACAT2), a cellular enzyme that converts free cholesterol (FC) into cholesteryl ester (CE), appeared to cause the formation of hepatic apoB-containing lipoproteins that preferentially trafficked cholesterol to the small intestine for TICE. We tested the hypothesis that the plasma concentration of TICE-competent, apoB-containing lipoproteins, could be increased by preloading the liver with cholesterol and then acutely depleting the cholesterol by knocking down hepatic ACAT2 with antisense oligonucleotides (ASO). After feeding a high cholesterol (0.2% wt/wt) diet for six weeks, C57BL/6 mice were treated with control non-targeting ASO or ACAT2 ASO for one or two weeks. After only one week of ACAT2 knockdown (ACAT2KD) hepatic ACAT2 protein expression was decreased nearly 80%. This translated into a 50% decrease in hepatic CE concentration in conjunction with a rarely seen 2-fold increase in hepatic FC concentration. Acute hepatic ACAT2KD increased plasma FC levels by 25%, which subsided after two weeks of treatment. The increased plasma FC was primarily associated with large and small LDL. After one week of hepatic ACAT2KD mice had a minor, non-significant increase in biliary cholesterol levels but had a 2-fold increase in fecal neutral sterol (FNS) excretion. In summary our data show that when CE is rapidly cleared from the liver, FNS levels dramatically increase with a transient increase in hepatic and plasma FC levels. We believe that in the ACAT2KD mice the increased plasma FC associated with LDL is feeding into TICE thus resulting in increased FNS.

Hepatic cholesteryl ester accumulation in lysosomal acid lipase deficiency: Non-invasive identification and treatment monitoring by magnetic resonance

Background & AimsLysosomal Acid Lipase (LAL) deficiency is a rare metabolic storage disease, caused by a marked reduction in activity of LAL, which leads to accumulation of cholesteryl esters (CE) and triglycerides (TG) in lysosomes in many tissues. We used 1H magnetic resonance (MR) spectroscopy to characterize the abnormalities in hepatic lipid content and composition in patients with LAL deficiency, and in ex vivo liver tissue from a LAL deficiency rat model. Secondly, we used MR spectroscopy to monitor the effects of an enzyme replacement therapy (ERT), sebelipase alfa (a recombinant human lysosomal acid lipase), on hepatic TG and CE content in the preclinical model.MethodsHuman studies employed cohorts of LAL-deficient patients and NAFLD subjects. Rat experimental groups comprised ex vivo liver samples of wild type, NAFLD, LAL-deficient, and LAL-deficient rats receiving 4 weeks of sebelipase alfa treatment. Hepatic 1H MR spectroscopy was performed using 3T (human) and 7T (preclinical) MRI scanners to quantify hepatic cholesterol and triglyceride content.ResultsCE accumulation was identified in LAL deficiency in both human and preclinical studies. A significant decrease in hepatic CE was observed in LAL-deficient rats following treatment with sebelipase alfa.ConclusionsWe demonstrate an entirely non-invasive method to identify and quantify the hepatic lipid signature associated with a rare genetic cause of fatty liver. The approach provides a more favorable alternative to repeated biopsy sampling for diagnosis and disease progression / treatment monitoring of patients with LAL deficiency and other disorders characterised by increased free cholesterol and/or cholesteryl esters.

Altered fatty acid concentrations in prefrontal cortex of schizophrenic patients

BackgroundDisturbances in prefrontal cortex phospholipid and fatty acid composition have been reported in patients with schizophrenia (SCZ), often as an incomplete lipid profile or a percent of total lipid concentration. In this study, we quantified absolute concentrations (nmol/g wet weight) and fractional concentrations (i.e. percent of total fatty acids) of several lipid classes and their constituent fatty acids in postmortem prefrontal cortex of SCZ patients (n = 10) and age-matched controls (n = 10). MethodsLipids were extracted, fractionated with thin layer chromatography and assayed. ResultsMean total lipid, phospholipid, individual phospholipids, plasmalogen, triglyceride and cholesteryl ester concentrations did not differ significantly between the groups. Compared to controls, SCZ brains showed significant increases in several monounsaturated and polyunsaturated fatty acid absolute concentrations in cholesteryl ester. Significant increases or decreases occurred in palmitoleic, linoleic, γ-linolenic and n-3 docosapentaenoic acid absolute concentrations in total lipids, triglycerides or phospholipids. Changes in fractional concentrations did not consistently reflect absolute concentration changes. ConclusionThese findings suggest disturbed prefrontal cortex fatty acid absolute concentrations, particularly within cholesteryl esters, as a pathological aspect of schizophrenia.

Fibrate treatment induced quantitative and qualitative HDL changes associated with an increase of SR-BI cholesterol efflux capacities in rabbits

Fibrates are widely used as lipid lowering drugs acting as peroxisome proliferator-activated receptors α (PPARα) agonists and modulating the expression of several genes involved in lipid and lipoprotein metabolism. Much less is known on the effect of fibrates in HDL structure and composition. Therefore, we examined whether fenofibrate induces quantitative and/or qualitative modifications in HDL metabolism in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. We first demonstrated that 3-week treatment with fenofibrate (250 mg/kg/day) induced an important increase in serum apolipoprotein A-I, HDL-cholesterol and HDL-phospholipids concentrations and a relative enrichment in HDL cholesteryl ester content. Moreover, the fatty acid profiles from fenofibrate-treated rabbits displayed a dramatic increase in the serum or HDL C18:3 ω6 to C18:2 ω6 ratio suggesting higher Δ6 desaturase activity. In addition, HDL from fenofibrate-treated animals exhibited higher relative proportions of sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. We then reported that fenofibrate induced major changes in the physical characteristics of HDL, mainly a higher size and a faster mobility on agarose gel electrophoresis. Finally, serum or HDL from treated rabbits exhibited higher capacity to promote cholesterol efflux from Scavenger receptor class B type I (SR-BI)-rich Fu5AH cells compared to controls. Our findings demonstrate that fenofibrate has beneficial effects in rabbits by increasing the mass of the circulating HDL pool and by modifying their composition transforming them as better acceptors of cellular cholesterol through SR-BI pathway. These effects of fenofibrate might contribute to its benefits on the prevention and treatment of atherosclerosis.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

Selenium Inhibits Renal Oxidation and Inflammation But Not Acute Kidney Injury in an Animal Model of Rhabdomyolysis

Acute kidney injury (AKI) is a manifestation of rhabdomyolysis (RM). Extracellular myoglobin accumulating in the kidney after RM promotes oxidative damage, which is implicated in AKI. To test whether selenium (Se) supplementation diminishes AKI and improves renal function. Dietary selenite increased Se in the renal cortex, as demonstrated by X-ray fluorescence microscopy. Experimental RM-stimulated AKI as judged by increased urinary protein/creatinine, clusterin, and kidney injury molecule-1 (KIM-1), decreased creatinine clearance (CCr), increased plasma urea, and damage to renal tubules. Concentrations of cholesterylester (hydro)peroxides and F₂-isoprostanes increased in plasma and renal tissues after RM, while aortic and renal cyclic guanidine monophosphate (cGMP; marker of nitric oxide (NO) bioavailability) decreased. Renal superoxide dismutase-1, phospho-P65, TNFα gene, MCP-1 protein, and the 3-chloro-tyrosine/tyrosine ratio (Cl-Tyr/Tyr; marker of neutrophil activation) all increased after RM. Dietary Se significantly decreased renal lipid oxidation, phospho-P65, TNFα gene expression, MCP-1 and Cl-Tyr/Tyr, improved NO bioavailability in aorta but not in the renal microvasculature, and inhibited proteinuria. However, CCr, plasma urea and creatinine, urinary clusterin, and histopathological assessment of AKI remained unchanged. Except for the Se++ group, renal angiotensin-receptor-1/2 gene/protein expression increased after RM with parallel increases in MEK1/2 inhibitor-sensitive MAPkinase (ERK) activity. We employed synchrotron radiation to identify Se distribution in kidneys, in addition to assessing reno-protection after RM. Se treatment has some potential as a therapeutic for AKI as it inhibits oxidative damage and inflammation and decreases proteinuria, albeit histopathological changes to the kidney and some plasma and urinary markers of AKI remain unaffected after RM.

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.

Altered lipid concentrations of liver, heart and plasma but not brain in HIV-1 transgenic rats

Disturbed lipid metabolism has been reported in antiretroviral-naive HIV-1-infected patients suggesting a direct effect of the virus on lipid metabolism. To test that the HIV-1 virus alone could alter lipid concentrations, we measured these concentrations in an HIV- 1 transgenic (Tg) rat model of human HIV-1 infection, which demonstrates peripheral and central pathology by 7-9 months of age. Concentrations were measured in high-energy microwaved heart, brain and liver from 7-9 month-old HIV-1 Tg and wildtype rats, and in plasma from non-microwaved rats. Plasma triglycerides and liver cholesteryl ester and total cholesterol concentrations were significantly higher in HIV-1 Tg rats than controls. Heart and plasma fatty acid concentrations reflected concentration differences in liver, which showed higher n-3 and n-6 polyunsaturated fatty acid (PUFA) concentrations in multiple lipid compartments. Fatty acid concentrations were increased or decreased in heart and liver phospholipid subfractions. Brain fatty acid concentrations differed significantly between the groups for minor fatty acids such as linoleic acid and n-3 docosapentaenoic acid. The profound changes in heart, plasma and liver lipid concentrations suggest a direct effect of chronic exposure to the HIV-1 virus on peripheral lipid (including PUFA) metabolism.

Low density lipoprotein receptor-related protein 1 expression correlates with cholesteryl ester accumulation in the myocardium of ischemic cardiomyopathy patients

Our hypothesis was that overexpression of certain lipoprotein receptors might be related to lipid accumulation in the human ischemic myocardium. Intramyocardial lipid overload contributes to contractile dysfunction and arrhythmias in cardiomyopathy. Thus, the purpose of this study was to assess the effect of hypercholesterolemic LDL and hypertrigliceridemic VLDL dose on LRP1 expression in cardiomyocytes, as well as the potential correlation between LRP1 expression and neutral lipid accumulation in the left ventricle tissue from ischemic cardiomyopathy patients. Cell culture experiments include control and LRP1-deficient cardiomyocytes exposed to lipoproteins under normoxic and hypoxic conditions. Explanted hearts from 18 ICM patients and eight non-diseased hearts (CNT) were included. Low density lipoprotein receptor-related protein 1 (LRP1), very low density lipoprotein receptor (VLDLR) and low density lipoprotein receptor (LDLR) expression was analyzed by real time PCR and Western blotting. Cholesteryl ester (CE), triglyceride (TG) and free cholesterol (FC) content was assess by thin layer chromatography following lipid extraction. Western blotting experiments showed that protein levels of LRP1, VLDLR and HIF-1α were significantly upregulated in ischemic hearts. Immunohistochemistry and confocal microscopy analysis showed that LRP1 and HIF-1α were upregulated in cardiomyocytes of ICM patients. In vitro studies showed that VLDL, LDL and hypoxia exerted an upregulatory effect on LRP1 expression and that LRP1 played a major role in cholesteryl ester accumulation from lipoproteins in cardiomyocytes. Myocardial CE accumulation strongly correlated with LRP1 levels in ischemic hearts. Taken together, our results suggest that LRP1 upregulation is key for myocardial cholesterol ester accumulation in ischemic human hearts and that LRP1 may be a target to prevent the deleterious effects of myocardial cholesterol accumulation in ischemic cardiomyopathy.

Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to trans fatty acids

The development of atherosclerosis and the inflammatory response were investigated in LDLr-KO mice on three high-fat diets (40% energy as fat) for 16 weeks: trans (TRANS), saturated (SAFA) or ω-6 polyunsaturated (PUFA) fats. The following parameters were measured: plasma lipids, aortic root total cholesterol (TC), lesion area (Oil Red-O), ABCA1 content and macrophage infiltration (immunohistochemistry), collagen content (Picrosirius-red) and co-localization of ABCA1 and macrophage (confocal microscopy) besides the plasma inflammatory markers (IL-6, TNF-α) and the macrophage inflammatory response to lipopolysaccharide from Escherichia coli (LPS). As expected, plasma TC and TG concentrations were lower on the PUFA diet than on TRANS or SAFA diets. Aortic intima macrophage infiltration, ABCA1 content, and lesion area on PUFA group were lower compared to TRANS and SAFA groups. Macrophages and ABCA1 markers did not co-localize in the atherosclerotic plaque, suggesting that different cell types were responsible for the ABCA1 expression in plaques. Compared to PUFA, TRANS and SAFA presented higher collagen content and necrotic cores in atherosclerotic plaques. In the artery wall, TC was lower on PUFA compared to TRANS group; free cholesterol was lower on PUFA compared to TRANS and SAFA; cholesteryl ester concentration did not vary amongst the groups. Plasma TNF-α concentration on PUFA and TRANS-fed mice was higher compared to SAFA. No difference was observed in IL-6 concentration amongst groups. Regarding the macrophage inflammatory response to LPS, TRANS and PUFA presented higher culture medium concentrations of IL-6 and TNF-α as compared to SAFA. The PUFA group showed the lowest amount of the anti-inflammatory marker IL-10 compared to TRANS and SAFA groups. In conclusion, PUFA intake prevented atherogenesis, even in a pro-inflammatory condition.

Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis

Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies (E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44-57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the 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 CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

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 &gt;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 (&gt;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.

Clinical studies in CKD

Introduction and Aims: Chronic kidney disease (CKD) is a major risk factor of cardiovascular disease and mortality in both the general population and post-myocardial patients. Kidney function gradually decreases with age (±1 ml/min/y), but in patients with a myocardial infarction (MI) this deterioration is accelerated (±2 ml/min/y). Epidemiological and experimental studies suggest that n-3 fatty acids supplementation may prevent or slow the decline of kidney function. We examined the effect of the marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) and of the plant-derived alpha-linolenic acid (ALA) on the decline of kidney function among patients who have had a first myocardial infaction. Methods: In a secondary analysis of the randomized, double blind, placebo-controlled Alpha Omega Trial, 2426 of 4937 (49%) patients (aged 60-80y; 79% men) were included of whom kidney function was measured both at baseline and at 41 months of follow-up. Margarine spreads were used to deliver 400 mg EPA-DHA/d, 2g ALA/d, both EPA-DHA and ALA, or a placebo for 40 months. We measured at baseline and end of follow-up both serum creatinine and Cystatin-C and calculated the estimated GFR (eGFR) from the CKD Epidemiology Collaboration (CKD-EPI) equations. Baseline characteristics were compared among the four treatment groups using ANOVA. The decline of kidney function in the three treatment groups ALA, EPA-DHA and EPA-DHA plus ALA were compared to the placebo group. Results: The 4 randomly assigned groups did not differ in baseline characteristics. Supplementation of ALA, EPA-DHA, EPA and DHA increased plasma cholesteryl ester concentrations of ALA, EPA, and DHA. At baseline kidney function did not differ between the four groups, mean (SD) creatinine and cystatin-C based eGFR were 76 (20) ml/min/1.73m2 and 80 (19) ml/min/1.73m2, respectively. During 41 months of follow-up the yearly decline of creatinine and cystatin-C based eGFR (SE) were in the placebo group (background): -2.28 (SE 0.20) and -1.31 (SE 0.12) ml/min/1.73m2/y, respectively. In comparison to the placebo group the mean difference (SE) of the yearly decline of creatinine and cystatine-C based eGFR (SE) were in the ALA supplementation group 0.05 (SE 0.28, P=0.86) and 0.23 (SE 0.18, P=0.20), in the EPA-DHA group 0.56 (SE 0.29, P=0.058) and 0.33 (SE 0.18, P=0.060) and in the EPA-DHA plus ALA group 0.24 (SE 0.29, P=0.40) and 0.28 (SE 0.18, P=0.10; Figure 1). Decline of kidney function (with error bars indicating SE) after 41 months, according to study group. P-value by three independent samples t-tests compared to the placebo group. Conclusions: In patients who had experienced a MI, low-dose supplementation with n-3 fatty acids did not statistically significantly reduce the decline in kidney function. However, there was a trend for EPA-DHA supplementation to slow down the decline of kidney function. These results should be confirmed in a study supplementing with higher doses of n-3 fatty acids.

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR(-/-) background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [(14)C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [(14)C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6C(lo)) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.