IntroductionLiver is an important organ in maintaining whole-body cholesterol homeostasis by mediating cholesterol synthesis, uptake, storage (in lipid droplets), transport (VLDL), and elimination (bile). Accumulation of excessive cholesterol in the peripheral tissues such as arteries drives pathological consequence including atherosclerosis (1Pathophysiology of coronary artery disease.). Reverse cholesterol transport (RCT) is a pathway by which cholesterol is removed from peripheral tissues and delivered to the liver, where it is excreted in bile, and subsequently into the feces in the form of bile acid and free cholesterol. High-density lipoprotein (HDL) acts as the acceptor of cholesterol effluxed from peripheral tissues, and the transport vehicle for delivery of cholesterol to the liver. The majority of effluxed cholesterol is esterified to cholesteryl ester (CE) by lecithin-cholesterol acyltransferase (LCAT) (2Rousset X. Vaisman B. Amar M. Sethi A.A. Remaley A.T. Lecithin: cholesterol acyltransferase--from biochemistry to role in cardiovascular disease.). Scavenger receptor-BI (SR-BI) then mediates selective uptake of CE from HDL particles into the liver. However, the detailed process downstream of CE uptake by SR-BI in hepatocytes is not fully understood.It has been reported that in the liver CE derived from HDL requires neutral cholesteryl ester hydrolase activity for conversion to free cholesterol (FC) (3Shimada A. Tamai T. Oida K. Takahashi S. Suzuki J. Nakai T. Miyabo S. Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).). FC can then be secreted into bile (either as free cholesterol or after conversion to bile acids) or esterified by acyl-CoA:cholesterol acyltransferase (ACAT) to CE, which is stored in lipid droplets or secreted in VLDL. Mobilization of CE stored in lipid droplets also requires participation of CE hydrolase(s). Despite the important roles of CE hydrolysis in the last step of RCT and in the liver cholesterol homeostasis, the CE hydrolase(s) that is/are involved in these biological processes in the liver still remains to be determined.The roles of carboxylesterases, including carboxylesterase1d (Ces1d, previously annotated as Ces3 or TGH, human ortholog CES1) in lipid metabolism have been studied extensively [reviewed in (4Lian J. Nelson R. Lehner R. Carboxylesterases in lipid metabolism: from mouse to human., 5Pharmacological intervention of liver triacylglycerol lipolysis: The good, the bad and the ugly.)]. In the liver, Ces1d/CES1 has been shown to participate in TG metabolism and the mobilization of preformed TG for VLDL assembly (6Lian J. Bahitham W. Panigrahi R. Nelson R. Li L. Watts R. Thiesen A. Lemieux M.J. Lehner R. Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis., 7Wei E. Ben Ali Y. Lyon J. Wang H. Nelson R. Dolinsky V.W. Dyck J.R. Mitchell G. Korbutt G.S. Lehner R. Loss of TGH/Ces3 in mice decreases blood lipids, improves glucose tolerance, and increases energy expenditure., 8Lian J. Wei E. Groenendyk J. Das S.K. Hermansson M. Li L. Watts R. Thiesen A. Oudit G.Y. Michalak M. Lehner R. Ces3/TGH Deficiency Attenuates Steatohepatitis.). Loss of Ces1d in mice enhances insulin sensitivity and protects from high-fat diet-induced liver steatosis by increasing FA oxidation and decreasing hepatic de novo lipogenesis (6Lian J. Bahitham W. Panigrahi R. Nelson R. Li L. Watts R. Thiesen A. Lemieux M.J. Lehner R. Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis., 8Lian J. Wei E. Groenendyk J. Das S.K. Hermansson M. Li L. Watts R. Thiesen A. Oudit G.Y. Michalak M. Lehner R. Ces3/TGH Deficiency Attenuates Steatohepatitis.).It has been proposed that CES1 catalyzes CE hydrolysis in the human macrophage (9Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice., 10Ghosh S. St Clair R.W. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.) and that Ces1d/CES1 catalyzes CE hydrolysis in the liver (11Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport., 12Bie J. Wang J. Yuan Q. Kakiyama G. Ghosh S.S. Ghosh S. Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr-/- mice., 13Bie J. Wang J. Marqueen K.E. Osborne R. Kakiyama G. Korzun W. Ghosh S.S. Ghosh S. Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.), thereby promoting cholesterol removal from the body by the RCT pathway. Excess cholesterol in peripheral tissues and macrophages is esterified to CE and stored in lipid droplets. This storage of CE is initially beneficial to avoid cell toxicity from FC, but excessive accumulation of CE in macrophages leads to the formation of foam cells. Foam cells accumulated in the intima of arterial walls promote the development of atherosclerotic lesions. Hydrolysis of CE in macrophages is the rate-limiting step of cholesterol efflux, which is the first step of RCT (14Regulation of lipid droplet cholesterol efflux from macrophage foam cells.). It is important to note that mouse macrophages do not express Ces1d, suggesting Ces1d does not play a critical role in mouse macrophage CE metabolism. Specific expression of CES1 in mouse macrophages was reported to reduce atherosclerosis in Ldlr−/− mice (9Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice.). However, the CE hydrolase activity of Ces1d/CES1 and the role of CES1 in macrophage CE turnover have been challenged (15Igarashi M. Osuga J. Uozaki H. Sekiya M. Nagashima S. Takahashi M. Takase S. Takanashi M. Li Y. Ohta K. Kumagai M. Nishi M. Hosokawa M. Fledelius C. Jacobsen P. Yagyu H. Fukayama M. Nagai R. Kadowaki T. Ohashi K. Ishibashi S. The critical role of neutral cholesterol ester hydrolase 1 in cholesterol removal from human macrophages., 16Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. Matsuzaka T. Shimano H. Yamada N. Aoki J. Morikawa R. Takanezawa Y. Arai H. Nagai R. Kadowaki T. Osuga J. Ishibashi S. Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis., 17Sakai K. Igarashi M. Yamamuro D. Ohshiro T. Nagashima S. Takahashi M. Enkhtuvshin B. Sekiya M. Okazaki H. Osuga J. Ishibashi S. Critical role of neutral cholesteryl ester hydrolase 1 in cholesteryl ester hydrolysis in murine macrophages.). In line with these studies, it has been also reported that CES1 knockdown in human macrophages did not reduce cholesterol efflux, but decreased cholesterol uptake by attenuating CD36 and scavenger receptor-A (SR-A) expression (18Ross M.K. Borazjani A. Mangum L.C. Wang R. Crow J.A. Effects of toxicologically relevant xenobiotics and the lipid-derived electrophile 4-hydroxynonenal on macrophage cholesterol efflux: silencing carboxylesterase 1 has paradoxical effects on cholesterol uptake and efflux., 19Mangum L.C. Hou X. Borazjani A. Lee J.H. Ross M.K. Crow J.A. Silencing carboxylesterase 1 in human THP-1 macrophages perturbs genes regulated by PPARgamma/RXR and RAR/RXR: down-regulation of CYP27A1-LXRalpha signaling.), thereby preventing atherosclerosis development.The controversial studies on the role of CES1 as a CE hydrolase in cholesterol efflux from macrophages also question the function of Ces1d/CES1 in liver cholesterol metabolism. In one study, overexpression of CES1 in the liver increased bile acid content in gallbladder bile, and enhanced the output of [3H]cholesterol from macrophages to bile and feces in the form of bile acid (11Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport.). In line with this, ablation of Ces1d expression in the liver of Ldlr-/- mice was reported to decrease HDL-to-feces RCT indicated by decreased flux of radioisotope derived from HDL-cholesterol to fecal cholesterol and bile acid, and increased atherosclerotic lesions in the aortic arch (13Bie J. Wang J. Marqueen K.E. Osborne R. Kakiyama G. Korzun W. Ghosh S.S. Ghosh S. Liver-specific cholesteryl ester hydrolase deficiency attenuates sterol elimination in the feces and increases atherosclerosis in ldlr-/- mice.). In contrast, in another study it was shown that ablation of Ces1d expression in Ldlr-/- mice alleviated Western-type diet (WTD)-induced atherosclerosis (20Lian J. Quiroga A.D. Li L. Lehner R. Ces3/TGH deficiency improves dyslipidemia and reduces atherosclerosis in Ldlr(-/-) mice.). These contradictory observations lead us to re-examine the role of Ces1d in hepatic cholesterol homeostasis.

By utilizing liver-specific Ces1d knockout mice, we assessed the effect of Ces1d inactivation on in vivo RCT and liver cholesterol storage. The results suggest that ablation of Ces1d in the liver did not alter cholesterol metabolism, and challenge the proposed role of Ces1d in hydrolysis of hepatic CE.

Material and methods AnimalsAll animal procedures were conducted in compliance with protocols approved by the University of Alberta’s Animal Case and Use Committee and in accordance of the Canadian Council on Animal Care policies and regulations. Ces1dflox/flox (Lox) and liver-specific Ces1d knockout (LKO) mice were generated previously (21Lian J. Wei E. Wang S.P. Quiroga A.D. Li L. Di Pardo A. van der Veen J. Sipione S. Mitchell G.A. Lehner R. Liver specific inactivation of carboxylesterase 3/triacylglycerol hydrolase decreases blood lipids without causing severe steatosis in mice.). Animals were maintained on a 12-h light (7 am - 7 pm)/12-h dark (7 pm - 7 am) cycle, controlled for temperature and humidity, and were fed a chow diet (5% fat (w/w) and 0.04% cholesterol) (PICO laboratory Rodent Diet). In a separate cohort, 10-week old male mice were fed a high-fat, high-cholesterol western-type diet (WTD, 42% kcal from fat, 0.2% cholesterol, Envigo TD 88137) for 2 weeks. Tissues were collected after 5 hours fasting. Cell culture and generation of McArdle RH-7777 cell lines stably expressing Ces1d

McArdle RH-7777 (McA) cells were obtained from ATCC and cultured in DMEM containing 50 units/ml penicillin/streptomycin, 10% horse serum, and 10% FBS at 37°C in humidified air containing 5% CO2. Wild-type McA cells were transfected with empty vector pCI-neo or with Ces1d-cDNA construct cloned into pCI-neo using Lipofectamine 2000. Transfected cells were grown in media containing 1.6 mg/ml G-418 for 5 days to select for neomycin resistance. Individual clones were isolated and analyzed for Ces1d protein by immunoblotting. Stable cell lines, designated pCI-neo and Ces1d, were thereafter maintained in media containing 0.4 mg/ml G-418.

 Immunoblot analyses

Proteins in cell lysates were resolved by SDS-polyacrylamide gels and transferred to PVDF membranes (catalog #IPVH00010; Millipore). Antibodies used in this study include anti-Ces1d (1:1,000 dilution, catalog # sc-374160; Santa Cruz), and anti-GAPDH (1:5,000 dilution, catalog # ab8245; Abcam). Immunoreactivity was detected by enhanced chemiluminescence and visualized by G:BOX system (SynGene, UK).

 Measurement of CE hydrolase activity in the microsomes of Ces1d expressing McA cellsBecause Ces1d is a disulfide-bonded glycoprotein localized in the lumen of the endoplasmic reticulum (ER) (22Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts., 23Alam M. Vance D.E. Lehner R. Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site., 24Gilham D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates., 25Wang H. Wei E. Quiroga A.D. Sun X. Touret N. Lehner R. Altered lipid droplet dynamics in hepatocytes lacking triacylglycerol hydrolase expression., 26Wang H. Gilham D. Lehner R. Proteomic and lipid characterization of apolipoprotein B-free luminal lipid droplets from mouse liver microsomes: implications for very low density lipoprotein assembly.), microsomes were isolated from cells for measurements of lipase activity. pCI-neo and Ces1d McA cells harvested from three 100mm dishes with 80% confluence in homogenate buffer (250mM sucrose, 20mM Tris, 1mM EDTA, pH 7.4) were homogenized using an isobiotec cell homogenizer (H&Y enterprise, US) with 0.1574-inch diameter ball using 3mL syringes to pass cells firmly through the chamber 40 times to disrupt the cells. Cell lysates were spun at 600g at 4°C for 5 minutes to isolate supernatants, which were then centrifugated for 15 minutes at 10,200g at 4°C to pellet heavy membranes. The supernatants were then centrifuged at 425,866g at 4°C for 15 minutes, and the microsomal pellet was re-suspended in 0.1M potassium phosphate buffer (pH 7.0), sonicated to release lumenal contents (including Ces1d), and used for the CE hydrolase activity assay. The CE hydrolase assay was performed as described previously (27Hajjar D.P. Minick C.R. Fowler S. Arterial neutral cholesteryl esterase. A hormone-sensitive enzyme distinct from lysosomal cholesteryl esterase.). Mixed micelles of cholesteryl-[14C]oleate, phosphatidylcholine (PC), and sodium taurocholate were prepared with the molar ratio of 1:4:2 in 0.1M potassium phosphate buffer (pH 7.0) using a sonicator (model W-385, Heat systems) at the setting of 2.5 for 2 x 1 minutes with 1 minute interval, followed by 4 x 30 seconds with 30 seconds interval. Micelles containing 0.1μmol cholesteryl-[14C]oleate (specific activity 400,000 DPM/μmol) were used as substrate for the assay with 100ug of microsomal fractions. The total reaction volume was 1mL in 0.1M potassium phosphate buffer (pH 7.0) containing 189μM sodium taurocholate. After 1 hour incubation at 37°C the reaction was stopped by addition of 3.25mL of methanol/chloroform/heptane 3.85:3.42:2.73 (v/v/v) and 50μl of 1M NaOH to 0.5mL of the reaction mixture. After centrifugation at 200g for 10 minutes, 1mL of the upper phase was used to measure radioactivity by liquid scintillation counting to assess the release of oleic acid from cholesteryl oleate. One unit (U) of enzyme activity corresponds to the release of 1μmol of labeled oleate from CE per minute.Cytosolic fractions of mouse white adipose tissue (WAT) were used as the positive control for hormone-sensitive lipase (HSL) CE hydrolase activity (28Hormone-sensitive lipase and neutral cholesteryl ester lipase.). In brief, mouse WAT was homogenized in homogenization buffer containing 1mM DTT to make 20% homogenate. Fat-free cytosolic fraction was obtained by centrifugation of the homogenate at 100,000g at 4°C for 45 min and recovery of the clear infranatant under the fat cake, and 100ug of the cytosolic protein containing hormone-sensitive lipase (HSL) was assessed for CE hydrolase activity as described above. 4-methylumbelliferyl heptanoate hydrolysis assayLipase activity in cell lysate and microsomal fraction was measured utilizing 4-methylumbelliferyl heptanoate (MUH) as the substrate as described previously (29Techniques to measure lipase and esterase activity in vitro.). The enzymatic reaction was initiated by the injection of 20 μL of 1mM MUH in 20 mM Tris/HCl (pH 8.0), 1 mM EDTA and 300 μM taurodeoxycholate to fractions containing 5 μg of protein in a 96-well plate in a final volume of 200μL. The plate was incubated at 37°C and the release of fluorescent 4-MU was detected with a Fluoroskan Ascent FL Type 374 (Thermo Labsystems) with excitation/emission wavelengths of 355/460nm. Fluorescence values generated with a standard solution of 4-methylumbelliferone (sodium salt) were used to quantify 4-MU release. Radiolabeling of HDL-CE

[3H]Cholesteryl oleate was incorporated into purified human HDL (Calbiochem, USA) using CETP activity in human lipoprotein deficient serum (LPDS). 5mg HDL and 0.5mCi [3H]cholesteryl oleate were added to 3ml LPDS and the volume was brought to 5ml with saline. The mixture was incubated overnight at 37°C with stirring. Labeled HDL was then isolated by ultracentrifugation (d=1.215). Lipid extraction and analysis was performed with a small aliquot of labeled HDL (HDL-[3H]CE) to confirm incorporation of the labeled CE into HDL. Lipids were separated by TLC, and 89% of radioactivity was confirmed to be associated with CE.

 In vivo RCT assessment

Male LKO and Lox mice maintained on chow diet were used in this study. HDL-[3H]CE (1.5X106 DPM) was administrated to each mouse via intravenous injection. Blood was collected 2 minutes (as the initial time-point), 1 hour, 10 hours, 24 hours, 36 hours and 48 hours after injection and radioactivity decay in the plasma was determined. Feces were collected for 48 hours. At 48 hours after injection, liver and gallbladder were collected after a 12-hour fast.

 Analytical ProceduresLiver lipids were extracted from liver homogenates in the presence of known amounts of phosphatidyldimethylethanolamine (PDME) as an internal standard by a modified Folch method (30Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.). High performance liquid chromatography (HPLC) was carried out to determine liver CE and FC concentrations on an Agilent 1100 instrument (Santa Clara, USA) equipped with quaternary pump and Alltech Evaporative Light-Scattering Detector 2000, using a modified version of the method of Abreu et al (31Abreu S. Solgadi A. Chaminade P. Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques.).

In the in vivo RCT assessment, lipids extracted from liver homogenates were separated by TLC. Radioactivity in CE and FC were determined by liquid scintillation counting.

HDL and apoB-containing lipoproteins in plasma were separated by phosphotungstic acid/MgCl2 precipitation method (32Assmann G. Schriewer H. Schmitz G. Hagele E.O. Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2.) and the radioactivity in each fraction was measured by liquid scintillation counting. Total cholesterol (TC) and FC concentrations in plasma and the HDL fraction were determined using a diagnostic kit (WAKO Diagnostics, US) according to manufacturer's instructions.

Biliary cholesterol and bile acid concentrations were determined using kits (Trinity Biotech, USA) according to manufacturer's instructions.

Feces collected for 48 hours were vacuum freeze dried, weighed, and ground into powder. Fecal neutral sterol and bile acid fractions were separated as described previously (33Annema W. Nijstad N. Tolle M. de Boer J.F. Buijs R.V. Heeringa P. van der Giet M. Tietge U.J. Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cholesterol transport during the acute phase response but not group IIA secretory phospholipase A(2)., 34Miettinen T.A. Ahrens Jr., E.H. Grundy S.M. Quantitative Isolation and Gas--Liquid Chromatographic Analysis of Total Dietary and Fecal Neutral Steroids.). Samples were heated at 80°C for 2 hours in alkaline methanol and then extracted 3 times with petroleum-ether by mixing for 30 seconds followed by centrifugation. Radioactivity associated with the neutral sterol fraction (top layer) and bile acid fraction (bottom layer) was quantified. Radioactivity recovered from each fraction was expressed relative to the injected tracer dose [total radioactivity in blood of each mouse at the initial time point after injection (2-minute)]. RNA isolation and real-time qPCR analysisTotal liver RNA was isolated using Trizol reagent (Invitrogen, USA). First-strand cDNA was synthesized from 2μg total RNA using Superscript ΙΙΙ reverse transcriptase (Invitrogen) primed by oligo (dt)12-18 (Invitrogen) and random primers (Invitrogen). Real-time qPCR was performed with Power SYBR® Green PCR Master Mix kit (Life Technologies, UK) using the StepOnePlus-Real time PCR System (Applied Biosystems, Canada). Data were analyzed with the StepOne software (Applied Biosystems). Standard curves were used to calculate mRNA abundance relative to that of a control gene, cyclophilin. Real-time qPCR primers are summarized in the Supplemental Table 1. All primers used to assess expression of liver carboxylesterases are listed in the Supplemental Table 2. All primers were synthesized by Integrated DNA Technologies (USA). Statistics

All values are expressed as mean ± SEM. Differences among group means were assessed by two-way ANOVA (decay curve), one-way ANOVA followed by Bonferroni post hoc test, and unpaired t-test for two group comparisons (GraphPad PRISM 8 software). Differences were considered statistically significant at *P<0.05, **P<0.01, ***P<0.001, P<0.0001.

DiscussionRCT is a pivotal pathway that removes excess cholesterol from peripheral tissues and transports it to the liver for excretion into bile and feces. This route is the major process by which HDL exerts protection against atherosclerosis (45Rader D.J. Alexander E.T. Weibel G.L. Billheimer J. Rothblat G.H. The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis.). Lipid poor pre-β-HDL secreted from liver and intestine initializes this process by taking up excess unesterified cholesterol from cells via ABCA1 mediated efflux, and mature HDL can further acquire unesterified cholesterol from cells via ABCG1 mediated efflux. Cholesterol acquired by HDL through efflux is esterified to CE by plasma LCAT. HDL-CE is then selectively taken up by the liver via SR-B1 (3Shimada A. Tamai T. Oida K. Takahashi S. Suzuki J. Nakai T. Miyabo S. Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35).), hydrolyzed, and the resulting cholesterol can be exported to bile either in its unesterified form or as a bile acid, or esterified by ACAT to CE, which can be stored in lipid droplets or secreted in VLDL. Hydrolysis of CE is the rate-limiting step of ABCA1/ABCG1-mediated cholesterol efflux from macrophages (14Regulation of lipid droplet cholesterol efflux from macrophage foam cells.) and hydrolysis of CE in the liver is necessary for cholesterol export into bile. Although CE hydrolase activity is critical for both the first step and the last step of RCT, the enzyme(s) responsible for the catalytic activity is/are still not fully elucidated. Ces1d (murine)/CES1 (human) has been reported to exhibit CE hydrolase activity in the macrophage (human) and liver and has been postulated to be involved in the RCT process (9Zhao B. Song J. Chow W.N. St Clair R.W. Rudel L.L. Ghosh S. Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr mice., 10Ghosh S. St Clair R.W. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase., 11Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport., 12Bie J. Wang J. Yuan Q. Kakiyama G. Ghosh S.S. Ghosh S. Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr-/- mice.). However, the role of Ces1d/CES1 as a CE hydrolase has been disputed (15Igarashi M. Osuga J. Uozaki H. Sekiya M. Nagashima S. Takahashi M. Takase S. Takanashi M. Li Y. Ohta K. Kumagai M. Nishi M. Hosokawa M. Fledelius C. Jacobsen P. Yagyu H. Fukayama M. Nagai R. Kadowaki T. Ohashi K. Ishibashi S. The critical role of neutral cholesterol ester hydrolase 1 in cholesterol removal from human macrophages., 16Okazaki H. Igarashi M. Nishi M. Tajima M. Sekiya M. Okazaki S. Yahagi N. Ohashi K. Tsukamoto K. Amemiya-Kudo M. Matsuzaka T. Shimano H. Yamada N. Aoki J. Morikawa R. Takanezawa Y. Arai H. Nagai R. Kadowaki T. Osuga J. Ishibashi S. Identification of a novel member of the carboxylesterase family that hydrolyzes triacylglycerol: a potential role in adipocyte lipolysis., 17Sakai K. Igarashi M. Yamamuro D. Ohshiro T. Nagashima S. Takahashi M. Enkhtuvshin B. Sekiya M. Okazaki H. Osuga J. Ishibashi S. Critical role of neutral cholesteryl ester hydrol