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• W2550897360 abstract "Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer. Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer. Lipid droplets (LDs) are cellular organelles that are important for energy and lipid metabolism (1Hashemi H.F. Goodman J.M. The life cycle of lipid droplets.Curr. Opin. Cell Biol. 2015; 33: 119-124Crossref PubMed Scopus (113) Google Scholar, 2Krahmer N. Farese Jr., R.V. Walther T.C. Balancing the fat: lipid droplets and human disease.EMBO Mol. Med. 2013; 5: 973-983Crossref PubMed Scopus (300) Google Scholar). LD accumulation is a hallmark of obesity and is linked to the metabolic syndrome and type II diabetes. LD accumulation is central to atherosclerosis development, in which macrophages in arterial walls accumulate cholesterol esters (CEs) in LDs to become foam cells. Finally, LDs accumulate in many carcinomas (3Straub B.K. Herpel E. Singer S. Zimbelmann R. Breuhahn K. Macher-Goeppinger S. Warth A. Lehmann-Koch J. Longerich T. Heid H. et al.Lipid droplet-associated PAT-proteins show frequent and differential expression in neoplastic steatogenesis.Mod. Pathol. 2010; 23: 480-492Crossref PubMed Scopus (105) Google Scholar), and LDs and lipid metabolism are connected to renal clear cell carcinoma and prostate cancer (4Hager M.H. Solomon K.R. Freeman M.R. The role of cholesterol in prostate cancer.Curr. Opin. Clin. Nutr. Metab. Care. 2006; 9: 379-385Crossref PubMed Scopus (124) Google Scholar, 5Schlaepfer I.R. Rider L. Rodrigues L.U. Gijón M.A. Pac C.T. Romero L. Cimic A. Sirintrapun S.J. Glodé L.M. Eckel R.H. et al.Lipid catabolism via CPT1 as a therapeutic target for prostate cancer.Mol. Cancer Ther. 2014; 13: 2361-2371Crossref PubMed Scopus (190) Google Scholar, 6Wu X. Daniels G. Lee P. Monaco M.E. Lipid metabolism in prostate cancer.Am. J. Clin. Exp. Urol. 2014; 2: 111-120PubMed Google Scholar, 7Tamura K. Makino A. Hullin-Matsuda F. Kobayashi T. Furihata M. Chung S. Ashida S. Miki T. Fujioka T. Shuin T. et al.Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism.Cancer Res. 2009; 69: 8133-8140Crossref PubMed Scopus (144) Google Scholar, 8Drabkin H.A. Gemmill R.M. Cholesterol and the development of clear-cell renal carcinoma.Curr. Opin. Pharmacol. 2012; 12: 742-750Crossref PubMed Scopus (40) Google Scholar). Among genes that encode LD proteins, LDAH is associated with prostate cancer. The SNP rs13385191 in intron 6 of LDAH is associated with increased prostate cancer risk (9Shui I.M. Lindström S. Kibel A.S. Berndt S.I. Campa D. Gerke T. Penney K.L. Albanes D. Berg C. Bueno-de-Mesquita H.B. et al.Prostate cancer (PCa) risk variants and risk of fatal PCa in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium.Eur. Urol. 2014; 65: 1069-1075Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Takata R. Akamatsu S. Kubo M. Takahashi A. Hosono N. Kawaguchi T. Tsunoda T. Inazawa J. Kamatani N. Ogawa O. et al.Genome-wide association study identifies five new susceptibility loci for prostate cancer in the Japanese population.Nat. Genet. 2010; 42: 751-754Crossref PubMed Scopus (237) Google Scholar, 11Long Q.Z. Du Y.F. Ding X.Y. Li X. Song W.B. Yang Y. Zhang P. Zhou J.P. Liu X.G. Replication and fine mapping for association of the C2orf43, FOXP4, GPRC6A and RFX6 genes with prostate cancer in the Chinese population.PLoS One. 2012; 7: e37866Crossref PubMed Scopus (34) Google Scholar, 12Lindström S. Schumacher F.R. Campa D. Albanes D. Andriole G. Berndt S.I. Bueno-de-Mesquita H.B. Chanock S.J. Diver W.R. Ganziano J.M. et al.Replication of five prostate cancer loci identified in an Asian population—results from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3).Cancer Epidemiol. Biomarkers Prev. 2012; 21: 212-216Crossref PubMed Scopus (22) Google Scholar). A rare A>G variant is associated with a difference in LDAH mRNA abundance, and prostate cancer risk is inversely correlated with its expression (13Penney K.L. Sinnott J.A. Tyekucheva S. Gerke T. Shui I.M. Kraft P. Sesso H.D. Freedman M.L. Loda M. Mucci L.A. et al.Association of prostate cancer risk variants with gene expression in normal and tumor tissue.Cancer Epidemiol. Biomarkers Prev. 2015; 24: 255-260Crossref PubMed Scopus (75) Google Scholar, 14Innocenti F. Cooper G.M. Stanaway I.B. Gamazon E.R. Smith J.D. Mirkov S. Ramirez J. Liu W. Lin Y.S. Moloney C. et al.Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue.PLoS Genet. 2011; 7: e1002078Crossref PubMed Scopus (172) Google Scholar). In addition, rs13385191 is associated with nonfatal outcome of prostate cancer (9Shui I.M. Lindström S. Kibel A.S. Berndt S.I. Campa D. Gerke T. Penney K.L. Albanes D. Berg C. Bueno-de-Mesquita H.B. et al.Prostate cancer (PCa) risk variants and risk of fatal PCa in the National Cancer Institute Breast and Prostate Cancer Cohort Consortium.Eur. Urol. 2014; 65: 1069-1075Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Lindström S. Schumacher F.R. Campa D. Albanes D. Andriole G. Berndt S.I. Bueno-de-Mesquita H.B. Chanock S.J. Diver W.R. Ganziano J.M. et al.Replication of five prostate cancer loci identified in an Asian population—results from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3).Cancer Epidemiol. Biomarkers Prev. 2012; 21: 212-216Crossref PubMed Scopus (22) Google Scholar). Linkage of the LDAH locus with prostate cancer suggests that loss of the lipid droplet-associated hydrolase (LDAH) function has a role in prostate tumorigenesis. The single polypeptide encoded by LDAH is predicted to be a serine hydrolase of the α/β-fold type (15Simon G.M. Cravatt B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.J. Biol. Chem. 2010; 285: 11051-11055Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 16Lenfant N. Hotelier T. Velluet E. Bourne Y. Marchot P. Chatonnet A. ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins: tools to explore diversity of functions.Nucleic Acids Res. 2013; 41: D423-D429Crossref PubMed Scopus (205) Google Scholar). Homologs in multiple species, including yeast (YPR147C), suggest a conserved function at LDs; however, LDAH's molecular function remains uncertain but has recently been investigated. Goo et al. reported that LDAH is a CE hydrolase (17Goo Y.H. Son S.H. Kreienberg P.B. Paul A. Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization.Arterioscler. Thromb. Vasc. Biol. 2014; 34: 386-396Crossref PubMed Scopus (37) Google Scholar). This finding is intriguing inasmuch as recent studies have linked accumulation of CEs in LDs to prostate and breast cancer aggressiveness (18Yue S. Li J. Lee S.Y. Lee H.J. Shao T. Song B. Cheng L. Masterson T.A. Liu X. Ratliff T.L. et al.Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness.Cell Metab. 2014; 19: 393-406Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 19de Gonzalo-Calvo D. López-Vilaró L. Nasarre L. Perez-Olabarria M. Vázquez T. Escuin D. Badimon L. Barnadas A. Lerma E. Llorente-Cortés V. Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.BMC Cancer. 2015; 15: 460Crossref PubMed Scopus (127) Google Scholar). Supporting a function in CE metabolism, two other SNPs in LDAH are associated with changes in LDL cholesterol (20Lettre G. Palmer C.D. Young T. Ejebe K.G. Allayee H. Benjamin E.J. Bennett F. Bowden D.W. Chakravarti A. Dreisbach A. et al.Genome-wide association study of coronary heart disease and its risk factors in 8,090 African Americans: the NHLBI CARe Project.PLoS Genet. 2011; 7: e1001300Crossref PubMed Scopus (248) Google Scholar, 21Shen H. Damcott C.M. Rampersaud E. Pollin T.I. Horenstein R.B. McArdle P.F. Peyser P.A. Bielak L.F. Post W.S. Chang Y.P. et al.Familial defective apolipoprotein B-100 and increased low-density lipoprotein cholesterol and coronary artery calcification in the old order amish.Arch. Intern. Med. 2010; 170: 1850-1855Crossref PubMed Scopus (58) Google Scholar). However, LDAH is near APOB on chromosome 2, and these SNPs were originally linked to APOB, a confounding factor because APOB is involved in cholesterol metabolism. Besides LDAH, other lipases have been implicated in CE hydrolysis. NCEH1 has been reported to hydrolyze CEs (22Igarashi M. Osuga J. Uozaki H. Sekiya M. Nagashima S. Takahashi M. Takase S. Takanashi M. Li Y. Ohta K. et al.The critical role of neutral cholesterol ester hydrolase 1 in cholesterol removal from human macrophages.Circ. Res. 2010; 107: 1387-1395Crossref PubMed Scopus (75) Google Scholar), but at least in mice, it has also been reported to hydrolyze ether lipid 2-acetyl monoalkylglycerol (23Buchebner M. Pfeifer T. Rathke N. Chandak P.G. Lass A. Schreiber R. Kratzer A. Zimmermann R. Sattler W. Koefeler H. et al.Cholesteryl ester hydrolase activity is abolished in HSL−/− macrophages but unchanged in macrophages lacking KIAA1363.J. Lipid Res. 2010; 51: 2896-2908Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The LD-localized hormone-sensitive lipase (HSL) contributes to CE hydrolysis (23Buchebner M. Pfeifer T. Rathke N. Chandak P.G. Lass A. Schreiber R. Kratzer A. Zimmermann R. Sattler W. Koefeler H. et al.Cholesteryl ester hydrolase activity is abolished in HSL−/− macrophages but unchanged in macrophages lacking KIAA1363.J. Lipid Res. 2010; 51: 2896-2908Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 24Kraemer F.B. Shen W.J. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis.J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 25Sekiya M. Osuga J. Yahagi N. Okazaki H. Tamura Y. Igarashi M. Takase S. Harada K. Okazaki S. Iizuka Y. et al.Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis.J. Lipid Res. 2008; 49: 1829-1838Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Whether HSL has a major role in CE hydrolysis in macrophages is debated because CE hydrolysis still occurs in its absence (26Contreras J.A. Hormone-sensitive lipase is not required for cholesteryl ester hydrolysis in macrophages.Biochem. Biophys. Res. Commun. 2002; 292: 900-903Crossref PubMed Scopus (31) Google Scholar). Lysosomal acid lipase also contributes to cellular CE metabolism and regulates macrophage cholesterol efflux, potentially through lipophagy (27Lohse P. Lohse P. Chahrokh-Zadeh S. Seidel D. Human lysosomal acid lipase/ cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity.J. Lipid Res. 1997; 38: 1896-1905Abstract Full Text PDF PubMed Google Scholar, 28Ouimet M. Marcel Y.L. Regulation of lipid droplet cholesterol efflux from macrophage foam cells.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 575-581Crossref PubMed Scopus (163) Google Scholar, 29Du H. Duanmu M. Witte D. Grabowski G.A. Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage.Hum. Mol. Genet. 1998; 7: 1347-1354Crossref PubMed Scopus (120) Google Scholar), but whether it has access to LDs under normal conditions is not clear. Thus, the enzymes that hydrolyze CEs at LDs are uncertain. In this study, we tested the reported role for LDAH in CE hydrolysis and the metabolism of other neutral lipids by generating and analyzing a knockout mouse model lacking this enzyme. CG9186 (dLDAH) secondary structure was predicted with JPred4 (30Drozdetskiy A. Cole C. Procter J. Barton G.J. JPred4: a protein secondary structure prediction server.Nucleic Acids Res. 2015; 43: W389-W394Crossref PubMed Scopus (1108) Google Scholar) and PSIPRED (31Buchan D.W. Minneci F. Nugent T.C. Bryson K. Jones D.T. Scalable web services for the PSIPRED Protein Analysis Workbench.Nucleic Acids Res. 2013; 41: W349-W357Crossref PubMed Scopus (1031) Google Scholar, 32Jones D.T. Protein secondary structure prediction based on position-specific scoring matrices.J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4460) Google Scholar). HeLa cells were cultured in DMEM with 10% FBS and PenStrep. S2 cell culture was performed as described (33Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J.X. Graham M. Christiano R. Frohlich F. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24: 384-399Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). HeLa and S2 cells were transfected using FuGENE HD (Promega, Madison, WI) and Effectene (Qiagen, Germantown, MD) transfection reagents, respectively, according to the manufacturer's instructions. LDs were induced and stained as described (33Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J.X. Graham M. Christiano R. Frohlich F. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24: 384-399Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 34Krahmer N. Guo Y. Wilfling F. Hilger M. Lingrell S. Heger K. Newman H.W. Schmidt-Supprian M. Vance D.E. Mann M. et al.Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase.Cell Metab. 2011; 14: 504-515Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 35Kory N. Thiam A.R. Farese R.V.J. Walther T.C. Protein crowding is a determinant of lipid droplet composition.Dev. Cell. 2015; 34: 351-363Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), S2 cells were induced with 1 mM oleic acid-BSA, and HeLa cells were induced with 0.5 mM oleic acid coupled to BSA. For colocalization experiments, a C-terminally tagged ADRP-YFP fusion construct or dsRed2-ER (Clontech, Mountain View, CA) was cotransfected into HeLa cells or GFP-Sec61β for S2 cells. For localization studies in mammalian cells we generated expression plasmids containing human full-length LDAH with N-terminal mCherry- or GFP-tag under the CMV promoter. For LD-targeting studies, we expressed Drosophila full-length CG9186 with C-terminal mCherry-tag, the LD domain alone (amino acids (aa) 152–201 of Drosophila CG9186 (CG9186aa152-201)), or Drosophila CG9186 with aa157–200 replaced by a AAAGGGGSGGGGS-linker (Δ aa157–200) under the actin promoter. Immunofluorescence and spinning-disk confocal microscopy (100 × 1.4 NA oil immersion objective [Olympus], iMIC [Till], CSU22 [Yokugawa], iXonEM 897 [Andor]) were as described (33Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J.X. Graham M. Christiano R. Frohlich F. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24: 384-399Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). LD area per cell was quantified as described (35Kory N. Thiam A.R. Farese R.V.J. Walther T.C. Protein crowding is a determinant of lipid droplet composition.Dev. Cell. 2015; 34: 351-363Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). All animal studies followed guidelines issued by Yale and Harvard universities' institutional animal care and use committees. Mice were housed at 12-h light/12-h dark cycle with ad libitum access to food and water unless indicated otherwise. The mouse strain used for this research project was created from embryonic stem cell clone 14003A-H3 (C57BL/6Ntac background, injected into B6(Cg)-Tyrc-2J/J blastocysts), obtained from the Knockout Mouse Project Repository (www.komp.org) and generated by Regeneron Pharmaceuticals. Forward primers S30636 (wild-type; 5′- CATCTCACCTCCTCTCCGTC-3′) or NeoF (knockout; 5′- TCATTCTCAGTATTGTTTTGCC-3′) and reverse primer SD (5′-CAGAGTCCTTCCCATGTCAC-3′) were used for genotyping. Velocigene targeted alleles were created as described (36Valenzuela D.M. Murphy A.J. Frendewey D. Gale N.W. Economides A.N. Auerbach W. Poueymirou W.T. Adams N.C. Rojas J. Yasenchak J. et al.High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.Nat. Biotechnol. 2003; 21: 652-659Crossref PubMed Scopus (469) Google Scholar). Mice with germ-line transmission of the knockout allele were backcrossed from a C57BL/6Ntac onto a C57BL/6J background for a minimum of three generations. Mice were, therefore, a mixture of these C57BL/6 strains. All animals were generated through breeding heterozygous animals. Major determinants of whole-body energy balance were evaluated by the Yale School of Medicine Mouse Metabolic Phenotyping Center using the Comprehensive Lab Animal Monitoring System with Oxymax (Columbus Labs), including VO2, VCO2, activity, feeding, and drinking behavior. Body composition was determined by proton-NMR with the Bruker Minispec. Glucose homeostasis was evaluated by glucose tolerance test, according to recommendations of the NIH-funded Mouse Metabolic Phenotyping Consortium (37Ayala J.E. Samuel V.T. Morton G.J. Obici S. Croniger C.M. Shulman G.I. Wasserman D.H. McGuinness O.P. NIH Mouse Metabolic Phenotyping Center Consortium Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice.Dis. Model. Mech. 2010; 3: 525-534Crossref PubMed Scopus (501) Google Scholar). Briefly, mice were fasted overnight before we collected a basal plasma sample for glucose and insulin measure. Mice were dosed intraperitoneally with 1 mg/g dextrose, and plasma was collected at set intervals for glucose and insulin measures. Glucose was measured by the glucose oxidase method using YSI, and insulin measured by radioimmunoassay. Histology slides were prepared by the Yale School of Medicine Research Histology core or the Harvard Medical School Rodent Histology core. For the cold-exposure experiments, animals were fasted overnight and placed at 4°C for indicated times. Temperatures were determined using a rectal thermometer. Blood glucose was measured as described below. A 60% high-fat diet (D12492) was obtained from Research Diets (New Brunswick, NJ). Lipids were extracted from tissue lysates (38Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), separated on silica TLC plates (Merck, Kenilworth, NJ) with n-heptane/isopropyl ether/acetic acid (60/40/4), and detected by cerium molybdate staining. Bands were identified by comparing with standards. Femurs and tibia were dissected, and muscle was removed. Bone marrow was flushed with 5 ml of DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) with a 5 ml syringe and a 25 gauge needle. Cells were centrifuge at 500 rpm for 10 min, resuspended in medium (DMEM/F12 + 20% HI-FBS + 20% L929 conditioned medium), and plated on petri dishes. On day 4, fresh medium was added to the plates. Experiments were performed on day 7. Cells were seeded in 6-well plates, and medium was changed to contain 1% FBS and loaded with 50 µg/ml of acetylated low-density lipoproteins (acLDLs) (Alfa Aesar, Ward Hill, MA) for 18 h. For quantification of total CEs, lipids were extracted. Cells were washed in PBS, and 750 µl of hexane:isopropanol (2:3) were added to each well and incubated rocking for 10 min at room temperature. The organic solvent phase was collected and dried. Lipids were resuspended in 40 µl of chloroform, spotted on a TLC plate, and developed in hexanes:ethylether:acetic acid (80:20:1). Cholesterol esters were quantified by charring with CuSO4 and densitometry. Values were normalized to protein content determined by BCA assay. To measure cholesterol ester turnover, cells were loaded with 50 µg/ml acLDL for 12 h and subsequently labeled with 0.25 µCi/ml 14C-oleate (Perkin-Elmer, Waltham, MA) for 6 h. The medium was changed, and 10 µg/ml Sandoz 58-035 ACAT inhibitor and 2 µg/ml methyl-β-cyclodextrin (both from Sigma Aldrich, St. Louis, MO) were added. Lipids were extracted after 0, 8, and 24 h and separated by TLC as described above. The CE band was scraped and quantified by scintillation counting. Blood glucose levels were measured using a FreeStyle Lite glucosemeter (Abbott Diabetes Care, Alameda, CA). Serum leptin levels and liver glycogen levels were determined by the Yale Mouse Metabolic Phenotyping Center Analytical Core. Testosterone and corticosterone assays were performed by the Vanderbilt University Medical Center Hormone Assay and Analytical Services Core. Lipidomics analysis of white adipose tissue (WAT), brown adipose tissue (BAT), and liver of 4-week ad libitum-fed animals fed a high-fat diet (HFD) was performed as described (39Saghatelian A. Trauger S.A. Want E.J. Hawkins E.G. Siuzdak G. Cravatt B.F. Assignment of endogenous substrates to enzymes by global metabolite profiling.Biochemistry. 2004; 43: 14332-14339Crossref PubMed Scopus (277) Google Scholar). For liquid chromatography-mass spectrometer analysis of lipids from livers of 22-week HFD ad libitum-fed animals, lipids were extracted from liver corresponding to 75 μg of protein by chloroform/methanol extraction (38Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Detected lipids were identified using LipidSearch (MKI, Tokyo, Japan). Peaks were defined through raw files, product ion, and precursor ion accurate masses. Lipid species were identified by database (>1,000,000 entries) search of positive and negative ion adducts. The accurate mass-extracted ion chromatograms were integrated for each identified lipid species and peak areas obtained for quantitation. An internal standard for phosphatidylinositol (PI 17:0–20:4; Avanti Polar Lipids, Alabaster, AL), which spiked prior to extraction, was used for normalization. WATs and livers from wild-type and Ldah knockout mice were collected and processed by filter-aided sample preparation as described (40Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5097) Google Scholar). Eluted peptides were analyzed by HPLC (EASY-nLC 1000, Thermo Scientific), combined with an Orbitrap mass spectrometer (Q Exactive HF, Thermo Scientific). Raw mass spectrometry data were processed by the MaxQuant software version 1.5.1.2, and statistical analyses were performed with the Perseus software (Max Planck Institute of Biochemistry, Munich, Germany (41Tyanova S. Temu T. Sinitcyn P. Carlson A. Hein M.Y. Geiger T. Mann M. Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data.Nat. Methods. 2016; 13: 731-740Crossref PubMed Scopus (3574) Google Scholar)). Mouse tissues were homogenized in buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 20 μg/ml leupeptine, 2 μg/ml antipain, 1 μg/ml pepstatin) followed by centrifugation at 20,000 g for 30 min at 4°C. The protein content of the 20,000 g infranatant was then determined by the Bio-Rad Protein Assay Kit with BSA as a standard. Measurement of in vitro triacylglycerol (TG) hydrolase activity was as described (42Schweiger M. Eichmann T.O. Taschler U. Zimmermann R. Zechner R. Lass A. Measurement of lipolysis.Methods Enzymol. 2014; 538: 171-193Crossref PubMed Scopus (115) Google Scholar). Briefly, 10 μg of WAT protein or 100 μg of liver protein in a total volume of 100 μl buffer A were incubated with 100 μl of a phospholipid-emulsified triolein substrate solution. The substrate for the measurement of TG-hydrolytic activity in WAT contained 1.67 mM triolein, 190 μM phosphatidylcholine/phosphatidylinositol (ratio 3:1), and 10 μCi/ml 3H-triolein and was prepared by sonication in 100 mM potassium phosphate buffer, pH 7.0 with 2% fatty acid-free BSA. For measurement of TG hydrolase activity in the liver, the substrate solution consisted of 0.32 mM triolein, 45 μM phosphatidylcholine/phosphatidylinositol (ratio 3/1), and 10 μCi/ml [9,10-3H] triolein and was prepared as described above. After 1 h at 37°C, released free fatty acids (FFAs) were extracted and quantified by liquid scintillation counting. The measurement of in vitro CE hydrolase activities in WAT and liver was performed according to the measurement of TG-hydrolase activity using a phospholipid-emulsified cholesteryl oleate substrate solution, which consisted of 0.45 mM cholesteryl oleate, 0.45 mM PC/PI (ratio 3:1), and 1 μCi / ml 14C-cholesteryl oleate. To measure hydrolase activity of LDAH protein in vitro, we used lysates of cells overexpressing LDAH or CG9186 for the lipid hydrolase activity assays. Tissues were lyzed in RIPA buffer with a dounce homogenizer and sonicated. For Western blot analysis, 50 µg protein of lysates were loaded. LDAH was detected using a polyclonal antibody directed against the C terminus of murine LDAH at a dilution 1:500 (17Goo Y.H. Son S.H. Kreienberg P.B. Paul A. Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization.Arterioscler. Thromb. Vasc. Biol. 2014; 34: 386-396Crossref PubMed Scopus (37) Google Scholar). The tubulin antibody was purchased from Sigma-Aldrich (Cat. no. T5168) and used at a dilution of 1:2000. The following primers were used in qRT-PCR analysis. Expression was normalized to the average of β-actin and cyclophilin levels. mLDAH: 5′-CTTCACGTGATGAAGCGAGT-3′ (forward primer), 5′-AGTTGGGAAGAGCAGAAAGG-3′ (reverse primer); mHSL: 5′-ACGAGACAGGCCTCAGTGTGA-3′ (forward primer), 5′-CCAC­G­CAACTCTGGGTCTATG-3′ (reverse primer); mATGL: 5′-GAGCCCCGGGGTGGAACAAGAT-3′ (forward primer), 5′-AAAAGGTGGT­GGGCAGGAGTAAGG-3′ (reverse primer); mβ-Actin: 5′-CATCGT­GGGCCGCTCTA-3′ (forward primer), 5′-CAC­CCACATAGGAGTC­CTTCTG-3′ (reverse primer); mCyclophilin: 5′-TGGAAGAGCA­CCAAGACAACA-3′ (forward primer), 5′-TGCCGGAGTCGACAATGAT-3′ (reverse primer). Statistical significance was tested using Student t-test. For experiments with multiple time points, a two-way ANOVA was used (GraphPad Prism Software). Value less than 0.05 would have been considered significant in all statistical analyses. In Drosophila S2 cells and Saccharomyces cerevisiae, LDAH homologs copurify with LD proteins (43Krahmer N. Hilger M. Kory N. Wilfling F. Stoehr G. Mann M. Farese Jr., R.V. Walther T.C. Protein correlation profiles identify lipid droplet proteins with high confidence.Mol. Cell. Proteomics. 2013; 12: 1115-1126Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 44Currie E. Guo X. Christiano R. Chitraju C. Kory N. Harrison K. Haas J. Walther T.C. Farese Jr, R.V. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.J. Lipid Res. 2014; 55: 1465-1477Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Drosophila CG9186 (referred to as dLDAH hereinafter) was highly enriched in the top fraction of a sucrose gradient used to separate cellular proteins, a purification profile typical of bona fide LD proteins, such as HSL (Fig. 1A). Consistent with this result, Drosophila and human LDAH localize to LDs (17Goo Y.H. Son S.H. Kreienberg P.B. Paul A. Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization.Arterioscler. Thromb. Vasc. Biol. 2014; 34: 386-396Crossref PubMed Scopus (37) Google Scholar, 45Thiel K. Heier C. Haberl V. Thul P.J. Oberer M. Lass A. Jäckle H. Beller M. The evolutionarily conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage.J. Cell Sci. 2013; 126: 2198-2212Crossref PubMed Scopus (37) Google Sc" @default.
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• W2550897360 title "Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism" @default.
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