Deletion of CGI-58 or Adipose Triglyceride Lipase Differently Affects Macrophage Function and Atherosclerosis

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice). CGI-58−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL−/− macrophages. In contrast to ATGL−/− macrophages, however, CGI-58−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency. Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice). CGI-58−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL−/− macrophages. In contrast to ATGL−/− macrophages, however, CGI-58−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency. Monocyte-derived macrophages are present in virtually all tissues, where they remove apoptotic cells and cellular debris generated by tissue remodelling and/or cellular necrosis. Under pathophysiological conditions, macrophages play a key role during atherogenesis by internalizing modified lipoproteins or lipoprotein remnants that have invaded the vessel wall to form cholesterol-rich foam cells. This (along with other disease-related functions for this immune cell) has prompted profound research into the role of the macrophage, and how its functions are regulated during disease progression (1McLaren J.E. Michael D.R. Ashlin T.G. Ramji D.P. Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy.Prog. Lipid Res. 2011; 50: 331-347Crossref PubMed Scopus (278) Google Scholar). Comparative gene identification-58 (CGI-58) is the coactivator of adipose TG lipase (ATGL), the major TG hydrolase for the initial and rate-limiting step in lipolysis (2Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). Lipolysis has been extensively studied in adipocytes, where under basal conditions CGI-58 binds to the surface of lipid droplets through interaction with perilipin1. Hormonal stimulation of lipolysis leads to phosphorylation of perilipin1 and hormone-sensitive lipase, resulting in the release of CGI-58 from perilipin1 to interact with and activate ATGL, which then converts TG to diacylglycerol and FA (3Zechner R. Zimmermann R. Eichmann T.O. Kohlwein S.D. Haemmerle G. Lass A. Madeo F. FAT SIGNALS–lipases and lipolysis in lipid metabolism and signaling.Cell Metab. 2012; 15: 279-291Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). Both mice and humans affected with ATGL or CGI-58 deficiency suffer from systemic TG accumulation, a condition called neutral lipid storage disease (NLSD) in humans (4Schweiger M. Lass A. Zimmermann R. Eichmann T.O. Zechner R. Neutral lipid storage disease: genetic disorders caused by mutations in adipose triglyceride lipase/PNPLA2 or CGI-58/ABHD5.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E289-E296Crossref PubMed Scopus (215) Google Scholar). Of note, specific phenotypical alterations are observed depending on whether ATGL or CGI-58 is defective. The most apparent difference is the severe epidermal skin defect observed in mice and humans with CGI-58 deficiency (2Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 5Radner F.P. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), which is absent in both species lacking ATGL (6Fischer J. Lefevre C. Morava E. Mussini J.M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Crossref PubMed Scopus (365) Google Scholar, 7Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar). This finding resulted in different classifications of the respective human diseases, namely NLSD with myopathy in ATGL deficiency (6Fischer J. Lefevre C. Morava E. Mussini J.M. Laforet P. Negre-Salvayre A. Lathrop M. Salvayre R. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy.Nat. Genet. 2007; 39: 28-30Crossref PubMed Scopus (365) Google Scholar), whereas CGI-58 deficiency leads to NLSD with ichthyosis (8Lefèvre C. Jobard F. Caux F. Bouadjar B. Karaduman A. Heilig R. Lakhdar H. Wollenberg A. Verret J.L. Weissenbach J. et al.Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.Am. J. Hum. Genet. 2001; 69: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Several ongoing studies using tissue-specific CGI-58 and ATGL-deficient (−/−) mice aim at studying the proteins' shared and individual contribution to lipid metabolism. In addition, functional differences observed in hepatocytes between mice with CGI-58 and ATGL deficiency argue for an ATGL-independent function of CGI-58 in this cell population (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Lord C.C. Brown J.M. Distinct roles for alpha-beta hydrolase domain 5 (ABHD5/CGI-58) and adipose triglyceride lipase (ATGL/PNPLA2) in lipid metabolism and signaling.Adipocyte. 2012; 1: 123-131Crossref PubMed Google Scholar) and maybe other tissues as well (11Zierler K.A. Zechner R. Haemmerle G. Comparative gene identification-58/alpha/beta hydrolase domain 5: more than just an adipose triglyceride lipase activator?.Curr. Opin. Lipidol. 2014; 25: 102-109Crossref PubMed Scopus (9) Google Scholar). Because CGI-58−/− mice die shortly after birth due to a severe skin barrier defect (5Radner F.P. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. et al.Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), we generated myeloid-specific CGI-58 (macCGI-58)−/− mice to investigate the consequences of CGI-58 deficiency in macrophages. In the present study, we examined: i) whether CGI-58−/− macrophages mimic the TG accumulation phenotype observed in ATGL−/− macrophages; ii) whether CGI-58 deficiency affects macrophage function; and iii) whether the altered phenotype culminates in increased atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. We have previously shown that loss of ATGL in macrophages affects macrophage phenotype and function, such as TG-rich lipid droplet accumulation, increased apoptosis (12Aflaki E. Radovic B. Chandak P.G. Kolb D. Eisenberg T. Ring J. Fertschai I. Uellen A. Wolinski H. Kohlwein S.D. et al.Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages.J. Biol. Chem. 2011; 286: 7418-7428Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and endoplasmic reticulum (ER) stress (13Aflaki E. Doddapattar P. Radovic B. Povoden S. Kolb D. Vujic N. Wegscheider M. Koefeler H. Hornemann T. Graier W.F. et al.C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.Cell Death Dis. 2012; 3: e280Crossref PubMed Scopus (42) Google Scholar), reduced migration (14Aflaki E. Balenga N.A. Luschnig-Schratl P. Wolinski H. Povoden S. Chandak P.G. Bogner-Strauss J.G. Eder S. Konya V. Kohlwein S.D. et al.Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis.Cell. Mol. Life Sci. 2011; 68: 3933-3947Crossref PubMed Scopus (52) Google Scholar), and decreased phagocytosis ability (15Chandak P.G. Radovic B. Aflaki E. Kolb D. Buchebner M. Frohlich E. Magnes C. Sinner F. Haemmerle G. Zechner R. et al.Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.J. Biol. Chem. 2010; 285: 20192-20201Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In addition, transplantation of ATGL−/− bone marrow into LDL receptor (LDLR)−/− mice revealed that the lack of ATGL in immune cells attenuates atherosclerosis susceptibility (16Lammers B. Chandak P.G. Aflaki E. Van Puijvelde G.H. Radovic B. Hildebrand R.B. Meurs I. Out R. Kuiper J. Van Berkel T.J. et al.Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 67-73Crossref PubMed Scopus (40) Google Scholar). Being the coactivator of ATGL, we predicted that the absence of CGI-58 in macrophages leads to TG-rich lipid droplet accumulation. We hypothesized that loss of the ATGL coactivator CGI-58 in myeloid cells affects macrophage function in vitro and in vivo and impacts atherosclerosis susceptibility. Mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58−/− mice) were generated by crossing CGI-58flox/flox mice (17Zierler K.A. Jaeger D. Pollak N.M. Eder S. Rechberger G.N. Radner F.P. Woelkart G. Kolb D. Schmidt A. Kumari M. et al.Functional cardiac lipolysis in mice critically depends on comparative gene identification-58.J. Biol. Chem. 2013; 288: 9892-9904Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (provided by Dr. Guenther Haemmerle, University of Graz, Austria) with transgenic mice that express Cre recombinase under the control of the murine M lysozyme promoter (18Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1563) Google Scholar) (LysMCre, C57BL/6 background; provided by Dr. Thomas Ruelicke, University of Veterinary Medicine, Vienna, Austria). CGI-58flox/flox [wild-type (Wt)] mice were used as controls. Experiments were performed with female animals. To investigate atherosclerosis susceptibility, we generated CGI-58flox/flox/ApoE−/− (designated as ApoE−/−) and macCGI-58/ApoE-DKO mice by crossing CGI-58flox/flox and macCGI-58-KO mice with ApoE−/− mice (Jackson Laboratory, Bar Harbor, ME). For genotyping, the following primers were used: CGI-58flox/flox-forward, 5′-GTCATGGTTGT­GG­GGAAATC-3′; CGI-58flox/flox-reverse, 5′-GACTGGAAG­GA­TTT­GA­GGGG-3′; Cre-mut, 5′-CCCAGAAATGCCAGATTACG-3′; Cre-comm, 5′-CTTGGGCTGCCAGAATTTCTC-3′; Cre-Wt, 5′-TTACAGTCGGCCAGGCTGAC-3′; ApoE-forward, 5′-GCCTAGCCGAG­GGA­GA­GCCG-3′; ApoE-reverse, 5′-TGTGACTTGGGAGCTCTGCAGC-3′; and ApoE-neo, 5′-GCCGCCCCGACTGCATCT-3′. Female mice were either fed a standard chow diet [containing 4% fat and 21% protein (R/M H; Ssniff, Soest, Germany)], challenged with a Western type diet (WTD) [TD88137mod; 21% fat, 0.2% cholesterol (Ssniff)] or a high-fat/high-cholesterol diet (HF/HCD) (E15126-34 EF R/M; 30% fat, 1% cholesterol) for 10–30 weeks starting at the age of 4–6 weeks. Mice were kept with water ad libitum on a regular light-dark cycle (12 h light, 12 h dark) in a clean environment. Body weights were measured weekly and plasma lipid parameters once a month. For atherosclerosis studies using antisense oligonucleotide (ASO)-mediated knockdown of CGI-58, 6-week-old male LDLR−/− mice were fed a diet enriched in 0.2% (w/w) cholesterol and 20% of energy as lard for 16 weeks in conjunction with weekly injections (50 mg/kg) of either a nontargeting control ASO or CGI-58 ASO, as previously described (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Plasma samples were collected by submandibular vein puncture at baseline (chow-fed animals, 6 weeks of age), and after 4, 8, and 16 weeks of diet and ASO treatment for subsequent lipid and lipoprotein analyses. The majority of animal experiments were performed according to the standards set by the Austrian Federal Ministry of Science and Research, Division of Genetic Engineering and Animal Experiments, Vienna, Austria (BMWF-66.010/0039-II/10b/2009, BMWF-66.010/0057-II/3b/2011). The ASO-mediated knockdown studies of CGI-58 were conducted in an American Association for Accreditation of Laboratory Animal Care-approved animal facility, and all experimental protocols were approved by the Institutional Animal Care and Use Committee at either the Wake Forest University School of Medicine or the Cleveland Clinic Lerner Research Institute. Peritoneal macrophages were collected after an ip injection of 2.5 ml 3% thioglycolate. After 3 days, the peritoneum was flushed with 10 ml PBS containing 1 mM EDTA. The cells were cultivated in DMEM (Gibco, Invitrogen, Carlsbad, CA) containing 10% lipoprotein-deficient serum (LPDS) and 1% penicillin/streptomycin for 2–3 h. Thereafter, the cells were washed three times with prewarmed PBS and the adherent cells (macrophages) were cultured in DMEM containing 25 mM glucose, 4 mM glutamine, 1 mM pyruvate, 10% LPDS, and 1% penicillin/streptomycin for 24 h. Bone marrow-derived macrophages were isolated from femur and tibia flushed with sterile PBS. Cells were cultured in DMEM containing 10% LPDS, 1% penicillin/streptomycin, and 10 ng/ml macrophage colony-stimulating factor for 7 days. To assess in vitro lipopolysaccharide (LPS)-induced acute-phase response, macrophages were treated with saline (control) or LPS (100 ng/ml) for 16 h. IL-6 concentrations in supernatants were determined by ELISA (Enzo Life Sciences, Lausen, Switzerland). For the studies using ASO-mediated knockdown, elicited peritoneal macrophages were collected 4 days after injection of 1 ml of 10% thioglycolate into the peritoneal cavities of C57BL/6 mice that had been treated with ASOs and fed a chow diet for 6 weeks, as previously described (19Brown J.M. Chung S. Sawyer J.K. Degirolamo C. Alger H.M. Nguyen T. Zhu X. Duong M.N. Wibley A.L. Shah R. et al.Inhibition of stearoyl-coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis.Circulation. 2008; 118: 1467-1475Crossref PubMed Scopus (126) Google Scholar). Following 2 h of culture, nonadherent cells were removed by washing three times with PBS, and remaining adherent macrophages were harvested for Western blotting using methods previously described (9Brown J.M. Betters J.L. Lord C. Ma Y. Han X. Yang K. Alger H.M. Melchior J. Sawyer J. Shah R. et al.CGI-58 knockdown in mice causes hepatic steatosis, but prevents diet-induced obesity and glucose intolerance.J. Lipid Res. 2010; 51: 3306-3315Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Blood was collected from 12 h-fasted mice or from 12 h-fasted/2 h-refed mice, and plasma was prepared by centrifugation at 5,200 g for 7 min at 4°C. Plasma TG, total cholesterol (TC), free cholesterol (FC), and nonesterified FA concentrations were measured enzymatically by commercially available kits (DiaSys, Holzheim, Germany; Wako Chemicals GmbH, Neuss, Germany). For atherosclerosis studies using ASO-mediated knockdown of CGI-58, total plasma concentrations of TC and TG were measured enzymatically by commercially available kits (Wako Chemicals, Richmond, VA). In addition, plasma lipoproteins were separated by fast protein liquid chromatography, and cholesterol concentrations in lipoprotein fractions were measured using an enzymatic assay as previously described (19Brown J.M. Chung S. Sawyer J.K. Degirolamo C. Alger H.M. Nguyen T. Zhu X. Duong M.N. Wibley A.L. Shah R. et al.Inhibition of stearoyl-coenzyme A desaturase 1 dissociates insulin resistance and obesity from atherosclerosis.Circulation. 2008; 118: 1467-1475Crossref PubMed Scopus (126) Google Scholar). Macrophages were plated for 2 h in serum-free DMEM. After washing the cells three times with PBS, lipids were extracted with 2 ml hexane:isopropanol (3:2, v:v) for 1 h at 4°C. One hundred microliters of 1% Triton X-100 in chloroform were added and the lipid extract was dried under a stream of nitrogen. The samples were dissolved in 100 μl ddH2O for 15 min at 37°C in a water bath. TG, TC, and FC concentrations were measured enzymatically by using 30 μl of the sample with the above mentioned kits. The readings were normalized to protein concentrations. Protein was quantitated using a Lowry assay (Bio-Rad Laboratories, Hercules, CA) after dissolving the proteins of cells in 2 ml NaOH (0.3 M) for 2 h at room temperature. FA composition in the TG fraction was quantitated by GC-flame ionization detection. Briefly, lipid extracts were separated by thin layer chromatography (hexane:diethylether:acetic acid, 70:30:1, v:v:v) and the band comigrating with tri-C16:0 TG was scraped, extracted with CHCl3/methanol (2:1, v:v), dried, and transesterified in BF3/toluene. Pentadecanoic acid was used as internal standard. Separation and quantitation were performed as previously described (20Sattler W. Puhl H. Hayn M. Kostner G.M. Esterbauer H. Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography: BF3/methanol transesterification of lyophilized samples instead of Folch extraction gives higher yields.Anal. Biochem. 1991; 198: 184-190Crossref PubMed Scopus (84) Google Scholar). Macrophages were plated on chamber slides in DMEM containing 10% LPDS and 1% penicillin/streptomycin for 24 h. Cells were washed three times with PBS and fixed with 10% formalin (30 min). Lipid droplets were visualized after Nile Red staining (2.5 μg/ml) by confocal laser scanning microscopy using an LSM 510 META microscope system (Carl Zeiss GmbH, Vienna, Austria). Pictures (×63 magnification) were taken at excitation 543 nm and signals were recorded using a 560 nm long pass filter. Macrophages were lysed with 100 μl of lysis buffer [100 mM potassium phosphate, 250 mM sucrose, 1 mM EDTA, 0.1 mM DTT (pH 7)], sonicated on ice twice for 10 s with 10 s interval, and protein concentrations were measured using a Lowry assay (BioRad Laboratories). The TG substrate contained 17 nmol triolein/assay and 2,000 cpm/nmol of [9,10-3H(N)]triolein (Perkin Elmer, Waltham, MA). The cholesteryl ester (CE) substrate contained 20 nmol cholesteryl oleate per assay and 1,000 cpm/nmol of cholesteryl [1-14C]oleate (Amersham Biosciences, Piscataway, NJ). Fifty micrograms of protein from cell lysates was mixed with 100 μl of substrate and incubated in a water bath for 1 h at 37°C. The reaction was stopped by the addition of 3.25 ml stop solution (methanol:chloroform:n-heptane, 10:9:7, v:v:v) and 1 ml of 0.1 M potassium carbonate and 0.1 M boric acid (pH 10.5) (21Schweiger M. Eichmann T.O. Taschler U. Zimmermann R. Zechner R. Lass A. Measurement of lipolysis.Methods Enzymol. 2014; 538: 171-193Crossref PubMed Scopus (114) Google Scholar). The tubes were vortexed for 10–15 s and centrifuged at 800 g for 20 min at 4°C. The radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting, and the release of FAs was calculated. Macrophages were incubated in 6-well plates with 300 μl medium, 2% FA-free BSA (Sigma-Aldrich, St. Louis, MO), and 2 units/ml of heparin for 1 h at 37°C under continuous shaking. For the substrate preparation per sample, 0.6 μCi [3H]triolein, 920 ng glycerol trioleate, and 0.1% Triton X-100 in chloroform were evaporated under a stream of nitrogen. Forty microliters of 1 M Tris-HCl (pH 8.6) and 80 μl ddH2O were added, and the mixture was sonicated six times (1 min on and 1 min off) on ice. Then 40 μl of heat-inactivated human serum containing ApoC-II as activator (obtained from a pool of donors, heated at 50°C for 1 h, and stored at 20°C) and 40 μl of 10% FA-free BSA were added to the substrate. Analysis was performed as previously described (15Chandak P.G. Radovic B. Aflaki E. Kolb D. Buchebner M. Frohlich E. Magnes C. Sinner F. Haemmerle G. Zechner R. et al.Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase.J. Biol. Chem. 2010; 285: 20192-20201Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Total RNA from macrophages was isolated using a PerfectPure RNA cultured cell kit (5Prime, Hamburg, Germany). RNA concentrations were measured at 260 nm on a NanoDrop instrument (Thermo Scientific, Wilmington, DE). Two micrograms of total RNA were reverse transcribed by using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR was performed on a LightCycler 480 (Roche Diagnostics, Rotkreuz, Switzerland) using the QuantifastTM SYBR® Green PCR kit (Qiagen, Hilden, Germany). Amplification of murine hypoxanthine-guanine phosphoribosyltransferase (HPRT) as housekeeping gene was performed on all samples as internal controls for variations in mRNA amounts. Expression profiles and associated statistical parameters were determined using the public domain program Relative Expression Software Tool-REST 2008 (22Pfaffl M.W. Horgan G.W. Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google Scholar). Primer sequences are listed in the supplementary material. Protein samples of lysed macrophages from the different genotypes (40 or 50 μg protein/lane) were separated by SDS-PAGE (15%). Proteins were transferred to polyvinylidene difluoride or nitrocellulose membranes. Blots were incubated with monoclonal anti-mouse antibodies against β-actin (1:20,000) (Santa Cruz, Heidelberg, Germany), ABHD5/CGI-58 (1:1,000) (Abnova GmbH, Heidelberg, Germany), and CCAAT/enhancer-binding protein homologous protein (CHOP) (1:1,000) (Cell Signaling Technology, Danvers, MA), or anti-rabbit polyclonal antibodies against Bax (1:1,000), cytochrome C (1:1,000), inositol-requiring enzyme 1α (IRE1α) (1:1,000), CHOP (1:1,000), and ATGL (1:200) (Cell Signaling Technology). HRP-conjugated goat anti-rabbit (1:5,000) or rabbit anti-mouse antibodies (1:1,000) (Dako, Glostrup, Denmark) were visualized by enhanced chemiluminescence detection (ClarityTM Western ECL substrate; Bio-Rad) using a ChemiDocTM MP imaging system (Bio-Rad). Macrophages were plated in XF96 polystyrene cell culture microplates (Seahorse Bioscience®, North Billerica, MA) at a density of 60,000 cells per well. After 24 h, cells were washed and preincubated for 30 min in XF assay medium supplemented with sodium pyruvate (1 mM) with or without glutamine (2 mM) and glucose (25 mM) at 37°C in a nonCO2 environment. The oxygen consumption rate (OCR) was subsequently measured every 7 min using an XF96 extracellular flux analyzer (Seahorse Bioscience®). A standard protocol with 15 min basal measurement followed by 10 μM oligomycin, addition of 0.3 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 2.5 μM antimycin A was performed. Oxygen consumption was either normalized to protein content (pmol O2/min × μg protein) or expressed as a percentage of the maximal mitochondrial respiration in the presence of 0.3 μM FCCP. Macrophages were plated in black 96-well μClear plates (Greiner Bio-One GmbH, Solingen, Germany). After 24 h of preincubation in DMEM, 10% LPDS, and 25 mM glucose, cells were incubated in DMEM, 10% LPDS, and 0, 6, or 25 mM glucose for 1 h, respectively. Cells were washed and incubated with 100 μl of fluorescein-labeled E. coli BioParticles (VybrantTM phagocytosis assay, Molecular Probes, Invitrogen; suspended in Hanks' balanced salt solution; 2 h). The suspension was removed and subsequently 100 μl of trypan blue was added (1 min) to quench the extracellular probe. After aspiration of trypan blue, the fluorescence was measured at 484 nm (excitation) and 535 nm (emission) on a Victor 1420 multilabel counter (PerkinElmer Life Sciences, Turku, Finland). Fluorescence was normalized to the protein content of each well. To analyze phagocytosis in vivo, mice were injected intraperitoneally with 200 μl of fluorescein-labeled E. coli BioParticles suspended in Hanks' balanced salt solution. After 2 h, macrophages were collected by flushing the peritoneal cavity with 10 ml PBS containing 1 mM EDTA and incubated in DMEM containing 25 mM glucose and 10% LPDS for 90 min. The cells were washed three times with PBS, and fluorescence was measured before and after adding trypan blue to obtain total and intracellular fluorescence, respectively. Experimental readings were normalized to protein content. Apoptosis was assayed by annexin V and propidium iodide (PI) costaining (Annexin-V-Fluor staining kit; Roche, Vienna, Austria). Two hundred thousand cells were washed twice with 200 μl PBS; 50 μl staining buffer was added and cells were incubated for 10 min. Macrophages were immediately analyzed on a FACScalibur flow cytometer (BD Biosciences, San José, CA). Animals were fasted for 6 h (6 AM to 12 PM) with free access to drinking water. Blood was taken from the tail vein before and 15, 30, 60, 120, and 180 min after an ip injection of glucose (2.0 g/kg body weight). Glucose concentrations from blood were determined using a portable glucometer (AccuCheck). We analyzed atherosclerotic lesions in the aortic root and aorta of ApoE−/− and macCGI-58/ApoE-DKO animals after 10 weeks of HF/HCD feeding. Mice were euthanized and the arterial tree was perfused in situ with PBS (100 mm Hg) for 10 min via a cannula in the left ventricular apex. Mice were perfused with 10% formalin (Carl Roth GmbH, Vienna, Austria) for 15 min. After fixing the hearts in 10% formalin, serial sections (8 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany). Images of the atherosclerotic lesion areas in Oil Red O-stained (Sigma-Aldrich) sections were taken with ScanScope T3 whole slide scanner (Aperio Technologies, Bristol, UK). Plaque areas were quantitated by ImageJ software. Mean lesion area was calculated from 10 consecutive Oil Red O-stained sections, starting at the appearance of the tricuspid valves. Sections were stained immunohistochemically for the presence of macrophages using a monoclonal rat anti-mouse Moma-2 antibody (1:600) (Acris, Hiddenhausen, Germany), as well as for collagen content using Masson's trichrome staining kit (Sigma-Aldrich). For en face analysis in macCGI-58/ApoE-DKO mice, aortas were dissected and plaques were stained with Oil Red O as described recently (23Kratzer A. Buche