ABCG1 regulates mouse adipose tissue macrophage cholesterol levels and ratio of M1 to M2 cells in obesity and caloric restriction
2015; Elsevier BV; Volume: 56; Issue: 12 Linguagem: Inglês
10.1194/jlr.m063354
ISSN1539-7262
AutoresWei Hao, Elizabeth J. Tarling, Timothy S. McMillen, Chongren Tang, Renee Leboeuf,
Tópico(s)Lipid metabolism and biosynthesis
ResumoIn addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue. In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue. Obesity remains highly prevalent in this country and is an important health issue because it poses an increased risk for several metabolic diseases including cancer and cardiovascular, liver, and gallbladder diseases (1.Azagury D.E. Lautz D.B. Obesity overview: epidemiology, health and financial impact, and guidelines for qualification for surgical therapy.Gastrointest. Endosc. Clin. N. Am. 2011; 21: 189-201Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 2.Banim P.J. Luben R.N. Bulluck H. Sharp S.J. Wareham N.J. Khaw K.T. Hart A.R. The aetiology of symptomatic gallstones quantification of the effects of obesity, alcohol and serum lipids on risk. Epidemiological and biomarker data from a UK prospective cohort study (EPIC-Norfolk).Eur. J. Gastroenterol. Hepatol. 2011; 23: 733-740Crossref PubMed Scopus (39) Google Scholar, 3.Malik V.S. Willett W.C. Hu F.B. Global obesity: trends, risk factors and policy implications.Nat. Rev. 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Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice.Biochim. Biophys. Acta. 2012; 1821: 425-434Crossref PubMed Scopus (17) Google Scholar). These ATMs have the appearance of foam cells similar to what is seen in atherosclerotic plaques and have been shown to contain neutral lipids based on histochemical stains (16.Shapiro H. Pecht T. Shaco-Levy R. Harman-Boehm I. Kirshtein B. Kuperman Y. Chen A. Bluher M. Shai I. Rudich A. Adipose tissue foam cells are present in human obesity.J. Clin. Endocrinol. Metab. 2013; 98: 1173-1181Crossref PubMed Scopus (91) Google Scholar). However, the chemical composition of these neutral lipids has not been carefully analyzed to our knowledge. Further, direct measures of ATM sterol content and in particular cholesterol have not been determined. In humans, adipose tissue contains the largest pool of free cholesterol in the body, estimated at 25% of total body cholesterol (18.Angel A. Fong B. 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In addition, adipocyte death is increased in obesity and during acute weight loss. Together, these events generate large quantities of free cholesterol that must be cleared from adipose tissue. Adipocytes can conduct cholesterol efflux as mediated by ABCA1 and scavenger receptor class B type I to lipid acceptors such as HDLs (27.Le Lay S. Ferre P. Dugail I. Adipocyte cholesterol balance in obesity.Biochem. Soc. Trans. 2004; 32: 103-106Crossref PubMed Scopus (69) Google Scholar, 28.Le Lay S. Robichon C. Le Liepvre X. Dagher G. Ferre P. Dugail I. Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3–L1 cells.J. Lipid Res. 2003; 44: 1499-1507Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 29.Zhang Y. McGillicuddy F.C. Hinkle C.C. O'Neill S. Glick J.M. Rothblat G.H. Reilly M.P. Adipocyte modulation of high-density lipoprotein cholesterol.Circulation. 2010; 121: 1347-1355Crossref PubMed Scopus (106) Google Scholar). However, these pathways may be severely reduced with adipose tissue inflammation as seen in obesity and during acute caloric restriction (29.Zhang Y. McGillicuddy F.C. Hinkle C.C. O'Neill S. Glick J.M. Rothblat G.H. Reilly M.P. Adipocyte modulation of high-density lipoprotein cholesterol.Circulation. 2010; 121: 1347-1355Crossref PubMed Scopus (106) Google Scholar). Thus, mechanisms whereby the bulk of adipose tissue cholesterol is removed from adipose tissue in obesity and during weight loss regimes remain unclear. We suggest that an important process for removal of free cholesterol from adipose tissue involves the cholesterol loading of ATMs from adipocytes via collision-based diffusion and/or pinocytosis of dead adipocytes. This would then be followed by cholesterol efflux from macrophages to cholesterol acceptors within adipose tissue and/or by macrophage egress from adipose tissue. Here, we show for the first time that ATMs taken from obese mice and mice undergoing acute body weight loss are enriched in cholesterol, supporting our hypothesis that ATMs participate in adipose tissue cholesterol modulation. We previously found that the expression of Abcg1 is markedly elevated in ATMs isolated from obese mice and that expression is further enhanced following caloric restriction (17.Edgel K.A. McMillen T.S. Wei H. Pamir N. Houston B.A. Caldwell M.T. Mai P.O. Oram J.F. Tang C. Leboeuf R.C. Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice.Biochim. Biophys. Acta. 2012; 1821: 425-434Crossref PubMed Scopus (17) Google Scholar). These data are in line with the finding that ABCG1 is one of the most upregulated genes in human adipose tissue samples after weight loss (30.Johansson L.E. Danielsson A.P. Parikh H. Klintenberg M. Norstrom F. Groop L. Ridderstrale M. 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Biol. 1997; 17: 2622-2629Crossref PubMed Scopus (31) Google Scholar), and peritoneal macrophages taken from obese mice (17.Edgel K.A. McMillen T.S. Wei H. Pamir N. Houston B.A. Caldwell M.T. Mai P.O. Oram J.F. Tang C. Leboeuf R.C. Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice.Biochim. Biophys. Acta. 2012; 1821: 425-434Crossref PubMed Scopus (17) Google Scholar). Here, we test the possibility that Abcg1 is a marker for cholesterol-enriched ATMs and that Abcg1 plays a key role in modulating ATM cholesterol levels in obesity and acute weight loss. ATMs were collected from mice for which the myeloid cells had been replaced by bone marrow transplantation (BMT) from Abcg1−/− mice and from WT mice undergoing autologous BMT. We found that Abcg1 expression affected the steady-state cholesterol levels of ATMs, with Abcg1−/− ATMs having the highest total cholesterol content. Other sterol species were not evaluated due to limitations in ATM cell quantities. We found that the ratio of M1 to M2 ATMs was altered with loss of Abcg1 expression, and that Abcg1 was most highly expressed by M2 rather than proinflammatory M1 macrophages suggesting that M2 cells have a unique role in adipose tissue sterol homeostasis. Overall, this study identifies ABCG1 as an important protein determining the relative levels of ATMs and cholesterol metabolism especially in M2 macrophages. Understanding the contribution of ABCG1 to ATM cholesterol homeostasis may provide new information for controlling obesity, weight loss, and related disorders such as lung, liver, and gallbladder diseases (38.Malur A. Huizar I. Wells G. Barna B.P. Malur A.G. Thomassen M.J. Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice.Biochem. Biophys. Res. Commun. 2011; 415: 288-293Crossref PubMed Scopus (15) Google Scholar, 39.Paschos P. Paletas K. 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BMT was conducted using C57BL/6 male mice as acceptors of either C57BL/6 bone marrow cells (WT-BMT) or cells from Abcg1−/− mice (Abcg1−/− BMT) using procedures as described below. Femurs taken from Abcg1−/− mice were placed in PBS and sent overnight on ice from the University of California, Los Angeles to the University of Washington where bone marrow cells were isolated. In addition, C57BL/6 male mice raised at the University of Washington (three to four generations from the Jackson Laboratory) were used as a reference strain for measures of body composition. All mice were housed in pathogen-free conditions, in a temperature- and humidity-controlled environment (12 h light/dark cycle). Animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of University of Washington. BMT was performed essentially as described (42.McMillen T.S. Heinecke J.W. LeBoeuf R.C. Expression of human myeloperoxidase by macrophages promotes atherosclerosis in mice.Circulation. 2005; 111: 2798-2804Crossref PubMed Scopus (147) Google Scholar). Briefly, male C57BL/6 mice (WT) at 13 weeks of age were irradiated (950 rads) and then engrafted with either WT or Abcg1−/− bone marrow cells (5 × 106 cells/mouse; n = 20 in each group) by retro-orbital injection. Mice were maintained on pelleted rodent chow (LabDiet 5053; Purina Mills, St. Louis, MO) and acidified water with neomycin (X-gen Pharmaceuticals, Big Flats, NY) for 4 weeks of recovery. Mice were then fed a high-fat diet (HFD) [D12492; Research Diets Inc., New Brunswick, NJ; 60% kcal (primarily lard) and 20% kcal carbohydrate (maltodextrin 10 and sucrose)] for 13 weeks. Mice were then randomly assigned to one of two groups for another 3 weeks of either continued ad libitum HFD feeding or to caloric restriction. For caloric restriction, mice were individually housed and given 60% of the HFD food consumption as monitored during weeks 10–12 as described (43.Pamir N. McMillen T.S. Li Y.I. Lai C.M. Wong H. LeBoeuf R.C. Overexpression of apolipoprotein A5 in mice is not protective against body weight gain and aberrant glucose homeostasis.Metabolism. 2009; 58: 560-567Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). At the end of all feeding studies, mice were fasted for 4 h in the morning, bled from the retro-orbital sinus into tubes containing 1 mM EDTA to obtain plasma, and euthanized by cervical dislocation, and tissues were collected for analyses. An aliquot of epididymal fat was immediately used to isolate ATMs (see below). In vivo body composition analysis of lean and fat mass content was performed before and 2 weeks after caloric restriction using quantitative magnetic resonance (EchoMRI™ 3-in-1 Animal Tissue Composition Analyzer; Echo Medical Systems, Houston, TX) (44.Taicher G.Z. Tinsley F.C. Reiderman A. Heiman M.L. Quantitative magnetic resonance (QMR) method for bone and whole-body-composition analysis.Anal. Bioanal. Chem. 2003; 377: 990-1002Crossref PubMed Scopus (196) Google Scholar, 45.Tinsley F.C. Taicher G.Z. Heiman M.L. Evaluation of a quantitative magnetic resonance method for mouse whole body composition analysis.Obes. Res. 2004; 12: 150-160Crossref PubMed Scopus (207) Google Scholar). Intraperitoneal glucose tolerance tests (IPGTTs) were conducted as described (43.Pamir N. McMillen T.S. Li Y.I. Lai C.M. Wong H. LeBoeuf R.C. Overexpression of apolipoprotein A5 in mice is not protective against body weight gain and aberrant glucose homeostasis.Metabolism. 2009; 58: 560-567Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 46.Pamir N. McMillen T.S. Kaiyala K.J. Schwartz M.W. LeBoeuf R.C. Receptors for tumor necrosis factor-alpha play a protective role against obesity and alter adipose tissue macrophage status.Endocrinology. 2009; 150: 4124-4134Crossref PubMed Scopus (64) Google Scholar). Epididymal fat pads were excised and minced in Dulbecco's PBS (DPBS with calcium chloride and magnesium; Life Technologies, Grand Island, NY). Tissue suspensions were centrifuged (500 g, 5 min) followed by incubation with 1 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 20 min with shaking. The cell suspension was filtered through a 100 μm filter and then centrifuged (300 g, 5 min) to separate adipocytes from the stromal vascular fraction (SVF) pellet. For fluorescence-activated cell sorting (FACS), the SVF pellet was resuspended and incubated in 0.5 ml RBC Lysis Buffer (eBioscience, San Diego, CA) for 5 min. The SVF was washed and pelleted in DPBS and then resuspended in sorting buffer (eBioscience). Cells were incubated with Fc Block (eBioscience) prior to staining with conjugated antibodies or isotype controls for 15 min at 4°C followed by two washes in 10× DPBS. Cells were resuspended in DPBS supplemented with propidium iodide to assess cell viability and then subjected to FACS (FACSAria; BD Biosciences, San Jose, CA). Viable cells were sorted directly into DPBS, pelleted, and frozen at −80°C for further analysis. Antibodies used in FACS were as follows: F4/80-FITC, CD11c-PerCP5.5, CD45-eFluor450 (eBioscience), and CD206-PECy7 (AbD Serotec; Bio-Rad Laboratories Inc., Hercules, CA). Cells were resuspended in 100 µl DPBS, and cholesterol-d7 (Sigma-Aldrich) was added as the internal standard. After saponification, the lipid fraction was extracted from the membrane preparation with hexane and dried under nitrogen gas. Total cholesterol levels were determined after derivatization using LC/MS/MS as described (47.Honda A. Yamashita K. Hara T. Ikegami T. Miyazaki T. Shirai M. Xu G. Numazawa M. Matsuzaki Y. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.J. Lipid Res. 2009; 50: 350-357Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Unesterified cholesterol was measured using the same procedure except without the saponification step, and the quantity of cholesterol moiety of cholesteryl ester was calculated indirectly using the following equation: cholesterol moiety of cholesteryl ester = total cholesterol − unesterified cholesterol. Bone marrow cells isolated from femurs of 10-week-old WT and Abcg1−/− mice were cultured in low-glucose DMEM (Thermo Scientific, Rockford, IL) supplemented with 10% FBS (Life Technologies), macrophage-colony stimulating factor [30% L-929-conditioned medium prepared as described (48.Gersuk G. Hiraoka A. Marr K.A. Human monocytes differentiate into macrophages under the influence of human KPB-M15 conditioned medium.J. Immunol. Methods. 2005; 299: 99-106Crossref PubMed Scopus (9) Google Scholar)] for differentiation into macrophages. By day 7, macrophages were polarized with 5 ng/ml lipopolysaccharide and 12 ng/ml IFNƔ (M1) or 10 ng/ml interleukin (IL) 4 (M2). Nonpolarized macrophages [bone marrow-derived macrophages (BMDMs)] were cultured in complete medium alone. At days 9 and 10, cells are used for mRNA extractions or chemotaxis assays. Macrophage migration was determined as described (49.Frevert C.W. Wong V.A. Goodman R.B. Goodwin R. Martin T.R. Rapid fluorescence-based measurement of neutrophil migration in vitro.J. Immunol. Methods. 1998; 213: 41-52Crossref PubMed Scopus (138) Google Scholar). Briefly, macrophages were incubated with 1 μg/ml Calcein AM (Invitrogen) for 30 min at 37°C. Cells were then washed with DPBS and resuspended in RPMI 1640 medium with 0.1% free fatty acid-free BSA (Sigma-Aldrich) to a final concentration of 1 × 106 cells/ml. Cell migration was assessed using the disposable 96-well ChemoTx System 101-3 (Neuro probe, Gaithersburg, MD) according to the manufacturer's instructions. Recombinant monocyte chemoattractant protein 1 (MCP-1) (Biolegend, San Diego, CA) at 50 ng/µl was used as chemoattractant to trigger cell migration. The fluorescence of cells was measured using Synergy4 microplate reader (Bio-Tek, Winooski, VT). Percentages of cell migration were calculated as migrated cells/loading control (cells loaded directly to the bottom of the well), corrected for spontaneous migration. Each sample was tested in 6 wells. Macrophages were suspended in DMEM medium containing 1 mg/ml BSA and 100 μg/ml acetylated LDL (ac-LDL) and incubated for 24 h prior to cell collection and usage for mRNA or migration studies. Mouse cells were homogenized in lysis buffer (0.1 M K2HPO4, 1 mM PMSF, 0.2% Triton X-100, and 0.1% protease inhibitor cocktail; P8340, Sigma). The homogenate was centrifuged (12,000 g for 10 min at 4°C), and the supernatant was collected. Equal amounts of protein (20 μg) were electrophoresed on 15% SDS polyacrylamide gels and then electro-transferred to a ProTran nitrocellulose membrane (Schleicher and Schuell, Riviera Beach, FL). Protein levels were measured by immunoblots using antibodies for ABCA1 (Novus Biological, Littleton, CO), ABCG1 (Cell Signaling Technology, Danvers, MA), and GAPDH (Abcam, Boston, MA), and then probed with horseradish peroxidase secondary antibody (Chemicon International, Temecula, CA). Blots were developed using SuperSignal pico ECL kit (Pierce, Thermo Scientific, Waltham, MA). Molecular band intensity was determined by densitometry using National Institutes of Health ImageJ software. Total RNA was extracted from adipose t
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