Hematopoietic ABCA1 deletion promotes monocytosis and worsens diet-induced insulin resistance in mice
2015; Elsevier BV; Volume: 57; Issue: 1 Linguagem: Inglês
10.1194/jlr.m064303
ISSN1539-7262
AutoresChongren Tang, Yuhua Liu, Wendy Yang, Carl Storey, Timothy S. McMillen, Barbara Houston, Jay W. Heinecke, Renee Leboeuf,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoLow-grade chronic inflammation plays an important role in the pathogenesis of obesity-induced insulin resistance. ABCA1 is essential for reverse cholesterol transport and HDL synthesis, and protects against macrophage inflammation. In the present study, the effects of ABCA1 deficiency in hematopoietic cells on diet-induced inflammation and insulin resistance were tested in vivo using bone marrow transplanted (BMT)-WT and BMT-ABCA1−/− mice. When challenged with a high-fat high-carbohydrate diabetogenic diet with added cholesterol (HFHSC), BMT-ABCA1−/− mice displayed enhanced insulin resistance and impaired glucose tolerance as compared with BMT-WT mice. The worsened insulin resistance and impaired glucose tolerance in BMT-ABCA1−/− mice were accompanied by increased macrophage accumulation and inflammation in adipose tissue and liver. Moreover, BMT-ABCA1−/− mice had significantly higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis when challenged with the HFHSC diet. In vitro studies indicated that macrophages from ABCA1−/− mice showed significantly increased inflammatory responses induced by saturated fatty acids. Taken together, these studies point to an important role for hematopoietic ABCA1 in modulating a feed-forward mechanism in obesity such that inflamed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of inflammation and associated disease processes. Low-grade chronic inflammation plays an important role in the pathogenesis of obesity-induced insulin resistance. ABCA1 is essential for reverse cholesterol transport and HDL synthesis, and protects against macrophage inflammation. In the present study, the effects of ABCA1 deficiency in hematopoietic cells on diet-induced inflammation and insulin resistance were tested in vivo using bone marrow transplanted (BMT)-WT and BMT-ABCA1−/− mice. When challenged with a high-fat high-carbohydrate diabetogenic diet with added cholesterol (HFHSC), BMT-ABCA1−/− mice displayed enhanced insulin resistance and impaired glucose tolerance as compared with BMT-WT mice. The worsened insulin resistance and impaired glucose tolerance in BMT-ABCA1−/− mice were accompanied by increased macrophage accumulation and inflammation in adipose tissue and liver. Moreover, BMT-ABCA1−/− mice had significantly higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis when challenged with the HFHSC diet. In vitro studies indicated that macrophages from ABCA1−/− mice showed significantly increased inflammatory responses induced by saturated fatty acids. Taken together, these studies point to an important role for hematopoietic ABCA1 in modulating a feed-forward mechanism in obesity such that inflamed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of inflammation and associated disease processes. Low-grade inflammation has emerged as an important contributor to the etiology of insulin resistance in obesity and type 2 diabetes (1.Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6135) Google Scholar). Adipose tissue macrophages appear to be responsible for much of the increase in inflammation in adipose tissue with obesity (1.Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6135) Google Scholar, 2.Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. et al.Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5185) Google Scholar, 3.Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr, A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7456) Google Scholar) and monocytosis plays a significant role in promoting adipose macrophage infiltration and inflammation in obesity (4.Nagareddy P.R. Kraakman M. Masters S.L. Stirzaker R.A. Gorman D.J. Grant R.W. Dragoljevic D. Hong E.S. Abdel-Latif A. Smyth S.S. et al.Adipose tissue macrophages promote myelopoiesis and monocytosis in obesity.Cell Metab. 2014; 19: 821-835Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 5.Luft V.C. Schmidt M.I. Pankow J.S. Couper D. Ballantyne C.M. Young J.H. Duncan B.B. Chronic inflammation role in the obesity-diabetes association: a case-cohort study.Diabetol. Metab. Syndr. 2013; 5: 31Crossref PubMed Scopus (71) Google Scholar, 6.Ohshita K. Yamane K. Hanafusa M. Mori H. Mito K. Okubo M. Hara H. Kohno N. Elevated white blood cell count in subjects with impaired glucose tolerance.Diabetes Care. 2004; 27: 491-496Crossref PubMed Scopus (75) Google Scholar). Interestingly, recent studies suggest that cellular cholesterol balance regulates the inflammatory status of macrophages (7.Yvan-Charvet L. Welch C. Pagler T.A. Ranalletta M. Lamkanfi M. Han S. Ishibashi M. Li R. Wang N. Tall A.R. Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.Circulation. 2008; 118: 1837-1847Crossref PubMed Scopus (342) Google Scholar, 8.Zhu X. Lee J.Y. Timmins J.M. Brown J.M. Boudyguina E. Mulya A. Gebre A.K. Willingham M.C. Hiltbold E.M. Mishra N. et al.Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.J. Biol. Chem. 2008; 283: 22930-22941Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The addition of a relatively small amount of dietary cholesterol results in a marked increase in the accumulation of macrophages in adipose tissue (9.Subramanian S. Han C.Y. Chiba T. McMillen T.S. Wang S.A. Haw 3rd, A. Kirk E.A. O'Brien K.D. Chait A. Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 685-691Crossref PubMed Scopus (144) Google Scholar). Mice with macrophage-specific transgenic expression of cholesterol ester hydrolase, which results in significant cholesterol ester and free reduction in macrophages, reduces macrophage infiltration and improves insulin sensitivity (10.Bie J. Zhao B. Song J. Ghosh S. Improved insulin sensitivity in high fat- and high cholesterol-fed Ldlr-/- mice with macrophage-specific transgenic expression of cholesteryl ester hydrolase: role of macrophage inflammation and infiltration into adipose tissue.J. Biol. Chem. 2010; 285: 13630-13637Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11.Zhao 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.J. Clin. Invest. 2007; 117: 2983-2992Crossref PubMed Scopus (109) Google Scholar). Moreover, deficiency of ABCA1 and ABCG1 promotes monocytosis (12.Murphy A.J. Dragoljevic D. Tall A.R. Cholesterol efflux pathways regulate myelopoiesis: a potential link to altered macrophage function in atherosclerosis.Front. Immunol. 2014; 5: 490Crossref PubMed Scopus (39) Google Scholar, 13.Yvan-Charvet L. Pagler T. Gautier E.L. Avagyan S. Siry R.L. Han S. Welch C.L. Wang N. Randolph G.J. Snoeck H.W. et al.ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation.Science. 2010; 328: 1689-1693Crossref PubMed Scopus (525) Google Scholar). These studies suggest that cholesterol metabolism plays an important role in macrophage infiltration and inflammation in peripheral tissues, including adipose tissue and liver. ABCA1 is a cell-membrane protein that is essential for HDL synthesis and reverse cholesterol transport. ABCA1 protects against cholesterol accumulation in macrophages by transporting cholesterol and phospholipids to apolipoproteins from cells (14.Oram J.F. ATP-binding cassette transporter A1 and cholesterol trafficking.Curr. Opin. Lipidol. 2002; 13: 373-381Crossref PubMed Scopus (137) Google Scholar, 15.Oram J.F. The ins and outs of ABCA.J. Lipid Res. 2008; 49: 1150-1151Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 16.Oram J.F. Heinecke J.W. ATP-binding cassette transporter A1: a cell cholesterol exporter that protects against cardiovascular disease.Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (422) Google Scholar). ABCA1 has been shown to be a crucial regulator of intracellular cholesterol stores and plays an important role in the functions of multiple tissues (17.Oram J.F. Vaughan A.M. ATP-Binding cassette cholesterol transporters and cardiovascular disease.Circ. Res. 2006; 99: 1031-1043Crossref PubMed Scopus (325) Google Scholar, 18.van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. et al.Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc. Natl. Acad. Sci. USA. 2002; 99: 6298-6303Crossref PubMed Scopus (320) Google Scholar, 19.Brunham L.R. Singaraja R.R. Duong M. Timmins J.M. Fievet C. Bissada N. Kang M.H. Samra A. Fruchart J.C. McManus B. et al.Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 548-554Crossref PubMed Scopus (89) Google Scholar). Leukocyte ABCA1 expression is associated with increased fasting glucose concentration, and reduced ABCA1 expression in leukocytes is associated with type 2 diabetes (19.Brunham L.R. Singaraja R.R. Duong M. Timmins J.M. Fievet C. Bissada N. Kang M.H. Samra A. Fruchart J.C. McManus B. et al.Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 548-554Crossref PubMed Scopus (89) Google Scholar, 20.Patel D.C. Albrecht C. Pavitt D. Paul V. Pourreyron C. Newman S.P. Godsland I.F. Valabhji J. Johnston D.G. Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function.PLoS One. 2011; 6: e22142Crossref PubMed Scopus (66) Google Scholar, 21.Albrecht C. Simon-Vermot I. Elliott J.I. Higgins C.F. Johnston D.G. Valabhji J. Leukocyte ABCA1 gene expression is associated with fasting glucose concentration in normoglycemic men.Metabolism. 2004; 53: 17-21Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). ABCA1 deficiency in pancreatic β cells reduces insulin release (22.Brunham L.R. Kruit J.K. Pape T.D. Timmins J.M. Reuwer A.Q. Vasanji Z. Marsh B.J. Rodrigues B. Johnson J.D. Parks J.S. et al.Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment.Nat. Med. 2007; 13: 340-347Crossref PubMed Scopus (337) Google Scholar); whereas, hematopoietic-specific deletion of ABCA1 promotes hematopoietic stem cell proliferation, monocytosis, macrophage inflammation, and diet-induced atherosclerosis in mice (18.van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. et al.Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc. Natl. Acad. Sci. USA. 2002; 99: 6298-6303Crossref PubMed Scopus (320) Google Scholar, 23.Yvan-Charvet L. Ranalletta M. Wang N. Han S. Terasaka N. Li R. Welch C. Tall A.R. Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice.J. Clin. Invest. 2007; 117: 3900-3908Crossref PubMed Scopus (445) Google Scholar). We and others have reported that ABCA1 deficiency in macrophages increases pro-inflammatory cytokine expression in response to pro-inflammatory insults such as lipopolysaccharides (LPSs) (7.Yvan-Charvet L. Welch C. Pagler T.A. Ranalletta M. Lamkanfi M. Han S. Ishibashi M. Li R. Wang N. Tall A.R. Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.Circulation. 2008; 118: 1837-1847Crossref PubMed Scopus (342) Google Scholar, 8.Zhu X. Lee J.Y. Timmins J.M. Brown J.M. Boudyguina E. Mulya A. Gebre A.K. Willingham M.C. Hiltbold E.M. Mishra N. et al.Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.J. Biol. Chem. 2008; 283: 22930-22941Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 24.Tang C. Liu Y. Kessler P.S. Vaughan A.M. Oram J.F. The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor.J. Biol. Chem. 2009; 284: 32336-32343Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Considering the clear role of macrophages in the propagation of inflammatory signaling in adipose tissue and liver, we hypothesized that knockout of ABCA1 in hematopoietic-derived cells would increase monocytosis, macrophage tissue accumulation, and insulin resistance in response to a diabetogenic diet. To address this hypothesis, we generated mice with ABCA1 deletion exclusively in hematopoietic cells using bone marrow transplantation. Our results reveal that when challenged with a high-fat high-carbohydrate diabetogenic diet with added cholesterol (HFHSC) (9.Subramanian S. Han C.Y. Chiba T. McMillen T.S. Wang S.A. Haw 3rd, A. Kirk E.A. O'Brien K.D. Chait A. Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 685-691Crossref PubMed Scopus (144) Google Scholar), mice deficient in ABCA1 in their hematopoietic compartment exhibit increased hematopoietic stem cell proliferation, myeloid cell expansion, monocytosis, macrophage accumulation, and inflammation in adipose tissue and liver, as well as more severe diet-induced insulin resistance. Our data suggest an important role for hematopoietic ABCA1 in a feed-forward mechanism in obesity such that inflamed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of inflammation and associated disease processes. All mice used in the study were males of the DBA/2 background, housed in specific pathogen-free micro-isolators and maintained on a 12 h light/dark cycle. ABCA1−/− DBA mice were a gift from Robert Aiello, Pfizer-Wyeth, and the generation and characterization of ABCA1−/− mice has been described previously (25.McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. et al.High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.Proc. Natl. Acad. Sci. USA. 2000; 97: 4245-4250Crossref PubMed Scopus (482) Google Scholar). For bone marrow (BM) transplantation experiments, whole BM from WT and ABCA1−/− mice was prepared by flushing the marrow cavities of the long bones with sterile saline. Cells (5 × 106) were injected retro-orbitally into lethally irradiated (11 Gy) WT recipient mice. After a 5 week recovery period, mice were placed on either standard chow or a HFHSC diet (0.15% cholesterol) (Bioserv F4997, Frenchtown, NJ) for up to 22 weeks. Body weights were measured weekly. Food intake was recorded after 10 weeks of diet. At euthanization, harvested tissues were snap-frozen in liquid nitrogen and stored at −70°C or were fixed with 10% neutral-buffered formalin and embedded in paraffin wax. All experimental procedures were undertaken with approval from the Institution Animal Care and Use Committee of the University of Washington. Metabolic variables in the mice were measured in blood samples obtained from the retro-orbital sinus after a 5 h fast. Cholesterol and triglycerides in plasma and fast-phase LC (FPLC) fractions were measured using colorimetric assay kits. Plasma insulin levels were measured using an ELISA kit (Millipore, Billerica, MA). Intra-peritoneal glucose and insulin tolerance tests were performed after a 5 h fast at weeks 20 and 21 of diet feeding, respectively, as previously described (9.Subramanian S. Han C.Y. Chiba T. McMillen T.S. Wang S.A. Haw 3rd, A. Kirk E.A. O'Brien K.D. Chait A. Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 685-691Crossref PubMed Scopus (144) Google Scholar). Body composition was performed on conscious immobilized mice using quantitative magnetic resonance (EchoMRI whole body composition analyzer; Echo Medical Systems, Houston, TX). Total RNA was extracted from whole adipose, liver, or cultured cells using a commercially available RNA extraction kit according to the manufacturer's protocol (Agilent Technologies, Santa Clara, CA). After spectroscopic quantification, 2 μg of RNA was reverse-transcribed, and the cDNA thus obtained was analyzed by real-time quantitative PCR. Primers specific for individual genes were purchased from Applied Biosystems (Assay-on-Demand; Life Technologies, Carlsbad, CA). GAPDH and L32 were used as the control housekeeping genes. Relative amounts of the target gene were calculated using the ΔΔCt formula. Paraffin-embedded epididymal fat and liver were sectioned, dewaxed, and rehydrated prior to antigen retrieval by boiling in 10 mM sodium citrate buffer (pH 6.5). Tissue samples were blocked with 5% normal rabbit serum for 40 min, followed by incubation for 2 h with 10 μg/ml rat anti-mouse F4/80 antibody (AbD Serotec). Biotin-conjugated secondary anti-rat antibody was applied for 1 h, followed by a 30 min incubation with Vectastain ABC solution (Vector Laboratories). The percentage of F4/80-positive cells for each sample was calculated as the number of nuclei of F4/80-expressing cells divided by the total number of nuclei per sample. Leukocytes and differential blood cell counts were quantified from whole blood using a hematology cell counter (HEMAVET 950FS). Adipose macrophages were quantified using flow cytometry, as previously described (9.Subramanian S. Han C.Y. Chiba T. McMillen T.S. Wang S.A. Haw 3rd, A. Kirk E.A. O'Brien K.D. Chait A. Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 685-691Crossref PubMed Scopus (144) Google Scholar). Briefly, epididymal white adipose tissue from mice was excised at the time of euthanization and minced in PBS. Minced samples were digested using LPS-depleted collagenase (type IV) cocktail (Sigma-Aldrich, St. Louis, MO) (1 mg/ml). The digested mixture was filtered through a 100 uM cell strainer, and stromal vascular cells were pelleted. The stromal vascular fraction (SVC) was then used for flow cytometry using anti-F4/80 antibody. Stained cells were analyzed on an LSR II flow cytometer (BD Biosciences) running FACSDiva software. Flow cytometry was used to quantify blood monocytes and hematopoietic stem cells, as previously described (13.Yvan-Charvet L. Pagler T. Gautier E.L. Avagyan S. Siry R.L. Han S. Welch C.L. Wang N. Randolph G.J. Snoeck H.W. et al.ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation.Science. 2010; 328: 1689-1693Crossref PubMed Scopus (525) Google Scholar). For all samples, cells were counted with a hemocytometer, and Fc receptors were blocked using anti-FcγRII/III antibody 2.4G2 prior to adding the fluorescent-tagged antibodies. For blood monocytes and myeloid counting, red blood cells were lysed using red blood cell lysis buffer (eBioscience). Monocytes were identified as CD45hiCD115hi and further identified into Ly6Chi and Ly6Clo subsets, myeloid cells were identified as CD11bhi/Gr-Ihi. For BM cell counting, BM cells were collected from leg bones, lysed to remove red blood cells, and filtered before use. Hematopoietic stem cells and multipotential progenitor cells (HSPCs) were identified as lineage−, Sac1+, and ckit+, and the myeloid progenitor cells (MPCs) were identified as lineage−-Sca1−- ckit+. Dead cells were excluded from all samples using fixable viability dye, eFluor 450 (eBioscience). All flow cytometry data were collected on the LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software 10.0 (Tree Star). Thioglycollate-elicited peritoneal macrophages were obtained, as previously described (26.Tang C. Oram J.F. The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes.Biochim. Biophys. Acta. 2009; 1791: 563-572Crossref PubMed Scopus (100) Google Scholar), and cultured in DMEM with 10% fetal bovine serum for 2 h. Unseeded cells were washed away, and the medium was changed to DMEM with 0.1% fatty acid-free bovine albumin. Cells were loaded with 50 μg/ml acetylated LDL overnight. For cytokine expression experiments, cells were pretreated with or without 10 μg/ml of apoA-I in DMEM and 0.1% fatty acid-free bovine albumin for 3 h, washed twice, treated with or without the indicated fatty acids (250 μM palmitate or mixed fatty acids) conjugated to 2% (w/v) fatty acid-free and low endotoxin BSA (Sigma-Aldrich) overnight, and then processed for RNA extractions. BM cells were collected from leg bones, lysed to remove red blood cells, and cultured in IMDM (Gibco; Invitrogen) for 2 h to remove adherent cells in order to enrich progenitor cells. The nonadherent cells were then cultured for 72 h in IMDM medium containing 6 ng/ml interleukin (IL)-3 (R&D Systems), 100 ng/ml stem cell factor (CSF; R&D Systems), and 2 ng/ml GM-CSF (R&D Systems). For proliferation measurements, cells were further incubated with 2 μCi/ml [3H]thymidine for 4 h and the radioactivity incorporated into the cells was determined by liquid scintillation counting. Data were analyzed using the GraphPad Prism 5 program (GraphPad Software Inc., La Jolla, CA) and are represented as means and SD. Student's t-test was used to detect differences within groups when applicable. One-way ANOVA was used to compare differences among all groups, and Bonferroni post hoc testing was used to detect differences among mean values of the groups. P < 0.05 was considered to be statistically significant. To assess the potential role of ABCA1 in regulating obesity-induced inflammation and insulin resistance in vivo, we generated bone marrow transplanted (BMT)-WT and BMT-ABCA1−/− chimeras by transplanting BM from ABCA1−/− and WT littermates into lethally irradiated WT recipients. After a 5 week recovery period, mice were either maintained on chow diet or switched to the HFHSC diet for up to 22 weeks. We found that there were no differences in body mass, adiposity, and macrophage accumulation between BMT-WT and BMT-ABCA1−/− mice (data not shown) on the control diet. While the HFHSC diet increased both body mass and adiposity, there were no significant differences between BMT-WT and BMT-ABCA1−/− mice (Fig. 1A, B). Consistent with the literature (18.van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. et al.Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc. Natl. Acad. Sci. USA. 2002; 99: 6298-6303Crossref PubMed Scopus (320) Google Scholar, 19.Brunham L.R. Singaraja R.R. Duong M. Timmins J.M. Fievet C. Bissada N. Kang M.H. Samra A. Fruchart J.C. McManus B. et al.Tissue-specific roles of ABCA1 influence susceptibility to atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 548-554Crossref PubMed Scopus (89) Google Scholar), plasma total cholesterol and HDL cholesterol were not significantly different between HFHSC diet-fed BMT-WT and BMT-ABCA1−/− mice (Fig. 1C). FPLC analysis indicated that the lipid profiles were similar between HFHSC diet-fed BMT-WT and BMT-ABCA1−/− mice (Fig. 1D). However, despite similar body mass, adiposity, and plasma lipid profiles, fasting plasma glucose levels (Fig. 2A) and the calculated HOMA_IR index (Fig. 2C) were significantly higher for the HFHSC diet-fed BMT-ABCA1−/− mice compared with those of HFHSC diet-fed BMT-WT mice. These data suggest that HFHSC diet-fed BMT-ABCA1−/− mice were more insulin resistant.Fig. 2Hematopoietic ABCA1 deletion worsened insulin resistance. BMT-WT and BMT-ABCA1−/− mice were fed the HFHSC diet for 22 weeks. Plasma fasting glucose (A) and plasma fasting insulin (B) were measured at week 21. C: Calculated HOMR-IR. D: Intraperitoneal glucose tolerance test. E: Intraperitoneal insulin tolerance test. F: Area under the curve (AUC) was calculated to measure the degree of the glucose tolerance impairment. G: AUC was calculated to measure the degree of the insulin tolerance impairment. *P < 0.05 versus BMT-WT mice; n = 10–15 per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We further performed intraperitoneal glucose and insulin tolerance tests in HFHSC diet-fed BMT-WT and BMT-ABCA1−/− mice. As shown in Fig. 2D–G, significantly worse glucose and insulin tolerance were noted in HFHSC diet-fed BMT-ABCA1−/− mice as compared with HFHSC diet-fed BMT-WT mice. In obesity, macrophages accumulated within adipose tissue promote insulin resistance (27.Osborn O. Olefsky J.M. The cellular and signaling networks linking the immune system and metabolism in disease.Nat. Med. 2012; 18: 363-374Crossref PubMed Scopus (1131) Google Scholar). To determine whether hematopoietic ABCA1 deficiency affects macrophage accumulation in adipose tissue, we examined the macrophage content of white adipose tissue. We found that immunohistochemical staining of F4/80 (Fig. 3A, B) and mRNA expression (data not shown) were significantly greater in adipose tissue of HFHSC diet-fed BMT-ABCA1−/− mice. Moreover, fluorescence-activated cell sorting analysis of the SVC showed that the percentage of F4/80-positive cells (Fig. 3C) was significantly higher in adipose tissue of HFHSC diet-fed BMT-ABCA1−/− mice, indicating increased macrophage accumulation. We subsequently examined the inflammatory profile of adipose tissue and found that adipose tissue and the SVC from HFHSC diet-fed BMT-ABCA1−/− mice had significantly higher expression of TNF-α, IL-1β, and IL-6 (Fig. 3D). Liver is another tissue that contributes to insulin resistance in obesity and contains a significant content of macrophages (Kupffer cells). We next examined the macrophage content of liver. We found that when fed the HFHSC diet, BMT-ABCA1−/− mice had significantly greater expression of F4/80 and CD68 in the liver (data not shown). Consistent with the increase of mRNA transcripts, immunohistochemical staining of F4/80 was significantly greater in the liver of HFHSC diet-fed BMT-ABCA1−/− mice (Fig. 4A, B), indicating higher macrophage accumulation. We subsequently examined the expression of inflammatory genes in the liver. In agreement with the observed increase in macrophage accumulation in the liver of HFHSC diet-fed BMT-ABCA1−/− mice, we also found a marked increase in the expression of inflammatory cytokines, TNF-α, IL-1β, and IL-6 (Fig. 4C). Consistent with the changes in insulin sensitivity, we also observed significantly decreased Akt phosphorylation in the liver of BMT-ABCA1−/− mice as compared with BMT-WT controls after stimulation with insulin (Fig. 4D). There is a strong association between obesity and leukocytosis, particularly of the myeloid lineage (6.Ohshita K. Yamane K. Hanafusa M. Mori H. Mito K. Okubo M. Hara H. Kohno N. Elevated white blood cell count in subjects with impaired glucose tolerance.Diabetes Care. 2004; 27: 491-496Crossref PubMed Scopus (75) Google Scholar, 28.Schmidt M.I. Duncan B.B. Sharrett A.R. Lindberg G. Savage P.J. Offenbacher S. Azambuja M.I. Tracy R.P. Heiss G. Markers of inflammation and prediction of diabetes mellitus in adults (Atherosclerosis Risk in Communities study): a cohort study.Lancet. 1999; 353: 1649-1652Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). A recent pioneering study has shown that MPC proliferation and monocytosis drive macrophage accumulation and insulin resistance in obesity (4.Nagareddy P.R. Kraakman M. Masters S.L. Stirzaker R.A. Gorman D.J. Grant R.W. Dragoljevic D. Hong E.S. Abdel-Latif A. Smyth S.S. et al.Adipose tissue macrophages promote myelopoiesis and monocytosis in obesity.Cell Metab. 2014; 19: 821-835Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). A possible explanation for the increased macrophage accumulation and insulin resistance in the HSHFC-fed BMT-ABCA1−/− mice is that they may have higher blood levels of monocytes and elevated MPC proliferation. Leukocytes and differential blood counts were quantified from whole blood using a hematology cell counter, and monocyte subset and BM progenitor cells were quantified using flow cytometry. As shown in Fig. 5A, on the control chow diet there were no differences in leukocytes, blood counts, monocyte subset, or BM progenitor cells between BMT-WT and BMT-ABCA1−/− mice. Consistent with previous findings (4.Nagareddy P.R. Kraakman M. Masters S.L. Stirzaker R.A. Gorman D.J. Grant R.W. Dragoljevic D. Hong E.S. Abdel-Latif A. Smyth S.S. et al.Adipose tissue macrophages promote myelopoiesis and monocytosis in obesity.Cell Metab. 2014; 19: 821-835Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), diet-induced obese BMT-WT mice showed increases in the numbers of ci
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