Hematopoietic ABCA1 deletion promotes monocytosis and worsens diet-induced insulin resistance in mice

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.

to the manufacturer's protocol (Agilent Technologies, Santa Clara, CA). After spectroscopic quantifi cation, 2 g of RNA was reverse-transcribed, and the cDNA thus obtained was analyzed by real-time quantitative PCR. Primers specifi c 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 ⌬ ⌬ Ct formula.

Immunohistochemistry
Paraffi n-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 g/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.

White blood cell counts
Leukocytes and differential blood cell counts were quantifi ed from whole blood using a hematology cell counter (HEMAVET 950FS).

Flow cytometry
Adipose macrophages were quantifi ed using fl ow cytometry, as previously described ( 9 ). Briefl y, 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 fi ltered through a 100 uM cell strainer, and stromal vascular cells were pelleted. The stromal vascular fraction (SVC) was then used for fl ow cytometry using anti-F4/80 antibody. Stained cells were analyzed on an LSR II fl ow cytometer (BD Biosciences) running FACSDiva software.
Flow cytometry was used to quantify blood monocytes and hematopoietic stem cells, as previously described ( 13 ). For all samples, cells were counted with a hemocytometer, and Fc receptors were blocked using anti-Fc ␥ RII/III antibody 2.4G2 prior to adding the fl uorescent-tagged antibodies . For blood monocytes and myeloid counting, red blood cells were lysed using red blood cell lysis buffer (eBioscience). Monocytes were identifi ed as CD45 hi CD115 hi and further identifi ed into Ly6C hi and Ly6C lo subsets, myeloid cells were identifi ed as CD11b hi /Gr-I hi . For BM cell counting, BM cells were collected from leg bones, lysed to remove red blood cells, and fi ltered before use. Hematopoietic stem cells and multipotential progenitor cells (HSPCs) were identifi ed as lineage Ϫ , Sac1+, and ckit+, and the myeloid progenitor cells (MPCs) were identifi ed as lineage Ϫ -Sca1 Ϫ -ckit +. Dead cells were excluded from all samples using fi xable viability dye, eFluor 450 (eBioscience). All fl ow cytometry data were collected on the LSR II fl ow cytometer (BD Biosciences) and analyzed using FlowJo software 10.0 (Tree Star).

Cell culture
Thioglycollate-elicited peritoneal macrophages were obtained, as previously described ( 26 ), 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 g/ml acetylated LDL overnight. For cytokine expression experiments, cells were pretreated with or without 10 g/ml of apoA-I in DMEM others have reported that ABCA1 defi ciency in macrophages increases pro-infl ammatory cytokine expression in response to pro-infl ammatory insults such as lipopolysaccharides (LPSs) ( 7,8,24 ). Considering the clear role of macrophages in the propagation of infl ammatory 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 ), mice defi cient in ABCA1 in their hematopoietic compartment exhibit increased hematopoietic stem cell proliferation, myeloid cell expansion, monocytosis, macrophage accumulation, and infl ammation 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 infl amed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of infl ammation and associated disease processes.

Animals and diet
All mice used in the study were males of the DBA/2 background, housed in specifi c pathogen-free micro-isolators and maintained on a 12 h light/dark cycle. ABCA1 Ϫ / Ϫ DBA mice were a gift from Robert Aiello, Pfi zer-Wyeth, and the generation and characterization of ABCA1 Ϫ / Ϫ mice has been described previously ( 25 ). For bone marrow (BM) transplantation experiments, whole BM from WT and ABCA1 Ϫ / Ϫ mice was prepared by fl ushing the marrow cavities of the long bones with sterile saline. Cells (5 × 10 6 ) were injected retroorbitally 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 Ϫ 70°C or were fi xed with 10% neutral-buffered formalin and embedded in paraffi n wax. All experimental procedures were undertaken with approval from the Institution Animal Care and Use Committee of the University of Washington.

Analytical procedures
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 ). Body composition was performed on conscious immobilized mice using quantitative magnetic resonance (EchoMRI whole body composition analyzer; Echo Medical Systems, Houston, TX).

Real-time quantitative PCR
Total RNA was extracted from whole adipose, liver, or cultured cells using a commercially available RNA extraction kit according

ABCA1
Ϫ / Ϫ 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 Ϫ / Ϫ mice (data not shown) on the control diet. While the HFHSC diet increased both body mass and adiposity, there were no signifi cant differences between BMT-WT and BMT-ABCA1 Ϫ / Ϫ mice ( Fig. 1A, B ). Consistent with the literature ( 18,19 ), plasma total cholesterol and HDL cholesterol were not signifi cantly different between HFHSC diet-fed BMT-WT and BMT-ABCA1 Ϫ / Ϫ mice ( Fig. 1C ). FPLC analysis indicated that the lipid profi les were similar between HFHSC diet-fed BMT-WT and BMT-ABCA1 Ϫ / Ϫ mice ( Fig.  1D ). However, despite similar body mass, adiposity, and plasma lipid profi les, fasting plasma glucose levels ( Fig. 2A ) and the calculated HOMA_IR index ( Fig. 2C ) were significantly higher for the HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice compared with those of HFHSC diet-fed BMT-WT mice. These data suggest that HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice were more insulin resistant. We further performed intraperitoneal glucose and insulin tolerance tests in HFHSC diet-fed BMT-WT and BMT-ABCA1 Ϫ / Ϫ mice. As shown in Fig. 2D-G , signifi cantly worse glucose and insulin tolerance were noted in HFHSC dietfed BMT-ABCA1 Ϫ / Ϫ mice as compared with HFHSC dietfed BMT-WT mice.

Hematopoietic ABCA1 deletion results in higher macrophage accumulation and infl ammatory cytokine expression in adipose tissue
In obesity, macrophages accumulated within adipose tissue promote insulin resistance ( 27 ). To determine whether hematopoietic ABCA1 deficiency affects macrophage and 0.1% fatty acid-free bovine albumin for 3 h, washed twice, treated with or without the indicated fatty acids (250 M 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 .

In vitro proliferation assay
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 Ci/ml [ 3 H]thymidine for 4 h and the radioactivity incorporated into the cells was determined by liquid scintillation counting.

Statistical analyses
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 signifi cant.

Hematopoietic ABCA1 deletion worsens diet-induced insulin resistance in vivo without affecting body mass and adiposity
To assess the potential role of ABCA1 in regulating obesity-induced infl ammation and insulin resistance in vivo, we generated bone marrow transplanted (BMT)-WT and BMT-ABCA1 Ϫ / Ϫ chimeras by transplanting BM from immunohistochemical staining of F4/80 was signifi cantly greater in the liver of HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice ( Fig. 4A, B ), indicating higher macrophage accumulation. We subsequently examined the expression of infl ammatory genes in the liver. In agreement with the observed increase in macrophage accumulation in the liver of HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice, we also found a marked increase in the expression of infl ammatory cytokines, TNF-␣ , IL-1 ␤ , and IL-6 ( Fig. 4C ). Consistent with the changes in insulin sensitivity, we also observed signifi cantly decreased Akt phosphorylation in the liver of BMT-ABCA1 Ϫ / Ϫ mice as compared with BMT-WT controls after stimulation with insulin ( Fig. 4D ).

Hematopoietic ABCA1 deletion results in myelopoiesis and monocytosis when challenged with the HFHSC diet
There is a strong association between obesity and leukocytosis, particularly of the myeloid lineage ( 6,28 ). A recent pioneering study has shown that MPC proliferation and monocytosis drive macrophage accumulation and insulin resistance in obesity ( 4 ). A possible explanation for the increased macrophage accumulation and insulin resistance in the HSHFC-fed BMT-ABCA1 Ϫ / Ϫ mice is that they may have higher blood levels of monocytes and elevated MPC proliferation. Leukocytes and differential blood counts 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 signifi cantly greater in adipose tissue of HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice. Moreover, fl uorescence-activated cell sorting analysis of the SVC showed that the percentage of F4/80-positive cells ( Fig. 3C ) was signifi cantly higher in adipose tissue of HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice, indicating increased macrophage accumulation. We subsequently examined the infl ammatory profi le of adipose tissue and found that adipose tissue and the SVC from HFHSC dietfed BMT-ABCA1 Ϫ / Ϫ mice had signifi cantly higher expression of TNF-␣ , IL-1 ␤ , and IL-6 ( Fig. 3D ).

Hematopoietic ABCA1 deletion results in elevated macrophage accumulation and infl ammatory cytokine expression in the liver
Liver is another tissue that contributes to insulin resistance in obesity and contains a signifi cant content of macrophages (Kupffer cells ). We next examined the macro phage content of liver. We found that when fed the HFHSC diet, BMT-ABCA1 Ϫ / Ϫ mice had significantly greater expression of F4/80 and CD68 in the liver (data not shown). Consistent with the increase of mRNA transcripts, progenitors displayed signifi cantly increased proliferation compared with BMT-WT progenitors in response to IL-3/ GM-CSF ( Fig. 6E ). These fi ndings suggest that increased proliferation and expansion of HSPCs may be responsible for the monocytosis and neutrophilia that develops in the BMT-ABCA1 Ϫ / Ϫ mice fed the HFHSC diet.

The ABCA1 pathway in macrophages reduces fatty acid-induced pro-infl ammatory cytokine expression
Obesity and type 2 diabetes are characterized by elevated fatty acid levels, and fatty acids can activate infl ammatory pathways, which have been suggested to contribute to insulin resistance. We and others have shown that macrophage ABCA1 deficiency increases LPS-induced proinfl ammatory cytokine expression. However, the role of the ABCA1 pathway in fatty acid-induced infl ammation in macrophages is unknown. Therefore, we investigated whether ABCA1 defi ciency in macrophages alters fatty acid-induced pro-infl ammatory cytokine expression, which may in turn infl uence insulin resistance. Peritoneal macrophages from ABCA1 Ϫ / Ϫ and WT mice were cholesterol loaded by acetylated LDL treatment and then incubated for 24 h with free fatty acids (250 M palmitate or mixed fatty acids). The expression levels of infl ammatory cytokines were measured by real-time quantitative PCR. As showed in Fig. 7A , ABCA1 defi ciency signifi cantly increased the fatty acid-induced expression of TNF-␣ and IL-6.
To further test whether stimulating the ABCA1 pathway attenuates fatty acid-induced pro-infl ammatory cytokine expression, we pretreated cholesterol-loaded peritoneal were quantifi ed from whole blood using a hematology cell counter, and monocyte subset and BM progenitor cells were quantifi ed using fl ow 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 Ϫ / Ϫ mice. Consistent with previous fi ndings ( 4 ), diet-induced obese BMT-WT mice showed increases in the numbers of circulating monocytes and neutrophils ( Fig. 5A , diet effect). Despite similar body weights as HFHSC diet-fed BMT-WT mice, blood counting indicated that HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice further increased the number of peripheral leukocytes, monocyte counts, neutrophils, and eosinophils, but had similar lymphocytes ( Fig. 5A ). Flow cytometry analysis indicated that there was a signifi cant increase in blood myeloid cells with a balanced increase in the "infl ammatory" Ly6C hi and "patrolling" Ly6C lo monocyte subsets in the HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice compared with HFHSC diet-fed BMT-WT mice ( Fig. 5B-E ).
The phenotype of the HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice suggested a myeloproliferative disorder phenotype, thus we quantifi ed BM hematopoietic stem cells and HSPCs and other MPCs in those mice. Importantly, both myeloid ( Fig. 6A, B ) and MPCs ( Fig. 6C, D ) were signifi cantly increased in HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice as compared with HFHSC diet-fed BMT-WT mice. Moreover, the lin Ϫ Sca+cKit+ population representing HSPCs showed a 2-fold increase in HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ mice ( Fig. 6C, D ). We further performed an in vitro BM proliferation assay and found that HFHSC diet-fed BMT-ABCA1 Ϫ / Ϫ   macrophages with apoA-I for 3 h, washed the cells, and then incubated the cells with free fatty acids for 24 h. As shown in Fig. 7B , pretreating cells with apoA-I, which activates ABCA1-mediated cholesterol effl ux and the anti-infl ammatory pathways, signifi cantly attenuated fatty acid-induced TNF-␣ and II-6 expression in WT but not in ABCA1 Ϫ / Ϫ cells. These fi ndings strongly suggest that activation of the ABCA1 pathway can signifi cantly reduce fatty acid-induced macrophage infl ammation.

DISCUSSION
We demonstrate that deletion of ABCA1 in hematopoietic cells worsens diet-induced hyperglycemia and insulin resistance. Importantly, the worsened insulin action in adipose tissue and liver of those mice occurs in conjunction with increased macrophage accumulation and enhanced infl ammatory cytokine expression in these tissues . In addition, deletion of ABCA1 in hematopoietic cells results in ABCA1 results in an increased macrophage infl ammatory response to LPS ( 7,8,24 ) and free fatty acids ( Fig. 7 ). Human studies also suggest an association between ABCA1 expression and chronic infl ammation ( 32 ). Further, it has been shown that hematopoietic cell-specifi c deletion of ABCA1 promotes hematopoietic stem cell proliferation and monocytosis ( 18,23 ). Taken together, these studies suggest that hematopoietic ABCA1 defi ciency may act as an enhancer of the feed-forward mechanism that promotes diet-induced insulin resistance. We are currently studying to determine whether increasing hematopoietic ABCA1 will weaken this malicious feed-forward mechanism and reduce diet-induced insulin resistance.
ABCA1 Ϫ / Ϫ -BM-derived macrophage mice demonstrated increased macrophage infi ltration, impairment in glucose tolerance, and increased insulin resistance on a HFHSC diet. This suggests that hematopoietic ABCA1 is crucial in preventing macrophage accumulation and insulin resistance in response to dietary fat and cholesterol. Our observations also indicate that loss of hematopoietic ABCA1 and the subsequent increase in macrophage accumulation in the liver and adipose tissue are suffi cient to infl uence whole-body glucose tolerance. However, in contrast to our study, a study by Zhu et al. ( 33 ) found no change in insulin sensitivity in mice lacking macrophage ABCA1, using the myeloid-specifi c M lysozyme promoter. Our BM transplantation studies involve the depletion of ABCA1 in all hematopoietic cells. In contrast, the myeloid-specifi c M lysozyme promoter used to drive Cre expression is restricted to macrophages and neutrophils ( 33 ). This suggests that the increased infl ammation and decreased insulin sensitivity observed in mice receiving ABCA1-null BM is attributable to loss of ABCA1 in other cell types or to loss of ABCA1 in cells in addition to macrophages. We did not observe any change in lymphocytes; instead, we found signifi cantly signifi cantly higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis when challenged with the HFHSC diet. We further demonstrate that ABCA1 defi ciency increases free fatty acid-induced pro-infl ammatory gene expression in macrophages. Our fi ndings are consistent with the important roles of macrophage infl ammation and monocytosis in promoting insulin resistance in dietinduced obesity ( 4,29,30 ). Our results suggest that hematopoietic ABCA1 is an important regulator of monocytosis and macrophage accumulation in diet-induced obesity.
Macrophages play a central role in diet-induced obesity and subsequent development of systemic insulin resistance. Adipose tissue macrophages from lean mice express many genes characteristic of alternatively activated macrophages that may protect adipose tissue from infl ammation; whereas, diet-induced obesity leads to a shift in the activation state to a pro-infl ammatory state contributing to insulin resistance ( 3,30,31 ). There is also a strong association between obesity and leukocytosis, particularly of the myeloid lineage ( 6,28 ). Pioneering studies by others ( 4 ) have shown that MPC proliferation and monocytosis drive macrophage accumulation and insulin resistance in obesity through a feed-forward mechanism, such that infl amed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of infl ammation and associated disease processes ( 4 ). Our results are consistent with this feed-forward mechanism, based on a model in which defi ciency of ABCA1 in macrophages increases tissue macrophage infl ammation, likely by increasing free-fatty acid-induced infl ammatory responses. This induces proliferation of the hyper-responsive BM hematopoietic progenitor cells due to ABCA1 defi ciency in those cells, ultimately resulting in monocytosis, more macrophage accumulation, and insulin resistance. Consistent with this model, we and others have shown that deletion of higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis. These data suggest that deletion of ABCA1 in mature myeloid cells or macrophages is not suffi cient, and that ABCA1 deletion in hematopoietic progenitor cells is required to promote diet-induced insulin resistance. Interestingly, the same divergent results were observed in studies of atherosclerosis between BM transplantation and myeloid-specifi c M lysozyme promoterdriven mice. There was no change in atherosclerosis in mice using myeloid-specifi c M lysozyme promoter, while BM transplantation showed increased atherosclerosis in lethally irradiated recipients of ABCA1-defi cient BM ( 18,19,34,35 ). These studies indicate a critical role for hematopoietic progenitor cell ABCA1 and its associated monocytosis in atherogenesis as well as insulin resistance. In summary, our results demonstrate that knockout of ABCA1 in hematopoietic cells appears to have no apparent impact on adipose and liver macrophage infl ammation in lean mice. However, when mice are made obese and there is a dramatic need to buffer against lipids, ABCA1 is very important for controlling monocytosis, adipose and liver macrophage infl ammation, and, in turn, insulin sensitivity . Thus, ABCA1 provides an important line of defense to prevent the deleterious effects of nutrient overload during obesity. This raises the exciting possibility of raising ABCA1 in hematopoietic-derived cells as a potential target for the therapeutic treatment of insulin resistance as well as atherosclerosis.