Myeloid cell-specific ABCA1 deletion does not worsen insulin resistance in HF diet-induced or genetically obese mouse models
2013; Elsevier BV; Volume: 54; Issue: 10 Linguagem: Inglês
10.1194/jlr.m038943
ISSN1539-7262
AutoresXuewei Zhu, Soonkyu Chung, Xin Bi, Chia‐Chi Chuang, Amanda Brown, Mingxia Liu, Jeongmin Seo, Helen Cuffe, Abraham K. Gebre, Elena Boudyguina, John S. Parks,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoObesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models. Obesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models. In the past decade, obesity-associated chronic low-grade inflammation has been recognized as a major cause of insulin resistance and type 2 diabetes. For example, overexpansion of adipose tissues increases the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) (1Hotamisligil G.S. Arner P. Caro J.F. Atkinson R.L. Spiegelman B.M. Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance.J. Clin. Invest. 1995; 95: 2409-2415Crossref PubMed Scopus (2962) Google Scholar), and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) (2Kanda H. Tateya S. Tamori Y. Kotani K. Hiasa K. Kitazawa R. Kitazawa S. Miyachi H. Maeda S. 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Wacher N.H. et al.Association of the ATP-binding cassette transporter A1 R230C variant with early-onset type 2 diabetes in a Mexican population.Diabetes. 2008; 57: 509-513Crossref PubMed Scopus (79) Google Scholar). Of interest, leukocyte ABCA1 gene expression is associated with fasting glucose concentrations in normoglycemic men, and reduced leukocyte ABCA1 gene expression is associated with type 2 diabetes (21Patel 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, 22Albrecht 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). To date, only one study has directly linked ABCA1 expression to hyperglycemia, reporting that pancreatic β cell-specific ABCA1 deletion results in defective insulin release from islet cells (23Brunham 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). Whether deletion of ABCA1 expression in other cell types, such as macrophages, alters glucose metabolism is unknown. Using myeloid cell (macrophage and neutrophil)-specific ABCA1 knockout (MSKO) mice, we demonstrated that macrophages from MSKO mice have a significant increase in FC and are more responsive to pro-inflammatory stimuli [e.g., lipopolysaccharide (LPS)] in vivo and in vitro compared with wild-type (WT) mice (24Zhu 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). This response was mediated through the TLR and myeloid differentiation primary-response protein 88 (MyD88)-dependent pathway and was independent of alterations in plasma lipid concentrations (24Zhu 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). Hypersensitivity to LPS is most likely due to increased lipid raft content and increased trafficking of TLR4 into plasma membrane lipid rafts in MSKO mouse macrophages (25Zhu X. Owen J.S. Wilson M.D. Li H. Griffiths G.L. Thomas M.J. Hiltbold E.M. Fessler M.B. Parks J.S. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.J. Lipid Res. 2010; 51: 3196-3206Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Furthermore, MSKO mice challenged with the Listeria monocytogenes cleared the bacterium better than WT mice (26Zhu X. Westcott M.M. Bi X. Liu M. Gowdy K.M. Seo J. Cao Q. Gebre A.K. Fessler M.B. Hiltbold E.M. et al.Myeloid cell-specific ABCA1 deletion protects mice from bacterial infection.Circ. Res. 2012; 111: 1398-1409Crossref PubMed Scopus (27) Google Scholar). These studies clearly demonstrated a regulatory role of myeloid ABCA1 in macrophage inflammation and innate immunity. Based on the documented relationship between macrophage inflammation and insulin resistance, we hypothesized that myeloid-specific ABCA1 deficiency may exacerbate obesity-induced chronic inflammation and insulin resistance in mice fed a HF diet or crossed into the leptin-deficient ob/ob background. However, MSKO and WT mice developed obesity, adipose inflammation, and insulin resistance to a similar extent, suggesting that myeloid ABCA1 expression does not significantly worsen obesity-induced chronic inflammation and insulin resistance. WT (ABCA1+/+) and MSKO (ABCA1-M/-M) mice were generated as described previously (24Zhu 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, 25Zhu X. Owen J.S. Wilson M.D. Li H. Griffiths G.L. Thomas M.J. Hiltbold E.M. Fessler M.B. Parks J.S. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.J. Lipid Res. 2010; 51: 3196-3206Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Mice were backcrossed into the C57BL/6 background for six generations before use in these studies and housed in a specific pathogen-free facility with a 12 h light/dark cycle. Experiments were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the experimental protocol was approved by the Wake Forest University Animal Care and Use Committee. At 8 weeks of age, male mice were switched from chow to a HF diet containing 45% of energy as lard and 0.015% cholesterol for 16–24 weeks. The HF diet was made by our institutional diet kitchen (see detailed diet composition in supplementary Table I). Body weight (BW) was measured biweekly. To generate MSKO-ob/ob mice, MSKO mice were first bred with heterozygous ob/ob (ob/+) mice. The resulting double heterozygous MSKO-ob/+ mice were intercrossed to generate ob/+ or MSKO-ob/+ mice, which were then intercrossed to generate ob/ob or MSKO-ob/ob mice, respectively. Ob/ob and MSKO-ob/ob male mice were fed chow for 14–15 weeks for the described studies. Peritoneal macrophages were harvested from mice by flushing the peritoneal cavity with cold PBS. The peritoneal cells were plated in RPMI media containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 1% Nutridoma SP media (Roche Applied Science). After a 2 h incubation, floating cells were removed by washing with PBS and adherent macrophages were used for experiments. Blood samples were taken from tail veins following a 4 h fast during the light cycle (chow phase) before, and at 2–4 week intervals after initiation of HF diet feeding. Plasma cholesterol (Wako) and TGs (Roche) were determined by enzymatic analysis according to the manufacturer's instructions. Plasma glucose levels were measured using a glucometer (Ascensia Contour, Bayer). Plasma insulin levels were measured by ELISA (Crystal Chem, Inc., Downers Grove, IL). For fed and overnight fasting plasma samples, blood was collected by tail vein at 9:00 AM in ad libitum fed mice (fed) or in mice fasted during the dark cycle for 15 h (5:00 PM–8:00 AM). Liver lipids were extracted with chloroform:methanol (2:1) and the extract was used for enzymatic assays (cholesterol, Wako; TG, Roche). Data were normalized to liver protein mass, measured by the Lowry protein assay. Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were done after 12–20 weeks of HF diet consumption or with ob/ob mice at 13–14 weeks of age. Briefly, for GTTs, mice were fasted overnight before intraperitoneal (ip) injection of 1 g glucose/kg BW. Blood was collected before and after injection (0, 15, 30, 60, and 120 min) to measure glucose concentrations using a commercial glucose monitor. One week later, the same groups of mice were used for ip injection of 1.5 U of regular human insulin/kg BW (HF diet-fed mice) or 3 U/kg BW (ob/ob mice) after a 5 h fast. Blood glucose concentrations were measured at 0, 15, 30, 60, and 120 min after injection. Total RNA in peritoneal macrophages and white adipose tissue was extracted using Trizol (Invitrogen) and RNeasy lipid tissue kits (Qiagen), respectively, according to the manufacturer's protocols. cDNA preparation and real-time PCR were conducted as described previously (24Zhu 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). Epididymal fat tissue sections were incubated with the primary antibodies to CD68 (abD Serotec) or cleaved caspase-3 (Cell Signaling), followed by the biotinylated secondary antibody. The staining was visualized using ABC reagent (ABC vector kit; Vector) and DAB substrate chromogen (Dako). The area of fat sections covered by CD68+ or cleaved caspase-3+ cells was measured using Image-Pro software to quantify the percentage of positive staining cells in adipose tissue. Stromal vascular cells were fractioned according to the procedure described by Brown et al. (27Brown J.M. Halvorsen Y.D. Lea-Currie Y.R. Geigerman C. McIntosh M. Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue.J. Nutr. 2001; 131: 2316-2321Crossref PubMed Scopus (124) Google Scholar). Briefly, epididymal fat was minced and enzymatically digested for 45 min with 5 ml/g tissue of collagenase type I (1 mg/ml, Worthington Biochemical Corporation) media. The digestion mixture was filtered through a 100 μm cell strainer and stromal vascular cells were pelleted by centrifugation (3,000 rpm for 10 min). The stromal vascular fraction (SVF) was then used for flow cytometry. After blocking the Fcγ receptor with purified anti-mouse CD16/CD32 antibody (Fcγ receptor III/II; BD Biosciences), SVF cells were incubated at 4°C for 30 min with isotype controls or the following Abs: PE-anti-F4/80 (BM8; BD Pharmingen), PE-Cy7-anti-CD11c (HL3; BD Pharmingen), and Alexa Fluor 647-anti-CD206 (MR5D3; AbD Serotec). Cell fluorescence was measured using a FACSCanto II flow cytometer and data analyzed with FlowJo software. Macrophages or stromal vascular cells isolated from adipose tissue were extracted with isopropanol (including 5α-cholestane as internal standard) at room temperature overnight and analyzed for cholesterol content by gas-liquid chromatography (27Brown J.M. Halvorsen Y.D. Lea-Currie Y.R. Geigerman C. McIntosh M. Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue.J. Nutr. 2001; 131: 2316-2321Crossref PubMed Scopus (124) Google Scholar). Data are presented as the mean ± SEM unless indicated otherwise. Differences were compared with two-tailed Student's t-test or one-way ANOVA using GraphPad Prism software. P < 0.05 was considered statistically significant. MSKO macrophages have increased plasma membrane FC and lipid rafts, resulting in increased response to TLR4 stimulation (LPS) compared with WT cells (24Zhu 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). Saturated fatty acids also activate TLR4 and induce chronic inflammation in the setting of obesity (5Hwang D. Modulation of the expression of cyclooxygenase-2 by fatty acids mediated through toll-like receptor 4-derived signaling pathways.FASEB J. 2001; 15: 2556-2564Crossref PubMed Scopus (125) Google Scholar, 6Lee J.Y. Sohn K.H. Rhee S.H. Hwang D. Saturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through Toll-like receptor 4.J. Biol. Chem. 2001; 276: 16683-16689Abstract Full Text Full Text PDF PubMed Scopus (993) Google Scholar, 7Shi H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. TLR4 links innate immunity and fatty acid-induced insulin resistance.J. Clin. Invest. 2006; 116: 3015-3025Crossref PubMed Scopus (2690) Google Scholar). We hypothesized that increased dietary saturated fatty acids (i.e., HF diet), functioning as TLR4 agonists, might increase inflammation during diet-induced obesity, worsening development of insulin resistance in MSKO versus WT mice. To test this possibility, we first measured cholesterol content in the resident peritoneal macrophages from obese mice. Consistent with our previous findings, macrophages from HF diet-fed MSKO mice (Fig. 1A, C) or chow-fed MSKO-ob/ob mice (Fig. 1B, D) had significantly more FC and CE accumulation compared with their WT counterparts. However, similar inflammatory cytokine gene expression was observed for macrophages from 24 week HF diet-fed WT and MSKO mice (Fig. 1E). Among the examined cytokines, only IL-6 was upregulated in macrophages from chow-fed MSKO-ob/ob versus ob/ob mice (Fig. 1F). In addition, the expression of TLR4 and its lipid-binding coreceptor CD36 (28Stewart C.R. Stuart L.M. Wilkinson K. van Gils J.M. Deng J. Halle A. Rayner K.J. Boyer L. Zhong R. Frazier W.A. et al.CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer.Nat. Immunol. 2010; 11: 155-161Crossref PubMed Scopus (1091) Google Scholar) did not differ between genotypes (Fig. 1G, H). These data suggest that despite significant increases in macrophage cholesterol accumulation resulting from ABCA1 deficiency, HF diet feeding was not sufficient to stimulate pro-inflammatory activation of macrophages in vivo. Obesity-associated low-grade chronic inflammation underlies the pathogenesis of type 2 diabetes and insulin resistance (29Weisberg 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 (7453) Google Scholar, 30Xu 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 (5183) Google Scholar). Macrophage ABCA1 dampens pro-inflammation via downregulating MyD88-dependent TLR signaling (24Zhu 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, 25Zhu X. Owen J.S. Wilson M.D. Li H. Griffiths G.L. Thomas M.J. Hiltbold E.M. Fessler M.B. Parks J.S. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.J. Lipid Res. 2010; 51: 3196-3206Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). We hypothesized that myeloid-specific ABCA1 deficiency may exacerbate chronic inflammation and insulin resistance associated with diet-induced or genetically obese mice. To test this hypothesis, we first fed the WT and MSKO male mice a HF diet containing 45% of energy as lard and 0.015% cholesterol. When challenged with the HF diet, WT and MSKO mice gained similar BW over the 24 week diet feeding period (Fig. 2A). Liver and fat mass were comparable between genotypes after 24 weeks of diet consumption (supplementary Fig. IA, B). We then assessed several key parameters of glucose homeostasis in WT and MSO mice. Blood glucose concentrations after a 4 h fast were comparable between both groups of mice (Fig. 2B). MSKO mice also exhibited similar plasma glucose clearance (GTT) compared with WT mice (Fig. 2C). Consistent with this finding, the ability of insulin to lower blood glucose concentrations during ITTs was equivalent in both groups of mice (Fig. 2D). We next measured plasma insulin concentrations in overnight-fasted and fed mice; again, no differences were observed between genotypes (Fig. 2E, F). Comparable glucose homeostasis between the two genotypes also occurred with prolonged diet feeding (e.g., 12–24 weeks) using different sets of mice. Adding cholesterol (0.2%) to the HF diet did not significantly alter the phenotype (data not shown). Similar outcomes were also observed for HF diet-fed female mice (data not shown). Together, our data suggest that myeloid cell-specific ABCA1 deletion does not affect the development of systemic insulin resistance in the presence of HF diet-induced obesity. Next, we tested our hypothesis using an early-onset genetic obesity mouse model by crossing MSKO mice into the ob/ob background. At 8 and 14 weeks of age, ob/ob and MSKO-ob/ob mice had comparable BWs (Fig. 3A, B). No differences in liver or fat mass were observed between genotypes (supplementary Fig. IC, D). A GTT was performed when mice were 13 weeks old, followed by an ITT the next week. Due to the low yield of the MSKO-ob/ob mice, we combined the data from three sets of mice by normalizing data to baseline values. Similar to the HF diet study, we saw no differences in the GTTs and ITTs for ob/ob and MSKO-ob/ob mice (Fig. 3C, D), indicating comparable systemic insulin resistance. Similar plasma insulin concentrations (Fig. 3E, F) further confirmed that insulin resistance did not differ between genotypes. We next examined the inflammatory status in fat, an important insulin target tissue. We first measured expression of F4/80 and CD68 (macrophage markers) in epididymal fat from HF diet-fed mice (17 weeks). Compared with chow, the HF diet significantly increased F4/80 and CD68 expression in adipose tissue, indicating macrophage infiltration into fat tissue (Fig. 3A). Unexpectedly, MSKO versus WT fat had significantly lower F4/80 expression and a trend toward less CD68 expression. Furthermore, immunohistochemistry staining of macrophages using CD68 antibody revealed that CD68+ cells in MSKO adipose tissues were greatly reduced compared with WT (Fig. 4B), indicating significantly less macrophage infiltration into fat. The comparable level of cleaved caspase-3 in adipose tissues ( supplementary Fig. IIA, B) rules out a major role for apoptosis in the differential accumulation of macrophages between genotypes. Interestingly, this difference was not observed with a longer period of HF diet feeding (24 weeks, supplementary Fig. IIIA). Obesity induces a phenotypic switch from M2-polarized state (alternatively activated) to an M1 (classically activated) pro-inflammatory state in adipose macrophages (31Lumeng C.N. Bodzin J.L. Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization.J. Clin. Invest. 2007; 117: 175-184Crossref PubMed Scopus (3335) Google Scholar). We examined expression of the M1 type of inflammatory cytokine/chemokine and M2 macrophage markers in fat. Feeding the HF diet for 17 weeks significantly induced pro-inflammatory cytokine (TNF-α) or chemokine (MCP-1) expression in adipose tissues compared with chow (Fig. 4C). However, no genotypic differences were observed between the chow-fed or H
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