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HDL and Glut1 inhibition reverse a hypermetabolic state in mouse models of myeloproliferative disorders

HDL and Glut1 inhibition reverse a hypermetabolic state in mouse models of myeloproliferative... A r t i c l e HDL and Glut1 inhibition reverse a hypermetabolic state in mouse models of myeloproliferative disorders 1 2,3 4,5 Emmanuel L. Gautier, Marit Westerterp, Neha Bhagwat, 2 4,5 4,5 6 Serge Cremers, Alan Shih, Omar Abdel-Wahab, Dieter Lütjohann, 1 4,5 2 Gwendalyn J. Randolph, Ross L. Levine, Alan R. Tall, 2,7 and Laurent Yvan-Charvet Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 Department of Medicine, Division of Molecular Medicine, Columbia University, New York, NY 10032 Department of Medical Biochemistry, Academic Medical Center of Amsterdam, University of Amsterdam, 1105 Amsterdam, Netherlands 4 5 Human Oncology and Pathogenesis Program; and Leukemia Service, Department of Medicine; Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Institute of Clinical Chemistry and Clinical Pharmacology, University Clinic Bonn, 53127 Bonn, Germany Institut National de la Santé et de la Recherche Médicale U1065, Centre Mediterraneen de Medecine Moleculaire (C3M), Avenir, 06204 Nice, France A high metabolic rate in myeloproliferative disorders is a common complication of neoplasms, but the underlying mechanisms are incompletely understood. Using three different mouse models of myeloproliferative disorders, including mice with defective cholesterol efflux pathways and two models based on expression of human leukemia dis - ease alleles, we uncovered a mechanism by which proliferating and inflammatory myeloid cells take up and oxidize glucose during the feeding period, contributing to energy dissipa- tion and subsequent loss of adipose mass. In vivo, lentiviral inhibition of Glut1 by shRNA prevented myeloproliferation and adipose tissue loss in mice with defective cholesterol efflux pathway in leukocytes. Thus, Glut1 was necessary to sustain proliferation and poten - tially divert glucose from fat storage. We also showed that overexpression of the human ApoA-I transgene to raise high-density lipoprotein (HDL) levels decreased Glut1 expression, dampened myeloproliferation, and prevented fat loss. These experiments suggest that inhibition of Glut-1 and HDL cholesterol–raising therapies could provide novel therapeutic approaches to treat the energy imbalance observed in myeloproliferative disorders. Chronic inflammatory diseases such as chronic metabolism, and enhanced energy expenditure CORRESPONDENCE Laurent Yvan-Charvet: infection, cancer, and heart failure are often in this state of chronic hypermetabolism is not [email protected] associated with adipose tissue loss (Delano and fully understood. 14 Moldawer, 2006). Adipose tissue loss in the Among chronic inflammatory diseases, he - Abbreviations used: 2-[ C]-DG, 2-[ C]-deoxyglucose; 2-NBDG, setting of chronic inflammatory diseases could matological malignancies such as leukemias and 2-[N-(7-nitrobenz-2-oxa-1, ultimately represent a major health problem myeloproliferative disorders are also associated 3-diazol-4-yl)amino]-2-deoxy- because of associated comorbidities such as with adipose tissue loss (Dingli et al., 2004). In d-glucose; ACL, ATP-citrate weakness, fatigue, and impaired immunity. Loss humans, among the most common activating lyase; HDL, high-density lipopro- tein; HSC, hematopoietic stem of adipose tissue is ultimately caused by an mutations in myeloid malignancies are muta- cell; PDP, pyruvate dehydrogenase imbalance between food intake and energy tions in the FMS-like tyrosine kinase 3 (Flt3) phosphoserine phosphatase; expenditure. Although food intake is often re- gene, which occur in 30% of cases of acute PFK, phosphofructokinase; RQ, respiratory quotient; SDH, duced in patients with chronic inflammatory myeloid leukemia as well as mutations in the succinate dehydrogenase; TCA, diseases, these patients exhibit a persistent state tricarboxylic acid; TMRE, © 2013 Gautier et al. This article is distributed under the terms of an Attribution– of inappropriately high metabolic rate, and tetramethylrhodamine ethyl ester. Noncommercial–Share Alike–No Mirror Sites license for the first six months after this substantially contributes to their adipose the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share loss (Delano and Moldawer, 2006). However, Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ by-nc-sa/3.0/). the link between chronic inflammation, tissue The Rockefeller University Press $30.00 J. Exp. Med. 2013 Vol. 210 No. 2 339-353 www.jem.org/cgi/doi/10.1084/jem.20121357 The Journal of Experimental Medicine thrombopoietin receptor (proto-oncogene c-Mpl) observed controls by indirect calorimetry. No significant changes in in 5–10% of patients with myelofibrosis. These mutations daily food intake, energy expenditure, or locomotor activity confer a fully penetrant myeloproliferative disorder in mice were observed in these mice (Table 1). Similar fat and cho- (Kelly et al., 2002; Pikman et al., 2006), providing a useful lesterol absorption and bile acid excretion were also observed / / / tool for studying the mechanism of adipose tissue loss associ- in Abca1 Abcg1 mice (not depicted). However, Abca1 / ated with myeloproliferative disorders. Abcg1 BM transplanted mice exhibited a higher respira- We have recently shown that mice lacking the cholesterol tory quotient (RQ) during the feeding period (dark phase; eu ffl x transporters ABCA1 and ABCG1 develop massive ex - Fig. 1 D), reflecting an 20% increase in glucose oxidation pansion and proliferation of hematopoietic stem and progeni- (Fig. 1 E) and exhibited hypoglycemia (Table 1). Injection i.p. tor cells, marked monocytosis, neutrophilia, and inlfi tration of a glucose bolus also revealed faster glucose utilization / / of various organs with myeloid cells, resembling a classical in Abca1 Abcg1 BM transplanted mice compared with myeloproliferative disorder (Hansson and Björkholm, 2010; WT transplanted controls (Fig. 1 F). We next examined the Yvan-Charvet et al., 2010). As we noticed a dramatic loss of uptake of the radiolabeled d-glucose analogue 2-[ C]- / / 14 / / adipose tissue in Abca1 Abcg1 BM chimeras, we hy- deoxyglucose (2-[ C]-DG) in Abca1 Abcg1 BM trans- pothesized that the myeloproliferative disorder of these mice planted mice in the fed state. No significant changes were might be responsible for their wasting syndrome and pro- observed in the total uptake of 2-[ C]-DG in the skeletal ceeded to investigate the underlying mechanisms. muscle and brain (Fig. 1 G), whereas, in line with their re- duced fat mass, total 2-[ C]-DG incorporation was reduced RESULTS in their adipose tissue (Fig. 1 G). In contrast, the total uptake Lack of ABCA1 and ABCG1 in hematopoietic cells of 2-[ C]-DG was increased by two- to threefold in the promotes adipose tissue atrophy heart, lung, spleen, and BM of these mice (Fig. 1 G) and by Determination of the fat and muscle mass of irradiated WT 1.5-fold when expressed as the rate constant for net tissue / / 14 recipients transplanted with Abca1 Abcg1 BM cells fed uptake of 2-[ C]-DG uptake (Fig. 1 H). Thus, we showed / / a chow diet revealed not only absence of adipose tissue that Abca1 Abcg1 BM transplanted mice have increased growth but also reduced epididymal fat mass at 24 wk after propensity to clear and use glucose. reconstitution compared with controls (Fig. 1 A). Gastroc- nemius muscle loss was also observed 30 wk after reconstitu- Inflammation diverts glucose from fat storage by promoting / / tion in these mice (Fig. 1 B). Subcutaneous and retroperitoneal adipose tissue insulin resistance in Abca1 Abcg1 adipose depots were also decreased by more than threefold at BM transplanted mice / / / 24 wk after reconstitution in Abca1 Abcg1 BM chime- Consistent with their myeloproliferative disorder, Abca1 / ras, consistent with their reduced plasma leptin levels (Table 1). Abcg1 BM chimeras exhibited a chronic ina fl mmatory state To test whether the adipose tissue atrophy of these mice was as reflected by a threefold increase in plasma TNF levels a direct consequence of their defective hematopoietic com- (Table 1). Microscopic examination of the adipose tissue of / / partment and myeloproliferative syndrome (Yvan-Charvet Abca1 Abcg1 BM transplanted mice revealed an in- et al., 2010), we generated hematopoietic stem cell (HSC)– creased inflammatory infiltrate reflected by crown-like struc - specific chimeric animals by transplanting lethally irradi - tures (Fig. 2 A). Because infiltrated leukocytes are a hallmark / / ated WT recipients with purified WT or Abca1 Abcg1 of insulin resistance in adipose tissue (Lumeng and Saltiel,  + + HSCs (Lin Sca cKit , LSK fraction). Mice transplanted 2011; Odegaard and Chawla, 2011), we wondered whether / / / / with Abca1 Abcg1 LSK cells reproduced the three- the adipose tissue infiltration observed in Abca1 Abcg1 hi hi fold increase in the Gr-1 /CD11b blood myeloid popu- BM chimeras would promote local insulin resistance and / / lation observed in Abca1 Abcg1 BM transplanted mice contribute to their reduced adipose tissue glucose uptake. To (Yvan-Charvet et al., 2010) and exhibited a twofold re- address this question, a bolus of insulin was injected into WT / / duction in fat mass 12 wk after reconstitution (Fig. 1 A). and Abca1 Abcg1 BM transplanted mice, and hallmarks Similar findings were observed in mice with specific knock - of insulin signaling (AKT phosphorylation and GLUT4 ad- out of these transporters in the hematopoietic lineage dressing to the cell membrane) were assessed. Western blot fl/fl fl/fl (Mx1-Cre Abca1 Abcg1 ) 10 wk after injections of analysis revealed reduced GLUT4 translocation from the PolyI:C to excise the STOP codon that prevents the expres- low-density microsome fraction to the plasma membrane in / / sion of the cre recombinase (Fig. 1 C). Together, these obser- response to insulin in the adipose tissue of Abca1 Abcg1 vations revealed that the expansion of adipose tissue is BM transplanted mice (Fig. 2 B). This was associated with severely compromised in mice lacking ABCA1 and ABCG1 reduced AKT phosphorylation, suggesting insulin resistance in their hematopoietic system. in adipocytes is secondary to local inflammatory cell infil - tration (Fig. 2 B). Accordingly, microscopic analysis of iso- ABCA1 and ABCG1 deficiency in leukocytes lated adipocytes revealed smaller adipocytes in WT recipients / / impacts glucose homeostasis transplanted with Abca1 Abcg1 BM cells (Fig. 2 A and We next compared the metabolic characteristics of chow-fed Table 1), whereas adipocyte cell numbers were comparable / / + Abca1 Abcg1 BM transplanted mice and their respective with controls (Table 1). Finally, depletion of Ly6C/G 340 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e / / Figure 1. Adipose tissue atrophy and enhanced glucose utilization in Abca1 Abcg1 BM chimeras. (A) Epididymal adipose tissue of WT / / / /  + + recipient mice transplanted with WT or Abca1 Abcg1 BM cells or WT and Abca1 Abcg1 HSC (Lin Sca cKit , LSK fraction) transplanted mice. (B) Gastrocnemius skeletal muscle mass of these mice. (C) Epididymal fat mass in mice with specific knockout of these transporters in the hematopoietic fl/fl fl/fl lineage (Mx1-Cre Abca1 Abcg1 ) 10 wk after three injections of 15 mg/kg PolyI:C to excise the STOP codon. (D and E) RQ (D) and glucose oxidation / / efc fi iency (E) measured by indirect calorimetry in chow-fed WT and Abca1 Abcg1 BM transplanted (BMT) mice 10 wk after reconstitution. (F) Glucose / / tolerance test was performed i.p. on chow-fed WT and Abca1 Abcg1 BM transplanted mice 12 wk after reconstitution. Blood glucose con- centrations were measured at the indicate time points. (G and H) Tissue uptake (G) and rate constant of 2-[ C]-DG (H) in 24-wk-old chow-fed WT and / / Abca1 Abcg1 BM transplanted mice at the end of the study period (40 min after i.v. injection of the radiolabeled tracer). All results are means ± SEM and are representative of an experiment of five to seven animals per group. *, P < 0.05 versus WT. JEM Vol. 210, No. 2 341 Table 1. Effect of leukocyte ABCA1 and ABCG1 deficiencies on body weight, plasma leptin levels, subcutaneous and retroperitoneal adipose depots, plasma glucose, insulin and TNF levels, epididymal adipose tissue cellularity, and energy metabolism Metabolic parameters BM transplantation / / WT Abca1 Abcg1 Body weight (g) 27.9 ± 0.9 27.4 ± 0.6 Plasma Leptin (ng/ml) 1.95 ± 0.4 0.7 ± 0.2* Subcutaneous fat mass (g) 0.52 ± 0.12 0.15 ± 0.03* Retroperitoneal fat mass (g) 0.28 ± 0.04* 0.09 ± 0.08* Plasma glucose (g/liter) 2.1 ± 0.1 1.7 ± 0.1* Plasma Insulin (ng/ml) 7.9 ± 0.8 6.1 ± 1.1 Plasma TNF (pg/ml) 3.1 ± 0.5 7.4 ± 0.6* Fat cell number (×10 ) 4.8 ± 0.9 4.6 ± 0.8 Fat cell weight (ng) 119 ± 14* 36 ± 5* Food intake (g/day) 3.28 ± 0.14 3.33 ± 0.13 0.75 Food intake (g/day/g ) 0.29 ± 0.01 0.29 ± 0.01 0.75 Energy expenditure, EE (W/kg ) 6.68 ± 0.25 7.11 ± 0.18 Locomotor activity (counts/14 min) 2,589 ± 544 2,560 ± 499 Values are mean ± SEM (n = 5 per group). *, P < 0.05 versus controls. myeloid cells with the anti–granulocyte receptor-1 (Gr-1) we next assessed the mRNA expression levels of several antibody RB6-8C5 partially reversed the reduced glucose members of this family, namely Glut1, Glut2, Glut3, Glut4, / / / / uptake in the adipose tissue of Abca1 Abcg1 mice (not and Glut6. Abca1 Abcg1 BM cells exhibited a two- depicted). Together, these results indicate that myeloprolif- fold up-regulation of the glucose transporter Glut1 mRNA eration and associated adipose tissue infiltration compromise expression, which was also the most highly expressed Glut adipose tissue function and likely contribute to the loss of fat member in leukocytes (Fig. 3 C). Flow cytometry analysis / / in Abca1 Abcg1 BM transplanted mice. Moreover, these showed a 30% increase in the cell surface expression of / / / / + findings revealed that Abca1 Abcg1 BM chimeras can Glut1 in vivo in Abca1 Abcg1 CD45 leukocytes (not efficiently clear glucose despite being insulin resistant. depicted), including monocytes, neutrophils, and lympho- cytes (Fig. 3 D), and this correlated with increased mito- chondrial membrane potential (Fig. 3 E). Together, these Glucose consumption by proliferating myeloid cells findings show that leukocytes have a major role in glucose contributes to whole body glucose dissipation / / / / uptake in Abca1 Abcg1 BM transplanted mice exhib- in Abca1 Abcg1 BM transplanted mice iting a myeloproliferative syndrome. This resulted in en- Although local inflammation promoted adipose tissue in - hanced whole body glucose oxidation that could explain the sulin resistance, this could not explain the enhanced glucose increased energy dissipation in these mice. uptake by noninsulin-sensitive tissues (Fig. 1 G) contrib- uting to enhanced whole body glucose oxidation during the feeding period (Fig. 1 E). Because these tissues exhibit Signaling via the IL-3/GM-CSF receptor common -subunit / / massive myeloid infiltration in Abca1 Abcg1 BM trans- enhances the expression of Glut1 and glycolytic enzymes / / planted mice (Yvan-Charvet et al., 2007; Out et al., 2008), in proliferating Abca1 Abcg1 leukocytes / we next investigated the uptake of glucose by Abca1 We next set out to better understand the mechanism leading / / / Abcg1 leukocytes. To estimate glucose utilization by flow to increased glucose uptake in Abca1 Abcg1 leukocytes. cytometry, we used the fluorescent d-glucose analogue Up-regulation of Glut-1 by oncogenes such as Ras or Src has 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy- been reported (Flier et al., 1987), and we recently showed / / d-glucose (2-NBDG) as a tool that reflects the glucose enhanced Ras-Erk signaling in Abca1 Abcg1 BM cells bound to the cells and its uptake. Leukocytes isolated from at basal state and in response to the hematopoietic growth / / Abca1 Abcg1 BM revealed a significant 30% increase factors IL-3 and GM-CSF (Yvan-Charvet et al., 2010). in 2-NBDG staining (Fig. 3 A). This increase was massive Therefore, we investigated the expression of Glut1 in re- in monocytes and neutrophils when the increased cell num- sponse to IL-3 in BM leukocytes. As shown in Fig. 3 F, Glut1 ber was taken into account (Fig. 3 B; Yvan-Charvet et al., mRNA levels were increased upon stimulation with IL-3 in 14 / / 2010), accounting for the total uptake of 2-[ C]-DG in WT BM cells and were further increased in Abca1 Abcg1 the BM (Fig. 1 G). As glucose uptake depends on the glu- cells. Inhibition of the Ras signaling pathway using a farnesyl cose transporter (Glut) family (Herman and Kahn, 2006), transferase inhibitor, known to prevent the anchorage of Ras 342 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e in the plasma membrane and to inhibit the proliferation of / / Abca1 Abcg1 BM cells (Yvan-Charvet et al., 2010), normalized Glut1 mRNA expression to WT levels (Fig. 3 F). Further characterization of signaling pathways downstream of the IL-3/GM-CSF receptor common -chain (Chang et al., 2003) showed that the phosphoinositide 3-kinase in- hibitor LY294002 prevented IL-3–induced Glut1 mRNA / / expression in Abca1 Abcg1 leukocytes (Fig. 3 F), similar to the ee ff ct of this inhibitor on the proliferation of these cells (Yvan-Charvet et al., 2010). Analysis of the rates of [ C]glucose oxidation revealed that in basal and IL-3–stimulated cond- i / / tions, Abca1 Abcg1 leukocytes exhibited a higher glyco- lytic rate (Fig. 3 G). This ee ff ct was inhibited by removal of membrane cholesterol by cyclodextrin (Fig. 3 G), consistent with the cholesterol-dependent regulation of IL-3R signal- ing (Yvan-Charvet et al., 2010). Analysis of genes of the gly- colytic and lipid synthetic pathways also revealed up-regulation of hexokinase 2 (Hk2), phosphofructokinase (PFK), and / / ATP-citrate lyase (ACL) mRNAs in Abca1 Abcg1 leu- kocytes that was dependent on the IL-3/GM-CSF receptor common -chain signaling pathway (Fig. 3 H). Fumarase and succinate dehydrogenase (SDH) mRNA expression was / / also increased in response to IL-3 in Abca1 Abcg1 leu- kocytes (Fig. 3 H). Together, our results point to the IL-3/ GM-CSF receptor common -chain/GLUT1 axis as a major determinant of the increased glucose uptake observed in / / Abca1 Abcg1 leukocytes. Metabolic profiling reveals increased glycolysis and oxidative phosphorylation in proliferating / / Abca1 Abcg1 leukocytes Further quantification of glycolytic metabolites by LC-MS showed higher glucose 6-phosphate/fructose 6-phosphate (G6P+F6P) and fructose 1,6-diphosphate (F1,6BP) levels Figure 2. Inlfi trated leukocytes modulate adipose tissue insulin sen - / / / / sitivity in Abca1 Abcg1 BM chimeras. (A) Parafn fi -embedded serial in basal and IL-3–stimulated Abca1 Abcg1 leukocytes / / sections obtained from the adipose tissue of WT and Abca1 Abcg1 (Fig. 4 A). Increased citric acid cycle metabolites (citrate, BM transplanted (BMT) mice fed a chow diet. Representative H&E stain- isocitrate, succinate, fumarate, and malate) and related mito- / / ing revealed an extensive myeloid cell infiltrate in Abca1 Abcg1 chondrial products (GTP and FAD) were also observed in BM transplanted mice compared with controls. Micrographs of iso- / / Abca1 Abcg1 leukocytes under basal and/or IL-3–stim- lated epididymal adipose cells confirmed reduced adipose cell size in ulated conditions (Fig. 4, B and C). Consistent with these / / Abca1 Abcg1 BM transplanted mice. Data representative of four to findings, a higher ATP to ADP ratio was observed in prolif - six animals per group are shown. Bars, 1 mm. (B) Western blot analysis of / / erating Abca1 Abcg1 leukocytes (Fig. 4 D). This was as- plasma membrane (PM) and low-density microsome (LDM) Glut4, phos- / / sociated with increased mitochondrial membrane potential pho-Akt, and total Akt in the adipose tissue of WT and Abca1 Abcg1 BM transplanted mice after acute i.p. injection of an insulin bolus. Quan- measured using a fluorescent tetramethylrhodamine ethyl titative results were obtained from two independent experiments. Values ester (TMRE) dye (not depicted) and increased SDH activity are mean ± SEM and expressed as percent expression of PM Glut4 over / / in Abca1 Abcg1 leukocytes (Fig. 4 E). HDM fraction or as arbitrary units (a.u.). *, P < 0.05 versus insulin-injected WT controls; , P < 0.05 versus saline-injected mice. / / The increased glycolysis in Abca1 Abcg1 leukocytes is required for proliferation / / To test whether high levels of glycolysis are required for not galactose enhanced the proliferation of Abca1 Abcg1 / / Abca1 Abcg1 leukocyte proliferation, we next cultured leukocytes to the level of high-glucose DMEM (DMEM cells with media containing galactose or glucose. Galactose rich), pointing to a prominent role of the glycolysis pathway enters glycolysis through the Leloir pathway, which occurs at in cell growth. However, a potential contribution of the pen- / / a significantly lower rate than glucose entry into glycolysis tose phosphate pathway to Abca1 Abcg1 leukocyte (Bustamante and Pedersen, 1977). Fig. 4 F shows that under proliferation could not be completely excluded from this glucose-free DMEM (DMEM poor), addition of glucose but experiment. We next challenged the mitochondria with either JEM Vol. 210, No. 2 343 / / Figure 3. Enhanced glycolytic activity in Abca1 Abcg1 leukocytes. (A) Ex vivo characterization of the glucose binding and/or uptake in WT / / +  + + and Abca1 Abcg1 BM leukocyte subpopulations (i.e., CD115 monocytes, CD115 GrI neutrophils, and TCRb lymphocytes) using a fluorescent d -glucose analogue (2-NBDG). (B) Glucose uptake normalized by the amount of BM leukocytes. (C) mRNA expression of glucose transporters (Gluts) in / / / / freshly isolated WT and Abca1 Abcg1 BM cells. (D) Cell surface expression of Glut1 was also quantified in WT and Abca1 Abcg1 BM neutro- phils, monocytes, and lymphocytes. (E) Mitochondrial membrane potential measured by fluorescent TMRE dye. All results are means ± SEM and are repre - sentative of two independent experiments (n = 5–6 animals per groups). *, P < 0.05 versus controls. MFI, mean fluorescence intensity. (F) Glut1 expression after BM cells were treated for 72 h with the indicated growth factors and in the presence or absence of 1 µM farnesyl transferase inhibitor (FTI) or 10 µM PI3K inhibitor (LY294002). Values were normalized to ribosomal 18S. (G) Effect of IL-3 treatment and cholesterol depletion by cyclodextrin (CD) on 14 / / [ C]glucose conversion into CO in WT and Abca1 Abcg1 BM cells. (H) mRNA expression of Hk2, PFK isoform p (PFKp), ACL, fumarase, and SDH sub- / / unit b (SDHb) in WT and Abca1 Abcg1 BM-derived cells untreated or treated for 72 h with IL-3. Values were normalized to ribosomal 18S. Results are means ± SEM of cultures from three independent mice. *, P < 0.05 versus WT controls; , P < 0.05 versus untreated condition. branched-chain amino acids (i.e., valine, leucine, and isoleu- leukocytes, suggesting the requirement of the pyruvate entry cine) or with cysteamine to fuel the tricarboxylic acid (TCA) into the TCA cycle for proliferation (Fig. 4 G). We next used cycle with acetyl-coA or coA, respectively, but these treat- the phosphoserine phosphatase inhibitor DL-AP3 (Hawkinson / / ments did not increase the proliferation of Abca1 Abcg1 et al., 1996), which blocks the rate-limiting step of the serine 344 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e biosynthetic pathway (Lunt and Vander Heiden, 2011) but responses (Fukuzumi et al., 1996; Gamelli et al., 1996). Con- also the pyruvate dehydrogenase phosphoserine phosphatases sistent with the previously observed enhancement in Toll-like / / (PDPs) known to switch metabolic flux from glycolysis to - receptor signaling in Abca1 Abcg1 macrophages (Yvan- ward oxidative phosphorylation. This treatment prevented Charvet et al., 2008), analysis of the rates of [ C]glucose / / the enhanced proliferation of Abca1 Abcg1 leukocytes oxidation in vitro in BM-derived macrophages revealed that / / (Fig. 4 G). Because inhibition of serine palmitoyltransferase in basal and LPS-stimulated conditions, Abca1 Abcg1 by myriocin, downstream of the serine biosynthetic pathway, macrophages exhibited a higher glucose consumption that was / / proportionately inhibited both WT and Abca1 Abcg1 associated with increased Glut1 mRNA levels (not depicted). leukocytes (Fig. 4 G), this suggested that the ee ff ct of DL-AP3 Interestingly, Glut1 inhibition by Fasentin reduced the ina fl m - / / was most likely related to inhibition of PDPs. Inhibition matory response induced by LPS in Abca1 Abcg1 mac- of diglyceride acyltransferase also proportionately inhibited rophages (Fig. 5 C). Although the mRNA levels of Hk2 / / both WT and Abca1 Abcg1 leukocyte proliferation and glucose-6-phosphate dehydrogenase followed the in - (Fig. 4 G), suggesting that despite enhanced lipid synthesis flammatory pattern (Fig. 5 D), glycolytic genes such as PFK (Fig. 5 A), neither ceramide or triglyceride biosynthesis and ACL were barely affected by LPS in either WT or / / was the limiting step for the proliferation of these cells. Abca1 Abcg1 macrophages (Fig. 5 D). This contrasted / / with proliferating Abca1 Abcg1 leukocytes (Fig. 4 I). / / / / Proliferating Abca1 Abcg1 leukocytes exhibit Thus, Abca1 Abcg1 macrophages could contribute enhanced mitochondrial metabolism not only to the enhanced glucose oxidation observed in / / To better understand the contribution of the mitochondrial Abca1 Abcg1 BM transplanted mice (Fig. 1 E), but / / metabolism, we next assessed the ability of Abca1 Abcg1 also to the local adipose tissue insulin resistance mediated by leukocytes to proliferate after treatment with membrane- enhanced inflammatory response (Fig. 2 B). Together, these permeable antioxidants. Although glutathione monoethyl findings revealed that Glut1 controls both the proliferative / / ester partially reduced the proliferation of all cells, tempol and inflammatory status of Abca1 Abcg1 leukocytes. / / abrogated the enhanced proliferation of Abca1 Abcg1 leukocytes (Fig. 4 H). Recently, Samudio et al. (2010) pro- Both Glut1 inhibition and ApoA-I overexpression prevent / / posed that leukemia cells uncouple fatty acid oxidation from fat loss in Abca1 Abcg1 BM transplanted mice ATP synthesis and rely on de novo fatty acid synthesis to sup- We next explored the in vivo relevance of reducing the prolif- port fatty acid oxidation. Consistent with this observation, eration and ina fl mmatory status of myeloid cells through Glut1 / / pharmacological inhibition of carnitine palmitoyltransferase I inhibition on the adipose tissue loss of Abca1 Abcg1 BM / / (CPT-1) with etomoxir prevented the hyperproliferative chimeras. Mice that received WT or Abca1 Abcg1 BM / / response of Abca1 Abcg1 leukocytes (Fig. 4 H). Finally, transduced with lentiviruses encoding Glut1 shRNA exhib- carnitine supplementation of the cells to promote mito- ited a 1.6-fold reduction in Glut1 mRNA expression (not chondrial efflux of excess acetyl moieties from both glu - depicted) and a 20–30% reduction in the cell surface expres - cose and fat oxidation (Muoio et al., 2012) prevented the sion of Glut1 in their BM cells 7 wk after reconstitution, and / / higher proliferation rate of Abca1 Abcg1 leukocytes this was sufficient to normalize the Glut1 cell surface expres - / / (Fig. 4 H), providing additional evidence that the mito- sion to WT levels in Abca1 Abcg1 BM transplanted chondrial metabolism was responsible for the enhanced mice (Fig. 5 E). This was associated with normalization of / / / / proliferation in Abca1 Abcg1 leukocytes. Together, the 2-NBDG uptake in Abca1 Abcg1 BM leukocytes / / these findings showed that Abca1 Abcg1 BM cells di- (Fig. 5 F) and with normalization of the leukocyte counts in rected the available glucose toward oxidative phosphory- these mice (Fig. 5 G). Remarkably, the adipose tissue loss of / / lation (Fig. 4 I), which could explain the enhanced glucose mice transplanted with Abca1 Abcg1 BM was rescued / / oxidation observed in Abca1 Abcg1 BM transplanted by transduction with Glut1 shRNA lentiviral particles (Fig. 5 H). mice (Fig. 1 E). We previously reported that the myeloproliferative syndrome / / and inflammatory phenotype of Abca1 Abcg1 BM chi- Glut1 inhibition prevents both IL-3–mediated meras was reversed by overexpression of the human apoA-I Tg myeloid proliferation and TLR4-mediated transgene (ApoA-I ; Yvan-Charvet et al., 2008, 2010). We macrophage inflammatory response now show that overexpression of the human apoA-I trans- / / The increased glucose conversion into lipids at basal or gene also reversed fat loss in Abca1 Abcg1 BM trans- / / after IL-3 activation in Abca1 Abcg1 leukocytes was planted mice (Fig. 5 I). Additionally, the apoA-I transgene / / abolished by Fasentin, a Glut1 inhibitor (Fig. 5 A; Wood normalized Glut1 cell surface expression in Abca1 Abcg1 et al., 2008), as was the proliferation of these cells (Fig. 5 B). BM cells (Fig. 5 J). Together, our results suggest that suppres- Together, these findings suggest that IL-3R  subunit sig- sion of Glut1 in myeloproliferative disorders may represent a naling enhances the Glut1-dependent glucose uptake of novel approach not only to treat leukocytosis but also associ- / / Abca1 Abcg1 leukocytes to promote their proliferation. ated adipose tissue loss. Additionally, we now show that the / / Enhanced glucose consumption and Glut1 expression were rescue of the myeloproliferative disease of Abca1 Abcg1 previously observed in macrophages during inflammatory BM chimeras by overexpression of the human apoA-I transgene JEM Vol. 210, No. 2 345 / / Figure 4. The IL-3R–dependent proliferation of Abca1 Abcg1 leukocytes is driven by increased mitochondrial metabolism. (A–D) Effect of IL-3 treatment on glycolytic metabolites (glucose 6-phosphate/fructose 6-phosphate and fructose 1,6-diphosphate; A), citric acid metabolites / / (B), related mitochondrial products (GTP and FAD; C), and ATP/ADP ratio (D) in WT and Abca1 Abcg1 BM cells determined by LC-MS. (E) SDH activity in these cells. Results are means ± SEM of cultures from three independent mice. *, P < 0.05 versus WT controls; , P < 0.05 versus treat- / / ment. (F) WT and Abca1 Abcg1 BM cells were grown for 48 h in 20 mM glucose DMEM (DMEM rich) or low-glucose DMEM (DMEM poor) supplemented with 20 mM glucose or 20 mM galactose in the presence of IL-3. Proliferation rates were determined after 2-h [ H]thymidine pulse labeling. (G and H) BM cells were grown for 48 h in liquid culture containing 10% FBS IMDM in the presence of the indicated chemical compounds and IL-3. Proliferation rates were determined after 2-h [ H]thymidine pulse labeling. Results are means ± SEM of an experiment performed in triplicate. *, P < 0.05 versus WT controls; , P < 0.05 versus untreated condition. BCAA, branched-chain amino acids; DGATi, diglyceride acyltransferase inhibitor. 346 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e (Yvan-Charvet et al., 2010) was in part associated with re- hallmark of chronic inflammatory diseases such as chronic duced Glut1 levels on leukocytes and prevented their adi - infection, cancer, and heart failure (Delano and Moldawer, pose loss. 2006), the underlying mechanisms remain poorly under- stood, leading to a lack of effective therapy. Most of the ApoA-I overexpression prevents the increased glucose emerging therapeutic approaches to prevent adipose tissue oxidation and adipose tissue atrophy in Flt3-ITD and loss focus on reducing the elevated circulating cytokine levels Mpl-W515L mutant–mediated myeloproliferative disorders (Argilés et al., 2011) in part because of their action on insulin As altered high-density lipoprotein (HDL) cholesterol ho- resistance and fat mobilization (Delano and Moldawer, 2006; meostasis has been previously observed in myeloprolifera- Das et al., 2011). However, traditional antiinflammatory ap - tive disorders (Fiorenza et al., 2000; Westerterp et al., proaches such as COX-2 inhibitors showed for instance lim- 2012), we further tested whether the increased glucose ited effectiveness in reversing the metabolic abnormalities oxidation and associated adipose tissue atrophy observed seen in cancer patients (Kumar et al., 2010). Thus, there is a / / in Abca1 Abcg1 BM transplanted mice could be ob- crucial need to better understand the mechanisms of adipose served as a more general phenotype in other mouse models of loss in this state of chronic hypermetabolism. Using three dif- myeloproliferative disorders and whether overexpression of ferent mouse models of myeloproliferative disorders, includ - the human apoA-I transgene would still be an efficient thera - ing two models based on expression of human leukemia peutic in these models. The Flt3-ITD and Mpl-W515L al- disease alleles (Flt3-ITD and Mpl-W515L), we uncovered a leles have been identified in patients with acute myeloid glucose steal mechanism by which proliferating and inflam - leukemia and myelofibrosis, respectively, and confer a fully matory myeloid cells take up and oxidize glucose during the penetrant myeloproliferative disorder in mice (Kelly et al., feeding period, contributing to energy dissipation and sub - 2002; Pikman et al., 2006). Accordingly, mice that received sequent loss of adipose mass. This reflected in part increased BM transduced with retroviruses encoding Flt3-ITD or Mpl- numbers of proliferating myeloid cells and tissues infiltrated W515L exhibited massive myeloid expansion compared with with inflammatory myeloid cells. control retrovirus (Fig. 6, A and B), an effect which was par - Although only partially understood, there is a relationship tially reversed by overexpression of the human ApoA-I trans- between leukemia-causing genes and cellular energy metabo- gene (Fig. 6, A and B). We also observed epididymal fat lism as the survival and proliferation of leukemic cells may re- mass atrophy and reduced plasma leptin levels in both models quire glucose for de novo lipid biosynthesis (DeBerardinis (Fig. 6 C and not depicted, respectively). Massive leukocyte et al., 2008; Lunt and Vander Heiden, 2011). The increased / / infiltration was also observed in their adipose tissue (not de - glucose utilization in hyperproliferating Abca1 Abcg1 / / picted). Similar to Abca1 Abcg1 BM transplanted mice, BM myeloid cells resulted in part from a Ras-dependent up- these features were associated with an increased RQ during regulation of Glut1 expression in response to enhanced IL-3 the dark phase (Fig. 6, D and E), reflecting an 20% increase signaling (Flier et al., 1987; Yvan-Charvet et al., 2010). Simi- in glucose oxidation during this period (Fig. 6 F). Interest- larly, Mpl and Flt3 are receptor tyrosine kinases coupled to Ras ingly, Mpl-W515L and Flt3-ITD mutant leukocytes exhib- signaling, and activating mutations in these receptors (Kelly ited a significant increase in Glut1 cell surface expression et al., 2002; Pikman et al., 2006) were shown to cause higher (Fig. 6 G). Remarkably, overexpression of the human ApoA-I Glut1 expression. In this context, it is tempting to parallel our transgene not only prevented the fat mass loss (Fig. 6 C) of myeloproliferative mouse models with other cancer models in mice bearing the Mpl-W515L and Flt3-ITD mutations, but which malignant cells rely on high levels of aerobic glycolysis also significantly reversed the increased Glut1 cell surface ex - as the major source of ATP to fuel cellular proliferation, known pression in Mpl-W515L and Flt3-ITD leukocytes (Fig. 6 G) as the Warburg ee ff ct (Vander Heiden et al., 2009). However, that was associated with increased RQ (Fig. 6, H and I) and the classical view of the Warburg ee ff ct, which involves defect glucose oxidation during the feeding period (Fig. 6 F). in mitochondrial oxidative phosphorylation, has been recently challenged, and mitochondrial metabolism may indeed be DISCUSSION functional in die ff rent types of tumor cells (Jose et al., 2011). Although there is growing evidence that weight loss, and We now provide both in vitro and in vivo evidence for in- especially the loss of muscle and adipose tissue mass, is a creased mitochondrial potential in leukocytes of our mouse (I) Scheme illustrating the induction of glycolysis through modulation of plasma membrane cholesterol by ABCA1 and ABCG1 deficiency and increased mitochondrial metabolism that produces ATP and synthesizes lipids for cell growth. Lack of these transporters promotes growth fac- tors dependent on IL-3R signaling–mediated glycolysis as reflected by (a) enhanced Hk2 and PFKp mRNA expression, (b) enhanced glycolytic metabolites content, (c) enhanced glucose oxidation (i.e., conversion into CO ), and (d) reversal of enhanced glucose oxidation by removal of cel- lular cholesterol by cyclodextrin. The pyruvate generated through glycolysis is directed into the TCA cycle and increases mitochondrial metabolism as reflected by (a) enhanced mRNA expression of fumarase and SDHb, (b) enhanced SDH activity, (c) enhanced mitochondrial metabolites and + / / mitochondrial membrane potential (H ), and (d) enhanced ATP/ADP ratio. Increased lipid synthesis was also observed in Abca1 Abcg1 BM cells as reflected by (a) enhanced mRNA expression of ACL and (b) enhanced glucose conversion into lipids. JEM Vol. 210, No. 2 347 / / Figure 5. Inhibition of Glut1 or overexpression of the human apoA-I transgene rescues adipose atrophy in Abca1 Abcg1 BM chimeras. 14 / / (A) Effect of IL-3 treatment on [ C]glucose conversion into lipids in WT and Abca1 Abcg1 BM cells in the presence or absence of 50 µM fasentin, a / / Glut1 inhibitor. (B) Effect of Fasentin on IL-3–mediated proliferation in WT and Abca1 Abcg1 BM cells. (C and D) Effect of Fasentin on LPS-mediated / / inflammatory (C) and glycolytic (D) responses in WT and Abca1 Abcg1 BM-derived macrophages. Values were normalized to ribosomal 18S. Results are means ± SEM of three independent experiments performed in triplicate. *, P < 0.05 versus WT mice; , P < 0.05 versus LPS treatment. (E and F) Quanti- fication of Glut1 cell surface expression (E) and 2-NBDG uptake by flow cytometry (F) in CD45 leukocytes isolated from the BM of WT recipients mice / / transplanted with WT or Abca1 Abcg1 BM transduced with a lentivirus encoding Glut1 shRNA 7 wk after reconstitution. (G and H) Peripheral leuko- cyte counts (G) and epididymal fat mass (H) of these mice at the end of the experiment. (I and J) Histograms showing epididymal fat mass loss (I) and Glut1 cell surface expression (J) in CD45 peripheral leukocytes in chow-fed transgenic recipient mice overexpressing the human apoA-I transgene trans- / / planted with Abca1 Abcg1 BM. Results are ± SEM of an experiment of four to six animals per group. *, P < 0.05 versus WT recipients transplanted with WT BM; , P < 0.05 versus control retrovirus. MFI, mean fluorescence intensity. model of myeloproliferative disorder. Surprisingly, not only metabolism of fatty acids (i.e., fats burn in the fire of car - inhibition of PDPs but also inhibition of CPT-1 prevented bohydrates; Samudio et al., 2010). Although the mechanisms / / the proliferation of Abca1 Abcg1 leukocytes. This em- orchestrating this phenomenon remain to be investigated, phasizes a scenario in which high rates of aerobic glycolysis it appears unlikely to be mediated by triglyceride or ce- in leukemia cells are necessary to support the mitochondrial ramide biosynthesis. Interestingly, the antiproliferative effect 348 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e Figure 6. HDL prevents the adipose tissue atrophy and enhanced glucose oxidation caused by Mpl-W515L and Flt3-ITD activating muta- +  + tions. (A) Representative dot plots and quantification of the splenic myeloid cells (CD115 monocytes and CD115 Gr1 neutrophils) of WT and ApoA-I transgenic recipient mice transplanted with Mpl-W515L– and Flt3-ITD–transduced BM cells. (B and C) Quantification of the myeloid cells (B) and epididy - mal fat mass (C) of these mice. (D, E, F, H, and I) RQ measured by indirect calorimetry (D, E, H, and I) and quantification of the nocturnal glucose oxidation of these mice (F). (G) Quantification of the cell surface expression of Glut1 in CD45 leukocytes of these mice. Results are mean ± SEM of an experiment of four to five animals per group. *, P < 0.05 versus control mice; , P < 0.05 versus respective WT recipients. MFI, mean fluorescence intensity. JEM Vol. 210, No. 2 349 of carnitine suggested that prevention of oxidative phos- leukocytes, reduced glucose oxidation during the feeding pe - phorylation by efflux of excess mitochondrial acetyl moi - riod, and prevented adipose tissue loss. eties (Muoio et al., 2012) might represent a novel strategy for In conclusion, our study elucidates a glucose steal mecha- the treatment of hematological malignancies. In conclusion, nism by proliferating and inflammatory myeloid cells that increased glucose oxidation and mitochondrial metabolism– contributes to depletion of adipose tissue in myeloprolifera- dependent proliferation of myeloid cells likely contributed tive disorders. This mechanism involved enhanced glucose to energy dissipation and subsequent loss of adipose mass. oxidation by myeloid cells that led to energy dissipation with Leukocytes and adipocytes can communicate through in- consequences on fat storage. Inhibition of glucose uptake by teraction between secreted inflammatory cytokines and their leukocytes with a Glut1 inhibitor or by treatments that in- cognate receptors (Lumeng and Saltiel, 2011; Odegaard and crease HDL levels could ultimately provide new therapeutic Chawla, 2011). The finding that highly infiltrated adipose approaches not only to limit cell proliferation but also to pre- depots were associated with impaired Glut4 translocation to vent the energy imbalance and fat atrophy observed in my- / the plasma membrane in response to insulin in Abca1 eloproliferative disorders and in other human malignancies. / Abcg1 BM chimeras was strongly implied by prior studies MATERIALS AND METHODS indicating insulin resistance in peripheral tissues secondary to / / / / Mice and treatments. WT, Abca1 , Abcg1 , and Abca1 Abcg1 increased tumor-derived cytokines (Delano and Moldawer, littermates in a mixed C57BL/6 × DBA background (Yvan-Charvet et al., 2006). Nevertheless, these findings provide strong evidences Tg 2007) were used for this study. Human apoA-1 transgenic (hapoA-1 ) mice that local inflammation driving adipose tissue insulin resis - were obtained from the Jackson Laboratory. BM transplantation into the ge- tance is not restricted to a role on fat mobilization in cancer- Tg netically uniform F1 generation obtained by crossing C57BL/6 WT, hapoA-1 associated adipose tissue loss (Das et al., 2011). This suggests mice with WT DBA mice (The Jackson Laboratory) was performed as pre- viously described (Yvan-Charvet et al., 2007). Animal protocols were ap- that myeloproliferative cells develop efficient strategies to proved by the Institutional Animal Care and Use Committee of Columbia divert glucose from its expected destination, i.e., fat storage University. Animals had ad libitum access to both food and water. as a mean to meet their energetic needs. In part, this could be related to a shunt of glucose to the pentose phosphate HSC transplantation. HSC transplantation was adapted from a previ- pathway that has been recently shown to modulate the in- + ously described protocol (Wagers et al., 2002). In brief, congenic CD45.1 flammatory response of activated immune cells (Ham, M., B6.SJL-Ptprca—Pep3b-/BoyJ were purchased from the Jackson Labora- tory and used to isolate Sca-1–depleted BM cells by FACS sorting. HSCs et al. 2008. The FASEB Journal Meeting. Abstr. 615.1; Blagih were isolated by FACS sorting of lineage-depleted BM from WT and and Jones, 2012). This could ultimately contribute not only / / Abca1 Abcg1 backcrossed for 10 generations on a C57/BL6 back- to the enhanced glucose oxidation observed in our mouse ground, based on the following cell surface markers: c-kit and Sca-1 (LSK, models of myeloproliferative disorders but also to the local  + + Lin Sca c-kit ). Lethally irradiated WT recipients were i.v. injected with adipose tissue insulin resistance mediated by enhanced in - 6 + 10 cells containing a mixture of CD45.1 Sca-1–depleted BM cells and + / / flammatory response. CD45.2 LSK cells from either WT or Abca1 Abcg1 mice in the ratio 1:2,000. Transplanted recipients were screened by flow cytometry for re - Finally, we showed that Glut1 blockade prevented both / / constitution of CD45.2 leukocytes in peripheral blood at 6 wk after trans- basal and IL-3–induced proliferation of Abca1 Abcg1 plant. More than 90% of leukocytes stained for the congenic marker CD45.2, leukocytes in vitro and prevented LPS-induced inflammatory thereby confirming the engraftment of LSK cells (Wagers et al., 2002). / / cytokine response in Abca1 Abcg1 macrophages. This translated in vivo into the rescue of leukocytosis and adipose Retroviral BM transplantation. The retroviral BM transplant assay was / / tissue infiltration of Abca1 Abcg1 BM transplanted mice performed as previously described (Pikman et al., 2006). In brief, control, Mpl-W515L and Flt3-ITD retroviral supernatants were titered and used to in response to Glut1 inhibition. Remarkably, this prevented transduce WT BM cells. In independent experiments, premade control and the adipose tissue loss of these mice, confirming the key Glut1 shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) were role of myeloid Glut1 in the diversion of energy stores from / / used to transduce WT and Abca1 Abcg1 BM cells. BM cells were cul- adipose to myeloid cells. Similarly, overexpression of the tured for 24 h in transplantation media (RPMI + 10% FBS + 6 ng/ml IL-3, human ApoA-I transgene, previously shown to raise plasma 10 ng/ml IL-6, and 10 ng/ml stem cell factor) and treated by spin infection HDL levels and prevent the myeloproliferative syndrome of with retroviral supernatants (1 ml supernatant per 4 × 10 cells in the pres- / / ence of polybrene) and centrifuged at 1,800 g for 90 min. The spin infection Abca1 Abcg1 BM transplanted mice (Yvan-Charvet was repeated 24 h later. After washing, the cells were used for BM transplan- et al., 2010), reduced Glut1 cell surface expression in leuko- tation into lethally irradiated WT recipient mice. cytes and prevented their fat loss. This reflected in part the removal of excess cholesterol from plasma membrane in Energy expenditure. Metabolic activity was performed by in vivo indi- / / Abca1 Abcg1 myeloid cells that prevented the IL-3R rect open circuit calorimetry at the Mouse Phenotyping Core of Columbia signaling (Yvan-Charvet et al., 2010) and subsequent in- University using a CaloSys calorimetry system (TSE Systems, Inc). Ani- mals were placed into experimental chambers with free access to food and crease in Glut1-dependent glucose uptake (Fig. 3 G). These water for a 4-d consecutive period. Food intake was recorded with an au - findings were further extended in two mouse models of my - tomated feeding monitor system through the study period. Constant air - eloproliferative disorders based on expression of human leu- flow (0.5 liter/min) was drawn through the chamber and monitored by a kemia disease alleles (Flt3-ITD and Mpl-W515L) in which mass-sensitive flow meter. To calculate oxygen consumption (VO ), carbon overexpression of the human ApoA-I transgene reduced my- dioxide production (VCO ), and RQ (ratio of VCO to VO ), gas con- 2 2 2 eloid expansion, decreased cell surface expression of Glut1 in centrations were monitored at the inlet and outlet of the scaled chambers. 350 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e Total metabolic rate (energy expenditure) was calculated from oxygen (GK1.5), CD8b (53-6.7), CD19 (eBio1D3), CD45R (B220, RA3-6B2), consumption and carbon dioxide production using Lusk’s equation and Gr-1 (Ly6G, RB6-8C5), Cd11b (Mac1, M1/70), Ter119 (Ly76) and expressed as watts per kilogram to the 0.75 power of body weight (Yvan- NK1.1 (Ly53, PK136), c-Kit (CD117, ACK2), and Sca-1 (D7) were all Charvet et al., 2005). Glucose and lipid oxidation were calculated as prev- i purchased from eBioscience. Glut1-FITC antibody was purchased from ously described (Yvan-Charvet et al., 2005). R&D Systems. These antibodies were used to sort HSCs and stain for leu- kocytes in peripheral blood and tissues. 14 14 In vivo 2-[ C]-DG uptake. Uptake of 2-[ C]-DG in peripheral tissues was measured as previously described (Rofe et al., 1988). In brief, 2 µCi BM harvest and treatment. Primary BM cells were resuspended in 2-[ C]-DG was i.v. injected, and blood samples were collected at 5, 10, 20, IMDM (Gibco) containing 10% FCS (STEMCELL Technologies) and cul- 30, and 40 min. Blood glucose was monitored through the study period tured for 1 h in tissue culture flasks to remove adherent cells, including mac - with a glucometer (Roche). After 40 min, adipose tissue, skeletal muscle, rophages. Suspended cells were then cultured for 72 h in the presence of 6 ng/ml heart, lung, spleen, and brain were rapidly dissected, weighed, and homog - IL-3 (R&D Systems). In some experiments, the farnesyl transferase inhibitor enized with 5% HClO solution. BM cells were collected from leg bones, (EMD Millipore) was used at the final concentration of 1 µM, fasentin and peripheral leukocytes were obtained after RBC lysis. The radioactivity (Sigma-Aldrich) at 50 µM, DL-AP3 (Tocris Bioscience) at 50 µM, myriocin incorporated in both 2-[ C]-DG and its 6-phosphate derivative was mea- (Sigma-Aldrich) at 10 µM, diglyceride acyltransferase inhibitor (EMD Milli- sured in the HClO extract and expressed as total radioactivity per tissue pore) at 40 µM, etomoxir (Sigma-Aldrich) at 40 µM, branched-chain amino weight. The rate constant of net tissue uptake of 2-[ C]-DG was calculated acids (leucine, isoleucine, and valine; Sigma-Aldrich) at 1 mM, glutathione as described previously (Rofe et al., 1988). In brief, the relative glucose monoethyl ester (EMD Millipore) at 10 mM, tempol (EMD Millipore) at uptake was calculated by dividing the area under the blood 2-[ C]-DG 4 mM, and carnitine (Sigma-Aldrich) at 1 mM. For proliferation assays, disappearance curve (cpm/min/ml) to the steady-state glucose concentra- cells were pulsed for 2 h with 2 µCi/ml [ H]thymidine, and the radioac- 14 6 tion (mM) multiplied by the tissue 2-[ C]-DG (cpm/g tissue or cpm/10 tivity incorporated into the cells was determined by standard procedures cells for the BM) at 40 min. using a liquid scintillation counter. In one experiment (Fig. 4 F), cells were resuspended in 20 mM glucose DMEM (DMEM rich; Gibco) or Blood parameters. Plasma leptin and insulin were determined by ELISA low-glucose DMEM (DMEM poor; Gibco) supplemented with 20 mM (Mouse Leptin Quantikine ELISA kit [R&D Systems]; Mouse insulin ELISA kit glucose or 20 mM galactose (Sigma-Aldrich) and cultured for 48 h in the [Crystal Chem, Inc.]). Plasma TNF was measured by Luminex assay (Cytokine presence of 6 ng/ml IL-3. SDH activity was determined by an ELISA Core Laboratory). Blood glucose was assayed with a glucometer (Roche). kit according to the manufacturer’s instructions (MitoSciences). BM- derived macrophages were isolated and cultured in 10% FBS in DMEM Histopathology. Mice were euthanized in accordance with the American supplemented with M-CSF for 5–10 d before the experiment. Where Veterinary Association Panel of Euthanasia. Adipose tissue was serially paraf- indicated, macrophages were incubated with 100 ng/ml LPS (Escherichia fin sectioned and stained with hematoxylin and eosin (H&E) for morpho - coli 0111:B4; Sigma-Aldrich). logical analysis as previously described (Yvan-Charvet et al., 2007). Glucose metabolism experiments. Isolated BM cells were cultured for Adipose tissue cellularity. Cellularity of epididymal adipose tissue was 24 h with or without 6 ng/ml IL-3 and 50 µM fasentin, a Glut1 inhibitor. determined as previously described (Yvan-Charvet et al., 2005). In brief, Where indicated, BM cells were differentiated into macrophages as described images of isolated adipocytes were acquired from a light microscope above. Cells were next incubated with 5.5 mM [ C]glucose in 2% BSA (IX-70; Olympus) fitted with a charge-coupled device camera (RS Photo - Krebs-Ringer bicarbonate buffer, pH 7.4. After 2 h, the generated CO metrics), and the measurement of 400 cell diameters was performed allow- and the C incorporation into lipids were quantified as previously described ing calculation of a mean fat cell weight. Tissue triglyceride content was (Yvan-Charvet et al., 2005). In some experiments, cellular cholesterol was measured from a sample of adipose tissue using a commercial kit (Sigma- depleted by 5 mM cyclodextrin for 1 h before growth factor treatment as Aldrich). Fat cell number was estimated by dividing the tissue lipid content previously described (Yvan-Charvet et al., 2010). by the fat cell weight. Directed metabolomic experiments. Isolated BM cells (10 cells) were Glucose tolerance tests. After 6 h of fasting, mice were injected i.p. with cultured with or without 6 ng/ml IL-3 for 24 h. The next day, suspended d-glucose (2 g/kg of body weight), and blood samples were obtained by tail cells were centrifuged at 1,000 rpm for 5 min, and pellets were rapidly bleeding at 0, 15, 30, 60, 90, and 120 min after injection (Yvan-Charvet washed (less than 10 s) with a mass spectrometry–compatible bue ff r (150 mM et al., 2005). Blood glucose was assayed with a glucometer (Roche). ammonium acetate solution) to prevent the presence of sodium and phos- phate in the residue and limit interference with LC-MS analyses. After a sec- ond step of centrifugation, pellets were immediately frozen in liquid nitrogen Flow cytometry analysis. BM cells were collected from leg bones or to quench metabolism according to the University of Michigan Molecular spleen, lysed to remove RBCs, and filtered before use. Freshly isolated cells Phenotyping Core facility’s instructions. Samples were shipped on dry ice to were stained with the appropriate antibodies for 30 min on ice. For periph- the Molecular Phenotyping Core facility where metabolites were extracted eral blood leukocytes analysis, 100 µl blood was collected into EDTA tubes by exposing the cells to a chilled mixture of 80% methanol, 10% chloroform, before RBC lysis, filtration, and staining for 30 min on ice. To assess the up - and 10% water. Glycolytic and citric acid metabolites were then analyzed by take of 2-NBDG, prestained peripheral blood leukocytes (Yvan-Charvet the Molecular Phenotyping Core facility using LC-MS as previously de- et al., 2010) were incubated with 10 µM 2-NBDG (Invitrogen) for 30 min, scribed (Yuneva et al., 2012). followed by flow cytometric detection of fluorescence produced by the cells (Zou et al., 2005). The mitochondrial membrane potential was analyzed with 25 nM fluorescent TMRE (AnaSpec) staining for 30 min on prestained Western blot analysis. 2-h-fasted mice were administrated insulin i.p. leukocytes. Viable cells, gated by light scatter or exclusion of CD45 cells, (0.4 U/mouse). After 5 min, freshly isolated adipose tissue was homoge- were analyzed on a four-laser LSRII cell analyzer (BD) or sorted on a FAC- nized as previously described (Yvan-Charvet et al., 2005), and cell ex- SAria Cell Sorter (BD), both running with DiVa software (BD). Data were tracts were either directly used to measure phospho-Akt (clone 587F11; analyzed using FlowJo software (Tree Star). Cell Signaling Technology) by Western blot analysis or fractionated by differential centrifugation to isolate plasma membranes and low-density Antibodies. Anti–mouse CD45 (clone 30F11), CD115 (AFS98), TCR- microsomes (Yvan-Charvet et al., 2010) and quantify Glut4 expression (H57-597), F4/80 (BM8), CD2 (RM2-5), CD3e (145-2C11), CD4 (clone 1F8; R&D Systems). JEM Vol. 210, No. 2 351 RNA analysis. Total RNA extraction, cDNA synthesis, and real-time Hawkinson, J.E., M. Acosta-Burruel, and P.L. Wood. 1996. The metabo- PCR were performed as described previously (Yvan-Charvet et al., 2005). tropic glutamate receptor antagonist L-2-amino-3-phosphonopropionic Ribosomal 18S RNA expression was used to account for variability in the acid inhibits phosphoserine phosphatase. Eur. J. Pharmacol. 307:219– initial quantities of mRNA. 225. http://dx.doi.org/10.1016/0014-2999(96)00253-1 Herman, M.A., and B.B. Kahn. 2006. Glucose transport and sensing in the maintenance of glucose homeostasis and metabolic harmony. J. Clin. Statistical analysis. Statistical signic fi ance was performed by two-tailed para - Invest. 116:1767–1775. http://dx.doi.org/10.1172/JCI29027 metric Student’s t test or by one-way ANOVA (four-group comparisons) with Jose, C., N. Bellance, and R. Rossignol. 2011. Choosing between gly- a Bonferroni multiple comparison post test (GraphPad Software). colysis and oxidative phosphorylation: a tumor’s dilemma? Biochim. Biophys. Acta. 1807:552–561. http://dx.doi.org/10.1016/j.bbabio.2010 We thank Dr. Kristie Gordon for assistance with flow cytometry, Pr. Pascal Ferre for .10.012 scientific discussion, and the French Cancer Research Association (ARC). Kelly, L.M., Q. Liu, J.L. Kutok, I.R. Williams, C.L. Boulton, and D.G. This work utilized Core Services supported by a National Institutes of Health Gilliland. 2002. FLT3 internal tandem duplication mutations associated (NIH) grant (DK089503) to the University of Michigan. 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HDL and Glut1 inhibition reverse a hypermetabolic state in mouse models of myeloproliferative disorders

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Abstract

A r t i c l e HDL and Glut1 inhibition reverse a hypermetabolic state in mouse models of myeloproliferative disorders 1 2,3 4,5 Emmanuel L. Gautier, Marit Westerterp, Neha Bhagwat, 2 4,5 4,5 6 Serge Cremers, Alan Shih, Omar Abdel-Wahab, Dieter Lütjohann, 1 4,5 2 Gwendalyn J. Randolph, Ross L. Levine, Alan R. Tall, 2,7 and Laurent Yvan-Charvet Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 Department of Medicine, Division of Molecular Medicine, Columbia University, New York, NY 10032 Department of Medical Biochemistry, Academic Medical Center of Amsterdam, University of Amsterdam, 1105 Amsterdam, Netherlands 4 5 Human Oncology and Pathogenesis Program; and Leukemia Service, Department of Medicine; Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Institute of Clinical Chemistry and Clinical Pharmacology, University Clinic Bonn, 53127 Bonn, Germany Institut National de la Santé et de la Recherche Médicale U1065, Centre Mediterraneen de Medecine Moleculaire (C3M), Avenir, 06204 Nice, France A high metabolic rate in myeloproliferative disorders is a common complication of neoplasms, but the underlying mechanisms are incompletely understood. Using three different mouse models of myeloproliferative disorders, including mice with defective cholesterol efflux pathways and two models based on expression of human leukemia dis - ease alleles, we uncovered a mechanism by which proliferating and inflammatory myeloid cells take up and oxidize glucose during the feeding period, contributing to energy dissipa- tion and subsequent loss of adipose mass. In vivo, lentiviral inhibition of Glut1 by shRNA prevented myeloproliferation and adipose tissue loss in mice with defective cholesterol efflux pathway in leukocytes. Thus, Glut1 was necessary to sustain proliferation and poten - tially divert glucose from fat storage. We also showed that overexpression of the human ApoA-I transgene to raise high-density lipoprotein (HDL) levels decreased Glut1 expression, dampened myeloproliferation, and prevented fat loss. These experiments suggest that inhibition of Glut-1 and HDL cholesterol–raising therapies could provide novel therapeutic approaches to treat the energy imbalance observed in myeloproliferative disorders. Chronic inflammatory diseases such as chronic metabolism, and enhanced energy expenditure CORRESPONDENCE Laurent Yvan-Charvet: infection, cancer, and heart failure are often in this state of chronic hypermetabolism is not [email protected] associated with adipose tissue loss (Delano and fully understood. 14 Moldawer, 2006). Adipose tissue loss in the Among chronic inflammatory diseases, he - Abbreviations used: 2-[ C]-DG, 2-[ C]-deoxyglucose; 2-NBDG, setting of chronic inflammatory diseases could matological malignancies such as leukemias and 2-[N-(7-nitrobenz-2-oxa-1, ultimately represent a major health problem myeloproliferative disorders are also associated 3-diazol-4-yl)amino]-2-deoxy- because of associated comorbidities such as with adipose tissue loss (Dingli et al., 2004). In d-glucose; ACL, ATP-citrate weakness, fatigue, and impaired immunity. Loss humans, among the most common activating lyase; HDL, high-density lipopro- tein; HSC, hematopoietic stem of adipose tissue is ultimately caused by an mutations in myeloid malignancies are muta- cell; PDP, pyruvate dehydrogenase imbalance between food intake and energy tions in the FMS-like tyrosine kinase 3 (Flt3) phosphoserine phosphatase; expenditure. Although food intake is often re- gene, which occur in 30% of cases of acute PFK, phosphofructokinase; RQ, respiratory quotient; SDH, duced in patients with chronic inflammatory myeloid leukemia as well as mutations in the succinate dehydrogenase; TCA, diseases, these patients exhibit a persistent state tricarboxylic acid; TMRE, © 2013 Gautier et al. This article is distributed under the terms of an Attribution– of inappropriately high metabolic rate, and tetramethylrhodamine ethyl ester. Noncommercial–Share Alike–No Mirror Sites license for the first six months after this substantially contributes to their adipose the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share loss (Delano and Moldawer, 2006). However, Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/ by-nc-sa/3.0/). the link between chronic inflammation, tissue The Rockefeller University Press $30.00 J. Exp. Med. 2013 Vol. 210 No. 2 339-353 www.jem.org/cgi/doi/10.1084/jem.20121357 The Journal of Experimental Medicine thrombopoietin receptor (proto-oncogene c-Mpl) observed controls by indirect calorimetry. No significant changes in in 5–10% of patients with myelofibrosis. These mutations daily food intake, energy expenditure, or locomotor activity confer a fully penetrant myeloproliferative disorder in mice were observed in these mice (Table 1). Similar fat and cho- (Kelly et al., 2002; Pikman et al., 2006), providing a useful lesterol absorption and bile acid excretion were also observed / / / tool for studying the mechanism of adipose tissue loss associ- in Abca1 Abcg1 mice (not depicted). However, Abca1 / ated with myeloproliferative disorders. Abcg1 BM transplanted mice exhibited a higher respira- We have recently shown that mice lacking the cholesterol tory quotient (RQ) during the feeding period (dark phase; eu ffl x transporters ABCA1 and ABCG1 develop massive ex - Fig. 1 D), reflecting an 20% increase in glucose oxidation pansion and proliferation of hematopoietic stem and progeni- (Fig. 1 E) and exhibited hypoglycemia (Table 1). Injection i.p. tor cells, marked monocytosis, neutrophilia, and inlfi tration of a glucose bolus also revealed faster glucose utilization / / of various organs with myeloid cells, resembling a classical in Abca1 Abcg1 BM transplanted mice compared with myeloproliferative disorder (Hansson and Björkholm, 2010; WT transplanted controls (Fig. 1 F). We next examined the Yvan-Charvet et al., 2010). As we noticed a dramatic loss of uptake of the radiolabeled d-glucose analogue 2-[ C]- / / 14 / / adipose tissue in Abca1 Abcg1 BM chimeras, we hy- deoxyglucose (2-[ C]-DG) in Abca1 Abcg1 BM trans- pothesized that the myeloproliferative disorder of these mice planted mice in the fed state. No significant changes were might be responsible for their wasting syndrome and pro- observed in the total uptake of 2-[ C]-DG in the skeletal ceeded to investigate the underlying mechanisms. muscle and brain (Fig. 1 G), whereas, in line with their re- duced fat mass, total 2-[ C]-DG incorporation was reduced RESULTS in their adipose tissue (Fig. 1 G). In contrast, the total uptake Lack of ABCA1 and ABCG1 in hematopoietic cells of 2-[ C]-DG was increased by two- to threefold in the promotes adipose tissue atrophy heart, lung, spleen, and BM of these mice (Fig. 1 G) and by Determination of the fat and muscle mass of irradiated WT 1.5-fold when expressed as the rate constant for net tissue / / 14 recipients transplanted with Abca1 Abcg1 BM cells fed uptake of 2-[ C]-DG uptake (Fig. 1 H). Thus, we showed / / a chow diet revealed not only absence of adipose tissue that Abca1 Abcg1 BM transplanted mice have increased growth but also reduced epididymal fat mass at 24 wk after propensity to clear and use glucose. reconstitution compared with controls (Fig. 1 A). Gastroc- nemius muscle loss was also observed 30 wk after reconstitu- Inflammation diverts glucose from fat storage by promoting / / tion in these mice (Fig. 1 B). Subcutaneous and retroperitoneal adipose tissue insulin resistance in Abca1 Abcg1 adipose depots were also decreased by more than threefold at BM transplanted mice / / / 24 wk after reconstitution in Abca1 Abcg1 BM chime- Consistent with their myeloproliferative disorder, Abca1 / ras, consistent with their reduced plasma leptin levels (Table 1). Abcg1 BM chimeras exhibited a chronic ina fl mmatory state To test whether the adipose tissue atrophy of these mice was as reflected by a threefold increase in plasma TNF levels a direct consequence of their defective hematopoietic com- (Table 1). Microscopic examination of the adipose tissue of / / partment and myeloproliferative syndrome (Yvan-Charvet Abca1 Abcg1 BM transplanted mice revealed an in- et al., 2010), we generated hematopoietic stem cell (HSC)– creased inflammatory infiltrate reflected by crown-like struc - specific chimeric animals by transplanting lethally irradi - tures (Fig. 2 A). Because infiltrated leukocytes are a hallmark / / ated WT recipients with purified WT or Abca1 Abcg1 of insulin resistance in adipose tissue (Lumeng and Saltiel,  + + HSCs (Lin Sca cKit , LSK fraction). Mice transplanted 2011; Odegaard and Chawla, 2011), we wondered whether / / / / with Abca1 Abcg1 LSK cells reproduced the three- the adipose tissue infiltration observed in Abca1 Abcg1 hi hi fold increase in the Gr-1 /CD11b blood myeloid popu- BM chimeras would promote local insulin resistance and / / lation observed in Abca1 Abcg1 BM transplanted mice contribute to their reduced adipose tissue glucose uptake. To (Yvan-Charvet et al., 2010) and exhibited a twofold re- address this question, a bolus of insulin was injected into WT / / duction in fat mass 12 wk after reconstitution (Fig. 1 A). and Abca1 Abcg1 BM transplanted mice, and hallmarks Similar findings were observed in mice with specific knock - of insulin signaling (AKT phosphorylation and GLUT4 ad- out of these transporters in the hematopoietic lineage dressing to the cell membrane) were assessed. Western blot fl/fl fl/fl (Mx1-Cre Abca1 Abcg1 ) 10 wk after injections of analysis revealed reduced GLUT4 translocation from the PolyI:C to excise the STOP codon that prevents the expres- low-density microsome fraction to the plasma membrane in / / sion of the cre recombinase (Fig. 1 C). Together, these obser- response to insulin in the adipose tissue of Abca1 Abcg1 vations revealed that the expansion of adipose tissue is BM transplanted mice (Fig. 2 B). This was associated with severely compromised in mice lacking ABCA1 and ABCG1 reduced AKT phosphorylation, suggesting insulin resistance in their hematopoietic system. in adipocytes is secondary to local inflammatory cell infil - tration (Fig. 2 B). Accordingly, microscopic analysis of iso- ABCA1 and ABCG1 deficiency in leukocytes lated adipocytes revealed smaller adipocytes in WT recipients / / impacts glucose homeostasis transplanted with Abca1 Abcg1 BM cells (Fig. 2 A and We next compared the metabolic characteristics of chow-fed Table 1), whereas adipocyte cell numbers were comparable / / + Abca1 Abcg1 BM transplanted mice and their respective with controls (Table 1). Finally, depletion of Ly6C/G 340 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e / / Figure 1. Adipose tissue atrophy and enhanced glucose utilization in Abca1 Abcg1 BM chimeras. (A) Epididymal adipose tissue of WT / / / /  + + recipient mice transplanted with WT or Abca1 Abcg1 BM cells or WT and Abca1 Abcg1 HSC (Lin Sca cKit , LSK fraction) transplanted mice. (B) Gastrocnemius skeletal muscle mass of these mice. (C) Epididymal fat mass in mice with specific knockout of these transporters in the hematopoietic fl/fl fl/fl lineage (Mx1-Cre Abca1 Abcg1 ) 10 wk after three injections of 15 mg/kg PolyI:C to excise the STOP codon. (D and E) RQ (D) and glucose oxidation / / efc fi iency (E) measured by indirect calorimetry in chow-fed WT and Abca1 Abcg1 BM transplanted (BMT) mice 10 wk after reconstitution. (F) Glucose / / tolerance test was performed i.p. on chow-fed WT and Abca1 Abcg1 BM transplanted mice 12 wk after reconstitution. Blood glucose con- centrations were measured at the indicate time points. (G and H) Tissue uptake (G) and rate constant of 2-[ C]-DG (H) in 24-wk-old chow-fed WT and / / Abca1 Abcg1 BM transplanted mice at the end of the study period (40 min after i.v. injection of the radiolabeled tracer). All results are means ± SEM and are representative of an experiment of five to seven animals per group. *, P < 0.05 versus WT. JEM Vol. 210, No. 2 341 Table 1. Effect of leukocyte ABCA1 and ABCG1 deficiencies on body weight, plasma leptin levels, subcutaneous and retroperitoneal adipose depots, plasma glucose, insulin and TNF levels, epididymal adipose tissue cellularity, and energy metabolism Metabolic parameters BM transplantation / / WT Abca1 Abcg1 Body weight (g) 27.9 ± 0.9 27.4 ± 0.6 Plasma Leptin (ng/ml) 1.95 ± 0.4 0.7 ± 0.2* Subcutaneous fat mass (g) 0.52 ± 0.12 0.15 ± 0.03* Retroperitoneal fat mass (g) 0.28 ± 0.04* 0.09 ± 0.08* Plasma glucose (g/liter) 2.1 ± 0.1 1.7 ± 0.1* Plasma Insulin (ng/ml) 7.9 ± 0.8 6.1 ± 1.1 Plasma TNF (pg/ml) 3.1 ± 0.5 7.4 ± 0.6* Fat cell number (×10 ) 4.8 ± 0.9 4.6 ± 0.8 Fat cell weight (ng) 119 ± 14* 36 ± 5* Food intake (g/day) 3.28 ± 0.14 3.33 ± 0.13 0.75 Food intake (g/day/g ) 0.29 ± 0.01 0.29 ± 0.01 0.75 Energy expenditure, EE (W/kg ) 6.68 ± 0.25 7.11 ± 0.18 Locomotor activity (counts/14 min) 2,589 ± 544 2,560 ± 499 Values are mean ± SEM (n = 5 per group). *, P < 0.05 versus controls. myeloid cells with the anti–granulocyte receptor-1 (Gr-1) we next assessed the mRNA expression levels of several antibody RB6-8C5 partially reversed the reduced glucose members of this family, namely Glut1, Glut2, Glut3, Glut4, / / / / uptake in the adipose tissue of Abca1 Abcg1 mice (not and Glut6. Abca1 Abcg1 BM cells exhibited a two- depicted). Together, these results indicate that myeloprolif- fold up-regulation of the glucose transporter Glut1 mRNA eration and associated adipose tissue infiltration compromise expression, which was also the most highly expressed Glut adipose tissue function and likely contribute to the loss of fat member in leukocytes (Fig. 3 C). Flow cytometry analysis / / in Abca1 Abcg1 BM transplanted mice. Moreover, these showed a 30% increase in the cell surface expression of / / / / + findings revealed that Abca1 Abcg1 BM chimeras can Glut1 in vivo in Abca1 Abcg1 CD45 leukocytes (not efficiently clear glucose despite being insulin resistant. depicted), including monocytes, neutrophils, and lympho- cytes (Fig. 3 D), and this correlated with increased mito- chondrial membrane potential (Fig. 3 E). Together, these Glucose consumption by proliferating myeloid cells findings show that leukocytes have a major role in glucose contributes to whole body glucose dissipation / / / / uptake in Abca1 Abcg1 BM transplanted mice exhib- in Abca1 Abcg1 BM transplanted mice iting a myeloproliferative syndrome. This resulted in en- Although local inflammation promoted adipose tissue in - hanced whole body glucose oxidation that could explain the sulin resistance, this could not explain the enhanced glucose increased energy dissipation in these mice. uptake by noninsulin-sensitive tissues (Fig. 1 G) contrib- uting to enhanced whole body glucose oxidation during the feeding period (Fig. 1 E). Because these tissues exhibit Signaling via the IL-3/GM-CSF receptor common -subunit / / massive myeloid infiltration in Abca1 Abcg1 BM trans- enhances the expression of Glut1 and glycolytic enzymes / / planted mice (Yvan-Charvet et al., 2007; Out et al., 2008), in proliferating Abca1 Abcg1 leukocytes / we next investigated the uptake of glucose by Abca1 We next set out to better understand the mechanism leading / / / Abcg1 leukocytes. To estimate glucose utilization by flow to increased glucose uptake in Abca1 Abcg1 leukocytes. cytometry, we used the fluorescent d-glucose analogue Up-regulation of Glut-1 by oncogenes such as Ras or Src has 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy- been reported (Flier et al., 1987), and we recently showed / / d-glucose (2-NBDG) as a tool that reflects the glucose enhanced Ras-Erk signaling in Abca1 Abcg1 BM cells bound to the cells and its uptake. Leukocytes isolated from at basal state and in response to the hematopoietic growth / / Abca1 Abcg1 BM revealed a significant 30% increase factors IL-3 and GM-CSF (Yvan-Charvet et al., 2010). in 2-NBDG staining (Fig. 3 A). This increase was massive Therefore, we investigated the expression of Glut1 in re- in monocytes and neutrophils when the increased cell num- sponse to IL-3 in BM leukocytes. As shown in Fig. 3 F, Glut1 ber was taken into account (Fig. 3 B; Yvan-Charvet et al., mRNA levels were increased upon stimulation with IL-3 in 14 / / 2010), accounting for the total uptake of 2-[ C]-DG in WT BM cells and were further increased in Abca1 Abcg1 the BM (Fig. 1 G). As glucose uptake depends on the glu- cells. Inhibition of the Ras signaling pathway using a farnesyl cose transporter (Glut) family (Herman and Kahn, 2006), transferase inhibitor, known to prevent the anchorage of Ras 342 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e in the plasma membrane and to inhibit the proliferation of / / Abca1 Abcg1 BM cells (Yvan-Charvet et al., 2010), normalized Glut1 mRNA expression to WT levels (Fig. 3 F). Further characterization of signaling pathways downstream of the IL-3/GM-CSF receptor common -chain (Chang et al., 2003) showed that the phosphoinositide 3-kinase in- hibitor LY294002 prevented IL-3–induced Glut1 mRNA / / expression in Abca1 Abcg1 leukocytes (Fig. 3 F), similar to the ee ff ct of this inhibitor on the proliferation of these cells (Yvan-Charvet et al., 2010). Analysis of the rates of [ C]glucose oxidation revealed that in basal and IL-3–stimulated cond- i / / tions, Abca1 Abcg1 leukocytes exhibited a higher glyco- lytic rate (Fig. 3 G). This ee ff ct was inhibited by removal of membrane cholesterol by cyclodextrin (Fig. 3 G), consistent with the cholesterol-dependent regulation of IL-3R signal- ing (Yvan-Charvet et al., 2010). Analysis of genes of the gly- colytic and lipid synthetic pathways also revealed up-regulation of hexokinase 2 (Hk2), phosphofructokinase (PFK), and / / ATP-citrate lyase (ACL) mRNAs in Abca1 Abcg1 leu- kocytes that was dependent on the IL-3/GM-CSF receptor common -chain signaling pathway (Fig. 3 H). Fumarase and succinate dehydrogenase (SDH) mRNA expression was / / also increased in response to IL-3 in Abca1 Abcg1 leu- kocytes (Fig. 3 H). Together, our results point to the IL-3/ GM-CSF receptor common -chain/GLUT1 axis as a major determinant of the increased glucose uptake observed in / / Abca1 Abcg1 leukocytes. Metabolic profiling reveals increased glycolysis and oxidative phosphorylation in proliferating / / Abca1 Abcg1 leukocytes Further quantification of glycolytic metabolites by LC-MS showed higher glucose 6-phosphate/fructose 6-phosphate (G6P+F6P) and fructose 1,6-diphosphate (F1,6BP) levels Figure 2. Inlfi trated leukocytes modulate adipose tissue insulin sen - / / / / sitivity in Abca1 Abcg1 BM chimeras. (A) Parafn fi -embedded serial in basal and IL-3–stimulated Abca1 Abcg1 leukocytes / / sections obtained from the adipose tissue of WT and Abca1 Abcg1 (Fig. 4 A). Increased citric acid cycle metabolites (citrate, BM transplanted (BMT) mice fed a chow diet. Representative H&E stain- isocitrate, succinate, fumarate, and malate) and related mito- / / ing revealed an extensive myeloid cell infiltrate in Abca1 Abcg1 chondrial products (GTP and FAD) were also observed in BM transplanted mice compared with controls. Micrographs of iso- / / Abca1 Abcg1 leukocytes under basal and/or IL-3–stim- lated epididymal adipose cells confirmed reduced adipose cell size in ulated conditions (Fig. 4, B and C). Consistent with these / / Abca1 Abcg1 BM transplanted mice. Data representative of four to findings, a higher ATP to ADP ratio was observed in prolif - six animals per group are shown. Bars, 1 mm. (B) Western blot analysis of / / erating Abca1 Abcg1 leukocytes (Fig. 4 D). This was as- plasma membrane (PM) and low-density microsome (LDM) Glut4, phos- / / sociated with increased mitochondrial membrane potential pho-Akt, and total Akt in the adipose tissue of WT and Abca1 Abcg1 BM transplanted mice after acute i.p. injection of an insulin bolus. Quan- measured using a fluorescent tetramethylrhodamine ethyl titative results were obtained from two independent experiments. Values ester (TMRE) dye (not depicted) and increased SDH activity are mean ± SEM and expressed as percent expression of PM Glut4 over / / in Abca1 Abcg1 leukocytes (Fig. 4 E). HDM fraction or as arbitrary units (a.u.). *, P < 0.05 versus insulin-injected WT controls; , P < 0.05 versus saline-injected mice. / / The increased glycolysis in Abca1 Abcg1 leukocytes is required for proliferation / / To test whether high levels of glycolysis are required for not galactose enhanced the proliferation of Abca1 Abcg1 / / Abca1 Abcg1 leukocyte proliferation, we next cultured leukocytes to the level of high-glucose DMEM (DMEM cells with media containing galactose or glucose. Galactose rich), pointing to a prominent role of the glycolysis pathway enters glycolysis through the Leloir pathway, which occurs at in cell growth. However, a potential contribution of the pen- / / a significantly lower rate than glucose entry into glycolysis tose phosphate pathway to Abca1 Abcg1 leukocyte (Bustamante and Pedersen, 1977). Fig. 4 F shows that under proliferation could not be completely excluded from this glucose-free DMEM (DMEM poor), addition of glucose but experiment. We next challenged the mitochondria with either JEM Vol. 210, No. 2 343 / / Figure 3. Enhanced glycolytic activity in Abca1 Abcg1 leukocytes. (A) Ex vivo characterization of the glucose binding and/or uptake in WT / / +  + + and Abca1 Abcg1 BM leukocyte subpopulations (i.e., CD115 monocytes, CD115 GrI neutrophils, and TCRb lymphocytes) using a fluorescent d -glucose analogue (2-NBDG). (B) Glucose uptake normalized by the amount of BM leukocytes. (C) mRNA expression of glucose transporters (Gluts) in / / / / freshly isolated WT and Abca1 Abcg1 BM cells. (D) Cell surface expression of Glut1 was also quantified in WT and Abca1 Abcg1 BM neutro- phils, monocytes, and lymphocytes. (E) Mitochondrial membrane potential measured by fluorescent TMRE dye. All results are means ± SEM and are repre - sentative of two independent experiments (n = 5–6 animals per groups). *, P < 0.05 versus controls. MFI, mean fluorescence intensity. (F) Glut1 expression after BM cells were treated for 72 h with the indicated growth factors and in the presence or absence of 1 µM farnesyl transferase inhibitor (FTI) or 10 µM PI3K inhibitor (LY294002). Values were normalized to ribosomal 18S. (G) Effect of IL-3 treatment and cholesterol depletion by cyclodextrin (CD) on 14 / / [ C]glucose conversion into CO in WT and Abca1 Abcg1 BM cells. (H) mRNA expression of Hk2, PFK isoform p (PFKp), ACL, fumarase, and SDH sub- / / unit b (SDHb) in WT and Abca1 Abcg1 BM-derived cells untreated or treated for 72 h with IL-3. Values were normalized to ribosomal 18S. Results are means ± SEM of cultures from three independent mice. *, P < 0.05 versus WT controls; , P < 0.05 versus untreated condition. branched-chain amino acids (i.e., valine, leucine, and isoleu- leukocytes, suggesting the requirement of the pyruvate entry cine) or with cysteamine to fuel the tricarboxylic acid (TCA) into the TCA cycle for proliferation (Fig. 4 G). We next used cycle with acetyl-coA or coA, respectively, but these treat- the phosphoserine phosphatase inhibitor DL-AP3 (Hawkinson / / ments did not increase the proliferation of Abca1 Abcg1 et al., 1996), which blocks the rate-limiting step of the serine 344 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e biosynthetic pathway (Lunt and Vander Heiden, 2011) but responses (Fukuzumi et al., 1996; Gamelli et al., 1996). Con- also the pyruvate dehydrogenase phosphoserine phosphatases sistent with the previously observed enhancement in Toll-like / / (PDPs) known to switch metabolic flux from glycolysis to - receptor signaling in Abca1 Abcg1 macrophages (Yvan- ward oxidative phosphorylation. This treatment prevented Charvet et al., 2008), analysis of the rates of [ C]glucose / / the enhanced proliferation of Abca1 Abcg1 leukocytes oxidation in vitro in BM-derived macrophages revealed that / / (Fig. 4 G). Because inhibition of serine palmitoyltransferase in basal and LPS-stimulated conditions, Abca1 Abcg1 by myriocin, downstream of the serine biosynthetic pathway, macrophages exhibited a higher glucose consumption that was / / proportionately inhibited both WT and Abca1 Abcg1 associated with increased Glut1 mRNA levels (not depicted). leukocytes (Fig. 4 G), this suggested that the ee ff ct of DL-AP3 Interestingly, Glut1 inhibition by Fasentin reduced the ina fl m - / / was most likely related to inhibition of PDPs. Inhibition matory response induced by LPS in Abca1 Abcg1 mac- of diglyceride acyltransferase also proportionately inhibited rophages (Fig. 5 C). Although the mRNA levels of Hk2 / / both WT and Abca1 Abcg1 leukocyte proliferation and glucose-6-phosphate dehydrogenase followed the in - (Fig. 4 G), suggesting that despite enhanced lipid synthesis flammatory pattern (Fig. 5 D), glycolytic genes such as PFK (Fig. 5 A), neither ceramide or triglyceride biosynthesis and ACL were barely affected by LPS in either WT or / / was the limiting step for the proliferation of these cells. Abca1 Abcg1 macrophages (Fig. 5 D). This contrasted / / with proliferating Abca1 Abcg1 leukocytes (Fig. 4 I). / / / / Proliferating Abca1 Abcg1 leukocytes exhibit Thus, Abca1 Abcg1 macrophages could contribute enhanced mitochondrial metabolism not only to the enhanced glucose oxidation observed in / / To better understand the contribution of the mitochondrial Abca1 Abcg1 BM transplanted mice (Fig. 1 E), but / / metabolism, we next assessed the ability of Abca1 Abcg1 also to the local adipose tissue insulin resistance mediated by leukocytes to proliferate after treatment with membrane- enhanced inflammatory response (Fig. 2 B). Together, these permeable antioxidants. Although glutathione monoethyl findings revealed that Glut1 controls both the proliferative / / ester partially reduced the proliferation of all cells, tempol and inflammatory status of Abca1 Abcg1 leukocytes. / / abrogated the enhanced proliferation of Abca1 Abcg1 leukocytes (Fig. 4 H). Recently, Samudio et al. (2010) pro- Both Glut1 inhibition and ApoA-I overexpression prevent / / posed that leukemia cells uncouple fatty acid oxidation from fat loss in Abca1 Abcg1 BM transplanted mice ATP synthesis and rely on de novo fatty acid synthesis to sup- We next explored the in vivo relevance of reducing the prolif- port fatty acid oxidation. Consistent with this observation, eration and ina fl mmatory status of myeloid cells through Glut1 / / pharmacological inhibition of carnitine palmitoyltransferase I inhibition on the adipose tissue loss of Abca1 Abcg1 BM / / (CPT-1) with etomoxir prevented the hyperproliferative chimeras. Mice that received WT or Abca1 Abcg1 BM / / response of Abca1 Abcg1 leukocytes (Fig. 4 H). Finally, transduced with lentiviruses encoding Glut1 shRNA exhib- carnitine supplementation of the cells to promote mito- ited a 1.6-fold reduction in Glut1 mRNA expression (not chondrial efflux of excess acetyl moieties from both glu - depicted) and a 20–30% reduction in the cell surface expres - cose and fat oxidation (Muoio et al., 2012) prevented the sion of Glut1 in their BM cells 7 wk after reconstitution, and / / higher proliferation rate of Abca1 Abcg1 leukocytes this was sufficient to normalize the Glut1 cell surface expres - / / (Fig. 4 H), providing additional evidence that the mito- sion to WT levels in Abca1 Abcg1 BM transplanted chondrial metabolism was responsible for the enhanced mice (Fig. 5 E). This was associated with normalization of / / / / proliferation in Abca1 Abcg1 leukocytes. Together, the 2-NBDG uptake in Abca1 Abcg1 BM leukocytes / / these findings showed that Abca1 Abcg1 BM cells di- (Fig. 5 F) and with normalization of the leukocyte counts in rected the available glucose toward oxidative phosphory- these mice (Fig. 5 G). Remarkably, the adipose tissue loss of / / lation (Fig. 4 I), which could explain the enhanced glucose mice transplanted with Abca1 Abcg1 BM was rescued / / oxidation observed in Abca1 Abcg1 BM transplanted by transduction with Glut1 shRNA lentiviral particles (Fig. 5 H). mice (Fig. 1 E). We previously reported that the myeloproliferative syndrome / / and inflammatory phenotype of Abca1 Abcg1 BM chi- Glut1 inhibition prevents both IL-3–mediated meras was reversed by overexpression of the human apoA-I Tg myeloid proliferation and TLR4-mediated transgene (ApoA-I ; Yvan-Charvet et al., 2008, 2010). We macrophage inflammatory response now show that overexpression of the human apoA-I trans- / / The increased glucose conversion into lipids at basal or gene also reversed fat loss in Abca1 Abcg1 BM trans- / / after IL-3 activation in Abca1 Abcg1 leukocytes was planted mice (Fig. 5 I). Additionally, the apoA-I transgene / / abolished by Fasentin, a Glut1 inhibitor (Fig. 5 A; Wood normalized Glut1 cell surface expression in Abca1 Abcg1 et al., 2008), as was the proliferation of these cells (Fig. 5 B). BM cells (Fig. 5 J). Together, our results suggest that suppres- Together, these findings suggest that IL-3R  subunit sig- sion of Glut1 in myeloproliferative disorders may represent a naling enhances the Glut1-dependent glucose uptake of novel approach not only to treat leukocytosis but also associ- / / Abca1 Abcg1 leukocytes to promote their proliferation. ated adipose tissue loss. Additionally, we now show that the / / Enhanced glucose consumption and Glut1 expression were rescue of the myeloproliferative disease of Abca1 Abcg1 previously observed in macrophages during inflammatory BM chimeras by overexpression of the human apoA-I transgene JEM Vol. 210, No. 2 345 / / Figure 4. The IL-3R–dependent proliferation of Abca1 Abcg1 leukocytes is driven by increased mitochondrial metabolism. (A–D) Effect of IL-3 treatment on glycolytic metabolites (glucose 6-phosphate/fructose 6-phosphate and fructose 1,6-diphosphate; A), citric acid metabolites / / (B), related mitochondrial products (GTP and FAD; C), and ATP/ADP ratio (D) in WT and Abca1 Abcg1 BM cells determined by LC-MS. (E) SDH activity in these cells. Results are means ± SEM of cultures from three independent mice. *, P < 0.05 versus WT controls; , P < 0.05 versus treat- / / ment. (F) WT and Abca1 Abcg1 BM cells were grown for 48 h in 20 mM glucose DMEM (DMEM rich) or low-glucose DMEM (DMEM poor) supplemented with 20 mM glucose or 20 mM galactose in the presence of IL-3. Proliferation rates were determined after 2-h [ H]thymidine pulse labeling. (G and H) BM cells were grown for 48 h in liquid culture containing 10% FBS IMDM in the presence of the indicated chemical compounds and IL-3. Proliferation rates were determined after 2-h [ H]thymidine pulse labeling. Results are means ± SEM of an experiment performed in triplicate. *, P < 0.05 versus WT controls; , P < 0.05 versus untreated condition. BCAA, branched-chain amino acids; DGATi, diglyceride acyltransferase inhibitor. 346 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e (Yvan-Charvet et al., 2010) was in part associated with re- hallmark of chronic inflammatory diseases such as chronic duced Glut1 levels on leukocytes and prevented their adi - infection, cancer, and heart failure (Delano and Moldawer, pose loss. 2006), the underlying mechanisms remain poorly under- stood, leading to a lack of effective therapy. Most of the ApoA-I overexpression prevents the increased glucose emerging therapeutic approaches to prevent adipose tissue oxidation and adipose tissue atrophy in Flt3-ITD and loss focus on reducing the elevated circulating cytokine levels Mpl-W515L mutant–mediated myeloproliferative disorders (Argilés et al., 2011) in part because of their action on insulin As altered high-density lipoprotein (HDL) cholesterol ho- resistance and fat mobilization (Delano and Moldawer, 2006; meostasis has been previously observed in myeloprolifera- Das et al., 2011). However, traditional antiinflammatory ap - tive disorders (Fiorenza et al., 2000; Westerterp et al., proaches such as COX-2 inhibitors showed for instance lim- 2012), we further tested whether the increased glucose ited effectiveness in reversing the metabolic abnormalities oxidation and associated adipose tissue atrophy observed seen in cancer patients (Kumar et al., 2010). Thus, there is a / / in Abca1 Abcg1 BM transplanted mice could be ob- crucial need to better understand the mechanisms of adipose served as a more general phenotype in other mouse models of loss in this state of chronic hypermetabolism. Using three dif- myeloproliferative disorders and whether overexpression of ferent mouse models of myeloproliferative disorders, includ - the human apoA-I transgene would still be an efficient thera - ing two models based on expression of human leukemia peutic in these models. The Flt3-ITD and Mpl-W515L al- disease alleles (Flt3-ITD and Mpl-W515L), we uncovered a leles have been identified in patients with acute myeloid glucose steal mechanism by which proliferating and inflam - leukemia and myelofibrosis, respectively, and confer a fully matory myeloid cells take up and oxidize glucose during the penetrant myeloproliferative disorder in mice (Kelly et al., feeding period, contributing to energy dissipation and sub - 2002; Pikman et al., 2006). Accordingly, mice that received sequent loss of adipose mass. This reflected in part increased BM transduced with retroviruses encoding Flt3-ITD or Mpl- numbers of proliferating myeloid cells and tissues infiltrated W515L exhibited massive myeloid expansion compared with with inflammatory myeloid cells. control retrovirus (Fig. 6, A and B), an effect which was par - Although only partially understood, there is a relationship tially reversed by overexpression of the human ApoA-I trans- between leukemia-causing genes and cellular energy metabo- gene (Fig. 6, A and B). We also observed epididymal fat lism as the survival and proliferation of leukemic cells may re- mass atrophy and reduced plasma leptin levels in both models quire glucose for de novo lipid biosynthesis (DeBerardinis (Fig. 6 C and not depicted, respectively). Massive leukocyte et al., 2008; Lunt and Vander Heiden, 2011). The increased / / infiltration was also observed in their adipose tissue (not de - glucose utilization in hyperproliferating Abca1 Abcg1 / / picted). Similar to Abca1 Abcg1 BM transplanted mice, BM myeloid cells resulted in part from a Ras-dependent up- these features were associated with an increased RQ during regulation of Glut1 expression in response to enhanced IL-3 the dark phase (Fig. 6, D and E), reflecting an 20% increase signaling (Flier et al., 1987; Yvan-Charvet et al., 2010). Simi- in glucose oxidation during this period (Fig. 6 F). Interest- larly, Mpl and Flt3 are receptor tyrosine kinases coupled to Ras ingly, Mpl-W515L and Flt3-ITD mutant leukocytes exhib- signaling, and activating mutations in these receptors (Kelly ited a significant increase in Glut1 cell surface expression et al., 2002; Pikman et al., 2006) were shown to cause higher (Fig. 6 G). Remarkably, overexpression of the human ApoA-I Glut1 expression. In this context, it is tempting to parallel our transgene not only prevented the fat mass loss (Fig. 6 C) of myeloproliferative mouse models with other cancer models in mice bearing the Mpl-W515L and Flt3-ITD mutations, but which malignant cells rely on high levels of aerobic glycolysis also significantly reversed the increased Glut1 cell surface ex - as the major source of ATP to fuel cellular proliferation, known pression in Mpl-W515L and Flt3-ITD leukocytes (Fig. 6 G) as the Warburg ee ff ct (Vander Heiden et al., 2009). However, that was associated with increased RQ (Fig. 6, H and I) and the classical view of the Warburg ee ff ct, which involves defect glucose oxidation during the feeding period (Fig. 6 F). in mitochondrial oxidative phosphorylation, has been recently challenged, and mitochondrial metabolism may indeed be DISCUSSION functional in die ff rent types of tumor cells (Jose et al., 2011). Although there is growing evidence that weight loss, and We now provide both in vitro and in vivo evidence for in- especially the loss of muscle and adipose tissue mass, is a creased mitochondrial potential in leukocytes of our mouse (I) Scheme illustrating the induction of glycolysis through modulation of plasma membrane cholesterol by ABCA1 and ABCG1 deficiency and increased mitochondrial metabolism that produces ATP and synthesizes lipids for cell growth. Lack of these transporters promotes growth fac- tors dependent on IL-3R signaling–mediated glycolysis as reflected by (a) enhanced Hk2 and PFKp mRNA expression, (b) enhanced glycolytic metabolites content, (c) enhanced glucose oxidation (i.e., conversion into CO ), and (d) reversal of enhanced glucose oxidation by removal of cel- lular cholesterol by cyclodextrin. The pyruvate generated through glycolysis is directed into the TCA cycle and increases mitochondrial metabolism as reflected by (a) enhanced mRNA expression of fumarase and SDHb, (b) enhanced SDH activity, (c) enhanced mitochondrial metabolites and + / / mitochondrial membrane potential (H ), and (d) enhanced ATP/ADP ratio. Increased lipid synthesis was also observed in Abca1 Abcg1 BM cells as reflected by (a) enhanced mRNA expression of ACL and (b) enhanced glucose conversion into lipids. JEM Vol. 210, No. 2 347 / / Figure 5. Inhibition of Glut1 or overexpression of the human apoA-I transgene rescues adipose atrophy in Abca1 Abcg1 BM chimeras. 14 / / (A) Effect of IL-3 treatment on [ C]glucose conversion into lipids in WT and Abca1 Abcg1 BM cells in the presence or absence of 50 µM fasentin, a / / Glut1 inhibitor. (B) Effect of Fasentin on IL-3–mediated proliferation in WT and Abca1 Abcg1 BM cells. (C and D) Effect of Fasentin on LPS-mediated / / inflammatory (C) and glycolytic (D) responses in WT and Abca1 Abcg1 BM-derived macrophages. Values were normalized to ribosomal 18S. Results are means ± SEM of three independent experiments performed in triplicate. *, P < 0.05 versus WT mice; , P < 0.05 versus LPS treatment. (E and F) Quanti- fication of Glut1 cell surface expression (E) and 2-NBDG uptake by flow cytometry (F) in CD45 leukocytes isolated from the BM of WT recipients mice / / transplanted with WT or Abca1 Abcg1 BM transduced with a lentivirus encoding Glut1 shRNA 7 wk after reconstitution. (G and H) Peripheral leuko- cyte counts (G) and epididymal fat mass (H) of these mice at the end of the experiment. (I and J) Histograms showing epididymal fat mass loss (I) and Glut1 cell surface expression (J) in CD45 peripheral leukocytes in chow-fed transgenic recipient mice overexpressing the human apoA-I transgene trans- / / planted with Abca1 Abcg1 BM. Results are ± SEM of an experiment of four to six animals per group. *, P < 0.05 versus WT recipients transplanted with WT BM; , P < 0.05 versus control retrovirus. MFI, mean fluorescence intensity. model of myeloproliferative disorder. Surprisingly, not only metabolism of fatty acids (i.e., fats burn in the fire of car - inhibition of PDPs but also inhibition of CPT-1 prevented bohydrates; Samudio et al., 2010). Although the mechanisms / / the proliferation of Abca1 Abcg1 leukocytes. This em- orchestrating this phenomenon remain to be investigated, phasizes a scenario in which high rates of aerobic glycolysis it appears unlikely to be mediated by triglyceride or ce- in leukemia cells are necessary to support the mitochondrial ramide biosynthesis. Interestingly, the antiproliferative effect 348 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e Figure 6. HDL prevents the adipose tissue atrophy and enhanced glucose oxidation caused by Mpl-W515L and Flt3-ITD activating muta- +  + tions. (A) Representative dot plots and quantification of the splenic myeloid cells (CD115 monocytes and CD115 Gr1 neutrophils) of WT and ApoA-I transgenic recipient mice transplanted with Mpl-W515L– and Flt3-ITD–transduced BM cells. (B and C) Quantification of the myeloid cells (B) and epididy - mal fat mass (C) of these mice. (D, E, F, H, and I) RQ measured by indirect calorimetry (D, E, H, and I) and quantification of the nocturnal glucose oxidation of these mice (F). (G) Quantification of the cell surface expression of Glut1 in CD45 leukocytes of these mice. Results are mean ± SEM of an experiment of four to five animals per group. *, P < 0.05 versus control mice; , P < 0.05 versus respective WT recipients. MFI, mean fluorescence intensity. JEM Vol. 210, No. 2 349 of carnitine suggested that prevention of oxidative phos- leukocytes, reduced glucose oxidation during the feeding pe - phorylation by efflux of excess mitochondrial acetyl moi - riod, and prevented adipose tissue loss. eties (Muoio et al., 2012) might represent a novel strategy for In conclusion, our study elucidates a glucose steal mecha- the treatment of hematological malignancies. In conclusion, nism by proliferating and inflammatory myeloid cells that increased glucose oxidation and mitochondrial metabolism– contributes to depletion of adipose tissue in myeloprolifera- dependent proliferation of myeloid cells likely contributed tive disorders. This mechanism involved enhanced glucose to energy dissipation and subsequent loss of adipose mass. oxidation by myeloid cells that led to energy dissipation with Leukocytes and adipocytes can communicate through in- consequences on fat storage. Inhibition of glucose uptake by teraction between secreted inflammatory cytokines and their leukocytes with a Glut1 inhibitor or by treatments that in- cognate receptors (Lumeng and Saltiel, 2011; Odegaard and crease HDL levels could ultimately provide new therapeutic Chawla, 2011). The finding that highly infiltrated adipose approaches not only to limit cell proliferation but also to pre- depots were associated with impaired Glut4 translocation to vent the energy imbalance and fat atrophy observed in my- / the plasma membrane in response to insulin in Abca1 eloproliferative disorders and in other human malignancies. / Abcg1 BM chimeras was strongly implied by prior studies MATERIALS AND METHODS indicating insulin resistance in peripheral tissues secondary to / / / / Mice and treatments. WT, Abca1 , Abcg1 , and Abca1 Abcg1 increased tumor-derived cytokines (Delano and Moldawer, littermates in a mixed C57BL/6 × DBA background (Yvan-Charvet et al., 2006). Nevertheless, these findings provide strong evidences Tg 2007) were used for this study. Human apoA-1 transgenic (hapoA-1 ) mice that local inflammation driving adipose tissue insulin resis - were obtained from the Jackson Laboratory. BM transplantation into the ge- tance is not restricted to a role on fat mobilization in cancer- Tg netically uniform F1 generation obtained by crossing C57BL/6 WT, hapoA-1 associated adipose tissue loss (Das et al., 2011). This suggests mice with WT DBA mice (The Jackson Laboratory) was performed as pre- viously described (Yvan-Charvet et al., 2007). Animal protocols were ap- that myeloproliferative cells develop efficient strategies to proved by the Institutional Animal Care and Use Committee of Columbia divert glucose from its expected destination, i.e., fat storage University. Animals had ad libitum access to both food and water. as a mean to meet their energetic needs. In part, this could be related to a shunt of glucose to the pentose phosphate HSC transplantation. HSC transplantation was adapted from a previ- pathway that has been recently shown to modulate the in- + ously described protocol (Wagers et al., 2002). In brief, congenic CD45.1 flammatory response of activated immune cells (Ham, M., B6.SJL-Ptprca—Pep3b-/BoyJ were purchased from the Jackson Labora- tory and used to isolate Sca-1–depleted BM cells by FACS sorting. HSCs et al. 2008. The FASEB Journal Meeting. Abstr. 615.1; Blagih were isolated by FACS sorting of lineage-depleted BM from WT and and Jones, 2012). This could ultimately contribute not only / / Abca1 Abcg1 backcrossed for 10 generations on a C57/BL6 back- to the enhanced glucose oxidation observed in our mouse ground, based on the following cell surface markers: c-kit and Sca-1 (LSK, models of myeloproliferative disorders but also to the local  + + Lin Sca c-kit ). Lethally irradiated WT recipients were i.v. injected with adipose tissue insulin resistance mediated by enhanced in - 6 + 10 cells containing a mixture of CD45.1 Sca-1–depleted BM cells and + / / flammatory response. CD45.2 LSK cells from either WT or Abca1 Abcg1 mice in the ratio 1:2,000. Transplanted recipients were screened by flow cytometry for re - Finally, we showed that Glut1 blockade prevented both / / constitution of CD45.2 leukocytes in peripheral blood at 6 wk after trans- basal and IL-3–induced proliferation of Abca1 Abcg1 plant. More than 90% of leukocytes stained for the congenic marker CD45.2, leukocytes in vitro and prevented LPS-induced inflammatory thereby confirming the engraftment of LSK cells (Wagers et al., 2002). / / cytokine response in Abca1 Abcg1 macrophages. This translated in vivo into the rescue of leukocytosis and adipose Retroviral BM transplantation. The retroviral BM transplant assay was / / tissue infiltration of Abca1 Abcg1 BM transplanted mice performed as previously described (Pikman et al., 2006). In brief, control, Mpl-W515L and Flt3-ITD retroviral supernatants were titered and used to in response to Glut1 inhibition. Remarkably, this prevented transduce WT BM cells. In independent experiments, premade control and the adipose tissue loss of these mice, confirming the key Glut1 shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) were role of myeloid Glut1 in the diversion of energy stores from / / used to transduce WT and Abca1 Abcg1 BM cells. BM cells were cul- adipose to myeloid cells. Similarly, overexpression of the tured for 24 h in transplantation media (RPMI + 10% FBS + 6 ng/ml IL-3, human ApoA-I transgene, previously shown to raise plasma 10 ng/ml IL-6, and 10 ng/ml stem cell factor) and treated by spin infection HDL levels and prevent the myeloproliferative syndrome of with retroviral supernatants (1 ml supernatant per 4 × 10 cells in the pres- / / ence of polybrene) and centrifuged at 1,800 g for 90 min. The spin infection Abca1 Abcg1 BM transplanted mice (Yvan-Charvet was repeated 24 h later. After washing, the cells were used for BM transplan- et al., 2010), reduced Glut1 cell surface expression in leuko- tation into lethally irradiated WT recipient mice. cytes and prevented their fat loss. This reflected in part the removal of excess cholesterol from plasma membrane in Energy expenditure. Metabolic activity was performed by in vivo indi- / / Abca1 Abcg1 myeloid cells that prevented the IL-3R rect open circuit calorimetry at the Mouse Phenotyping Core of Columbia signaling (Yvan-Charvet et al., 2010) and subsequent in- University using a CaloSys calorimetry system (TSE Systems, Inc). Ani- mals were placed into experimental chambers with free access to food and crease in Glut1-dependent glucose uptake (Fig. 3 G). These water for a 4-d consecutive period. Food intake was recorded with an au - findings were further extended in two mouse models of my - tomated feeding monitor system through the study period. Constant air - eloproliferative disorders based on expression of human leu- flow (0.5 liter/min) was drawn through the chamber and monitored by a kemia disease alleles (Flt3-ITD and Mpl-W515L) in which mass-sensitive flow meter. To calculate oxygen consumption (VO ), carbon overexpression of the human ApoA-I transgene reduced my- dioxide production (VCO ), and RQ (ratio of VCO to VO ), gas con- 2 2 2 eloid expansion, decreased cell surface expression of Glut1 in centrations were monitored at the inlet and outlet of the scaled chambers. 350 HDL and Glut1 in wasting syndrome | Gautier et al. A r t i c l e Total metabolic rate (energy expenditure) was calculated from oxygen (GK1.5), CD8b (53-6.7), CD19 (eBio1D3), CD45R (B220, RA3-6B2), consumption and carbon dioxide production using Lusk’s equation and Gr-1 (Ly6G, RB6-8C5), Cd11b (Mac1, M1/70), Ter119 (Ly76) and expressed as watts per kilogram to the 0.75 power of body weight (Yvan- NK1.1 (Ly53, PK136), c-Kit (CD117, ACK2), and Sca-1 (D7) were all Charvet et al., 2005). Glucose and lipid oxidation were calculated as prev- i purchased from eBioscience. Glut1-FITC antibody was purchased from ously described (Yvan-Charvet et al., 2005). R&D Systems. These antibodies were used to sort HSCs and stain for leu- kocytes in peripheral blood and tissues. 14 14 In vivo 2-[ C]-DG uptake. Uptake of 2-[ C]-DG in peripheral tissues was measured as previously described (Rofe et al., 1988). In brief, 2 µCi BM harvest and treatment. Primary BM cells were resuspended in 2-[ C]-DG was i.v. injected, and blood samples were collected at 5, 10, 20, IMDM (Gibco) containing 10% FCS (STEMCELL Technologies) and cul- 30, and 40 min. Blood glucose was monitored through the study period tured for 1 h in tissue culture flasks to remove adherent cells, including mac - with a glucometer (Roche). After 40 min, adipose tissue, skeletal muscle, rophages. Suspended cells were then cultured for 72 h in the presence of 6 ng/ml heart, lung, spleen, and brain were rapidly dissected, weighed, and homog - IL-3 (R&D Systems). In some experiments, the farnesyl transferase inhibitor enized with 5% HClO solution. BM cells were collected from leg bones, (EMD Millipore) was used at the final concentration of 1 µM, fasentin and peripheral leukocytes were obtained after RBC lysis. The radioactivity (Sigma-Aldrich) at 50 µM, DL-AP3 (Tocris Bioscience) at 50 µM, myriocin incorporated in both 2-[ C]-DG and its 6-phosphate derivative was mea- (Sigma-Aldrich) at 10 µM, diglyceride acyltransferase inhibitor (EMD Milli- sured in the HClO extract and expressed as total radioactivity per tissue pore) at 40 µM, etomoxir (Sigma-Aldrich) at 40 µM, branched-chain amino weight. The rate constant of net tissue uptake of 2-[ C]-DG was calculated acids (leucine, isoleucine, and valine; Sigma-Aldrich) at 1 mM, glutathione as described previously (Rofe et al., 1988). In brief, the relative glucose monoethyl ester (EMD Millipore) at 10 mM, tempol (EMD Millipore) at uptake was calculated by dividing the area under the blood 2-[ C]-DG 4 mM, and carnitine (Sigma-Aldrich) at 1 mM. For proliferation assays, disappearance curve (cpm/min/ml) to the steady-state glucose concentra- cells were pulsed for 2 h with 2 µCi/ml [ H]thymidine, and the radioac- 14 6 tion (mM) multiplied by the tissue 2-[ C]-DG (cpm/g tissue or cpm/10 tivity incorporated into the cells was determined by standard procedures cells for the BM) at 40 min. using a liquid scintillation counter. In one experiment (Fig. 4 F), cells were resuspended in 20 mM glucose DMEM (DMEM rich; Gibco) or Blood parameters. Plasma leptin and insulin were determined by ELISA low-glucose DMEM (DMEM poor; Gibco) supplemented with 20 mM (Mouse Leptin Quantikine ELISA kit [R&D Systems]; Mouse insulin ELISA kit glucose or 20 mM galactose (Sigma-Aldrich) and cultured for 48 h in the [Crystal Chem, Inc.]). Plasma TNF was measured by Luminex assay (Cytokine presence of 6 ng/ml IL-3. SDH activity was determined by an ELISA Core Laboratory). Blood glucose was assayed with a glucometer (Roche). kit according to the manufacturer’s instructions (MitoSciences). BM- derived macrophages were isolated and cultured in 10% FBS in DMEM Histopathology. Mice were euthanized in accordance with the American supplemented with M-CSF for 5–10 d before the experiment. Where Veterinary Association Panel of Euthanasia. Adipose tissue was serially paraf- indicated, macrophages were incubated with 100 ng/ml LPS (Escherichia fin sectioned and stained with hematoxylin and eosin (H&E) for morpho - coli 0111:B4; Sigma-Aldrich). logical analysis as previously described (Yvan-Charvet et al., 2007). Glucose metabolism experiments. Isolated BM cells were cultured for Adipose tissue cellularity. Cellularity of epididymal adipose tissue was 24 h with or without 6 ng/ml IL-3 and 50 µM fasentin, a Glut1 inhibitor. determined as previously described (Yvan-Charvet et al., 2005). In brief, Where indicated, BM cells were differentiated into macrophages as described images of isolated adipocytes were acquired from a light microscope above. Cells were next incubated with 5.5 mM [ C]glucose in 2% BSA (IX-70; Olympus) fitted with a charge-coupled device camera (RS Photo - Krebs-Ringer bicarbonate buffer, pH 7.4. After 2 h, the generated CO metrics), and the measurement of 400 cell diameters was performed allow- and the C incorporation into lipids were quantified as previously described ing calculation of a mean fat cell weight. Tissue triglyceride content was (Yvan-Charvet et al., 2005). In some experiments, cellular cholesterol was measured from a sample of adipose tissue using a commercial kit (Sigma- depleted by 5 mM cyclodextrin for 1 h before growth factor treatment as Aldrich). Fat cell number was estimated by dividing the tissue lipid content previously described (Yvan-Charvet et al., 2010). by the fat cell weight. Directed metabolomic experiments. Isolated BM cells (10 cells) were Glucose tolerance tests. After 6 h of fasting, mice were injected i.p. with cultured with or without 6 ng/ml IL-3 for 24 h. The next day, suspended d-glucose (2 g/kg of body weight), and blood samples were obtained by tail cells were centrifuged at 1,000 rpm for 5 min, and pellets were rapidly bleeding at 0, 15, 30, 60, 90, and 120 min after injection (Yvan-Charvet washed (less than 10 s) with a mass spectrometry–compatible bue ff r (150 mM et al., 2005). Blood glucose was assayed with a glucometer (Roche). ammonium acetate solution) to prevent the presence of sodium and phos- phate in the residue and limit interference with LC-MS analyses. After a sec- ond step of centrifugation, pellets were immediately frozen in liquid nitrogen Flow cytometry analysis. BM cells were collected from leg bones or to quench metabolism according to the University of Michigan Molecular spleen, lysed to remove RBCs, and filtered before use. Freshly isolated cells Phenotyping Core facility’s instructions. Samples were shipped on dry ice to were stained with the appropriate antibodies for 30 min on ice. For periph- the Molecular Phenotyping Core facility where metabolites were extracted eral blood leukocytes analysis, 100 µl blood was collected into EDTA tubes by exposing the cells to a chilled mixture of 80% methanol, 10% chloroform, before RBC lysis, filtration, and staining for 30 min on ice. To assess the up - and 10% water. Glycolytic and citric acid metabolites were then analyzed by take of 2-NBDG, prestained peripheral blood leukocytes (Yvan-Charvet the Molecular Phenotyping Core facility using LC-MS as previously de- et al., 2010) were incubated with 10 µM 2-NBDG (Invitrogen) for 30 min, scribed (Yuneva et al., 2012). followed by flow cytometric detection of fluorescence produced by the cells (Zou et al., 2005). The mitochondrial membrane potential was analyzed with 25 nM fluorescent TMRE (AnaSpec) staining for 30 min on prestained Western blot analysis. 2-h-fasted mice were administrated insulin i.p. leukocytes. Viable cells, gated by light scatter or exclusion of CD45 cells, (0.4 U/mouse). After 5 min, freshly isolated adipose tissue was homoge- were analyzed on a four-laser LSRII cell analyzer (BD) or sorted on a FAC- nized as previously described (Yvan-Charvet et al., 2005), and cell ex- SAria Cell Sorter (BD), both running with DiVa software (BD). Data were tracts were either directly used to measure phospho-Akt (clone 587F11; analyzed using FlowJo software (Tree Star). Cell Signaling Technology) by Western blot analysis or fractionated by differential centrifugation to isolate plasma membranes and low-density Antibodies. Anti–mouse CD45 (clone 30F11), CD115 (AFS98), TCR- microsomes (Yvan-Charvet et al., 2010) and quantify Glut4 expression (H57-597), F4/80 (BM8), CD2 (RM2-5), CD3e (145-2C11), CD4 (clone 1F8; R&D Systems). JEM Vol. 210, No. 2 351 RNA analysis. Total RNA extraction, cDNA synthesis, and real-time Hawkinson, J.E., M. Acosta-Burruel, and P.L. Wood. 1996. The metabo- PCR were performed as described previously (Yvan-Charvet et al., 2005). tropic glutamate receptor antagonist L-2-amino-3-phosphonopropionic Ribosomal 18S RNA expression was used to account for variability in the acid inhibits phosphoserine phosphatase. Eur. J. Pharmacol. 307:219– initial quantities of mRNA. 225. http://dx.doi.org/10.1016/0014-2999(96)00253-1 Herman, M.A., and B.B. Kahn. 2006. Glucose transport and sensing in the maintenance of glucose homeostasis and metabolic harmony. J. Clin. Statistical analysis. Statistical signic fi ance was performed by two-tailed para - Invest. 116:1767–1775. http://dx.doi.org/10.1172/JCI29027 metric Student’s t test or by one-way ANOVA (four-group comparisons) with Jose, C., N. Bellance, and R. Rossignol. 2011. Choosing between gly- a Bonferroni multiple comparison post test (GraphPad Software). colysis and oxidative phosphorylation: a tumor’s dilemma? Biochim. Biophys. Acta. 1807:552–561. http://dx.doi.org/10.1016/j.bbabio.2010 We thank Dr. Kristie Gordon for assistance with flow cytometry, Pr. Pascal Ferre for .10.012 scientific discussion, and the French Cancer Research Association (ARC). Kelly, L.M., Q. Liu, J.L. Kutok, I.R. Williams, C.L. Boulton, and D.G. This work utilized Core Services supported by a National Institutes of Health Gilliland. 2002. FLT3 internal tandem duplication mutations associated (NIH) grant (DK089503) to the University of Michigan. 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The Journal of Experimental Medicine – Pubmed Central

Published: Feb 11, 2013

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